WO2016035816A1 - Method for inducing differentiation of pluripotent stem cells into cardiomyocytes, and culture medium additive, differentiation-induction regulator, culture medium, culture medium preparation kit, and kit for inducing differentiation of pluripotent stem cells into cardiomyocytes suitable for said method - Google Patents

Method for inducing differentiation of pluripotent stem cells into cardiomyocytes, and culture medium additive, differentiation-induction regulator, culture medium, culture medium preparation kit, and kit for inducing differentiation of pluripotent stem cells into cardiomyocytes suitable for said method Download PDF

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WO2016035816A1
WO2016035816A1 PCT/JP2015/074947 JP2015074947W WO2016035816A1 WO 2016035816 A1 WO2016035816 A1 WO 2016035816A1 JP 2015074947 W JP2015074947 W JP 2015074947W WO 2016035816 A1 WO2016035816 A1 WO 2016035816A1
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medium
induction
differentiation
cardiomyocyte
inducing
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French (fr)
Japanese (ja)
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篤彦 内藤
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国立大学法人東京大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention relates to a method for inducing differentiation of cardiomyocytes from pluripotent stem cells, a medium additive added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells suitable for the method, and from pluripotent stem cells to cardiac muscle
  • a differentiation-inducing regulator added to a medium for inducing cell differentiation, a medium for inducing cardiomyocyte differentiation from pluripotent stem cells, a medium preparation kit for inducing cardiomyocyte differentiation from pluripotent stem cells, and
  • the present invention relates to a kit for inducing differentiation of cardiomyocytes from pluripotent stem cells.
  • iPS cells induced pluripotent stem cells
  • cardiomyocytes differentiated from human iPS cells are expected to be applied to various uses such as regenerative medicine, drug discovery research, and drug safety tests.
  • regenerative medicine for example, it is expected to restore lost heart function by transplanting myocardial cells differentiated from self iPS cells or banked iPS cells into the heart.
  • drug discovery research for example, the development of new drugs for cardiovascular diseases and intractable diseases using iPS cells of healthy individuals and cardiomyocytes differentiated from disease-specific iPS cells prepared from patients with intractable diseases is used for analysis and screening. Is expected to be.
  • cardiomyocytes are expected to be used for screening for cardiotoxicity during development or for developed drugs.
  • cardiomyocytes In order for cardiomyocytes to be industrially used for various applications such as regenerative medicine, drug discovery research, and drug safety testing, it is necessary to stably obtain cardiomyocytes in large quantities from pluripotent stem cells.
  • the method for inducing differentiation of cardiomyocytes is technically easy and is composed of a small number of procedures and must be an inexpensive method.
  • the cardiomyocytes show an action potential having a large and clear plateau phase, and stably show a large peak potassium current (300 pA or more), a peak calcium current (1 nA or more), and a peak sodium current (6.5 nA or more). High quality is preferable.
  • Non-Patent Document 1 a method for inducing differentiation of cardiomyocytes by an embryoid body formation method has been proposed (see, for example, Non-Patent Document 1).
  • the cells are once cultured in a flat state in the absence of the feeder cells, and after detaching again, the suspension culture is performed (see FIG. 1).
  • StemPro (registered trademark) 34 manufactured by Invitrogen is used as a medium for suspension culture
  • L-glutamine is 2 mM
  • transferrin is 150 ⁇ g / mL
  • ascorbic acid is 50 ⁇ g / mL
  • 0.5 ng of bone morphogenetic protein 4 (hereinafter sometimes referred to as “BMP4”) is used as a differentiation induction regulator from the beginning of suspension culture until the first day.
  • BMP4 was 10 ng / mL
  • basic fibroblast growth factor hereinafter sometimes referred to as “bFGF”
  • activin A was 6 ng / mL.
  • mL was added, and from day 4 to day 8, vascular endothelial growth factor (hereinafter sometimes referred to as “VEGF”) was 10 ng / mL, and 150 ng / mL of protein Dickkopf1 (hereinafter sometimes referred to as “Dkk-1”) is added, and after 8 days, VEGF is 10 ng / mL, Dkk-1 is 150 ng / mL, and bFGF is 5 ng / mL.
  • VEGF vascular endothelial growth factor
  • Dkk-1 protein Dickkopf1
  • cardiomyocytes can be obtained in a large amount because differentiation into cardiomyocytes can be induced by suspension culture (three-dimensional culture).
  • cytokines activin A, BMP4, Dkk-1, bFGF, and VEGF
  • StemPro registered trademark
  • No. 34 uses 2-mercaptoethanol, which is a toxic substance
  • Human-ExCyte which is a lipid component whose component is not clear
  • human serum albumin that is expensive and has a large lot-to-lot difference.
  • cardiomyocytes cannot be obtained at low cost.
  • Non-Patent Document 2 A method for inducing differentiation of cardiomyocytes without using cytokines has also been proposed (see, for example, Non-Patent Document 2).
  • the above proposal after feeder-less culture, the cells are isolated into single cells and then seeded at a high density to perform adhesion culture (see FIG. 2).
  • low molecular weight compounds such as CHIR99021 and IWP2 are used instead of expensive cytokines with large lot-to-lot differences.
  • the above proposal is performed by adhesion culture, and there is a problem that it is difficult to obtain a large amount of cardiomyocytes, and it is differentiated unless the cells can be adapted to feeder-less culture.
  • the timing of exchanging the culture medium is very strict, and there is a problem that it is technically difficult, for example, when the culture medium is replaced, the cells are easily detached.
  • Non-Patent Document 3 a method of inducing differentiation of cardiomyocytes by using both adhesion culture and suspension culture has been proposed (see Non-Patent Document 3, for example).
  • suspension culture is performed after feeder cell culture, and then further suspension culture is performed via adhesion culture (see FIG. 3).
  • low molecular compounds such as CHIR99021, BIO, and KY02111 are used instead of expensive cytokines with large lot-to-lot differences, it is considered that cardiomyocytes can be obtained stably and inexpensively.
  • the above-mentioned proposal uses both adhesion culture and suspension culture, it consists of a plurality of technically difficult procedures and has a problem that it is very complicated.
  • the present invention is a method for inducing differentiation of cardiomyocytes from pluripotent stem cells capable of producing high-quality cardiomyocytes in large quantities, stably, inexpensively and simply, and suitable for the method.
  • An object of the present invention is to provide a medium additive, a differentiation induction regulator, a medium, a medium preparation kit, and a kit for inducing differentiation of cardiomyocytes from pluripotent stem cells.
  • Means for solving the problems are as follows. That is, ⁇ 1> Embryoid body formation step of suspension culture of pluripotent stem cells to form embryoid bodies, Mesoderm induction step of culturing the embryoid body in suspension and inducing mesoderm, A suspension culture of the embryoid body after the mesoderm induction, and a cardiomyocyte induction step of inducing cardiomyocytes,
  • the cardiomyocyte induction step includes a first cardiomyocyte induction process and a second cardiomyocyte induction process;
  • the medium in the embryoid body formation step is any one of a medium in which a ROCK inhibitor is added to a first differentiation induction medium, and a medium in which a ROCK inhibitor is added to a one-part differentiation induction medium.
  • the medium in the mesoderm induction step is any one of a medium obtained by adding a Wnt signal activator to a second differentiation induction medium and a medium obtained by adding a Wnt signal activator to a one-part differentiation induction medium.
  • the second differentiation induction medium includes a Wnt signal inhibitor, a transforming growth factor ⁇ (hereinafter sometimes referred to as “TGF- ⁇ ”) signal inhibitor, and an estrogen.
  • the medium in the second cardiomyocyte induction treatment is either a medium in which an estrogen-like agent is added to the second differentiation-inducing medium, or a medium in which an estrogen-like agent is added to a one-part differentiation induction medium.
  • the first differentiation induction medium is a medium obtained by adding insulin, transferrin, albumin, and 1-thioglycerol to a basal medium;
  • the second differentiation-inducing medium is an Iskov-modified Dulbecco medium that contains albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL- ⁇ -tocopherol acetate, N-acetyl-L-cysteine, 1-thiol.
  • the one-part differentiation induction medium is transferred to Iscov modified Dulbecco medium with transferrin, albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL- ⁇ -tocopherol acetate, N-acetyl-L-cysteine, 1
  • Iscov modified Dulbecco medium with transferrin, albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL- ⁇ -tocopherol acetate, N-acetyl-L-cysteine, 1
  • a method for inducing differentiation of cardiomyocytes from pluripotent stem cells characterized in that the medium is a medium supplemented with thioglycerol and ascorbic acid.
  • a medium additive added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells A medium additive comprising at least one of albumin and 1-thioglycerol.
  • a differentiation-inducing regulator added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells A differentiation-inducing regulator comprising at least one selected from the group consisting of a ROCK inhibitor, a Wnt signal activator, a Wnt signal suppressor, a TGF- ⁇ signal inhibitor, and an estrogen-like agent .
  • ⁇ 4> A medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
  • the basal medium is at least one selected from the group consisting of Dulbecco's modified Eagle medium / nutrient mixture F-12 ham, Dulbecco's modified Eagle medium, Iskov's modified Dulbecco medium, RPMI 1640 medium, and ⁇ MEM medium. It is.
  • a medium preparation kit for inducing differentiation of cardiomyocytes from pluripotent stem cells The culture medium additive according to ⁇ 2>, The differentiation-inducing regulator according to ⁇ 3>, A medium comprising at least one basal medium selected from the group consisting of Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 Ham, Dulbecco's Modified Eagle Medium, Iskov's Modified Dulbecco Medium, RPMI 1640 Medium, and ⁇ MEM Medium
  • This is a preparation kit.
  • a kit for inducing differentiation of cardiomyocytes from pluripotent stem cells comprising at least one of the medium according to ⁇ 4> and the medium preparation kit according to ⁇ 5>. is there.
  • FIG. 1 is a diagram for explaining an example of a conventional method for inducing differentiation of cardiomyocytes from pluripotent stem cells.
  • FIG. 2 is a diagram for explaining another example of a conventional method for inducing differentiation of cardiomyocytes from pluripotent stem cells.
  • FIG. 3 is a diagram for explaining another example of a conventional method for inducing differentiation of cardiomyocytes from pluripotent stem cells.
  • FIG. 4 is a diagram for explaining an example of a method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
  • FIG. 5 is a diagram showing the results of Test Example 1.
  • FIG. 6 is a diagram showing the results of clone 1 in Test Example 2.
  • FIG. 5 is a diagram showing the results of Test Example 1.
  • FIG. 6 is a diagram showing the results of clone 1 in Test Example 2.
  • FIG. 7 is a diagram showing the results of clone 2 in Test Example 2.
  • FIG. 8 is a diagram showing the results of Test Example 3.
  • FIG. 9 is a diagram showing the results of clone 1 in Test Example 4.
  • FIG. 10 is a diagram showing the results of clone 2 in Test Example 4.
  • FIG. 11 is a diagram -1 showing the results of Test Example 5.
  • FIG. 12 is a diagram 2 showing the results of Test Example 5.
  • FIG. 13 is a diagram showing the results of Test Example 6.
  • FIG. 14 is a diagram showing the results when bFGF was added in Test Example 7.
  • FIG. 15 is a diagram showing the results when CHIR99021 in Test Example 7 is added.
  • FIG. 16 is a diagram showing the results of Test Example 8.
  • FIG. 17 is a diagram showing the results of Test Example 9.
  • FIG. 18 is a diagram showing the results of Test Example 10.
  • FIG. 19 is a diagram showing the results of Test Example 11.
  • FIG. 20 is a diagram showing the results of Test Example 12.
  • FIG. 21 is a diagram showing the results of Test Example 13.
  • FIG. 22 is a diagram showing the results of Test Example 14.
  • FIG. 23 is a diagram showing the results of Test Example 15.
  • FIG. 24 is a diagram showing the results of measuring the ratio of beating embryoid bodies in Test Example 16.
  • 25A is a diagram illustrating an example of action potentials measured by the patch clamp method of Test Example 16.
  • FIG. 25B is a diagram showing an example of a sodium current measured by the patch clamp method of Test Example 16.
  • FIG. 25C is a diagram illustrating an example of a potassium current measured by the patch clamp method of Test Example 16.
  • FIG. 25A is a diagram illustrating an example of action potentials measured by the patch clamp method of Test Example 16.
  • FIG. 25B is a diagram showing an example of a sodium current measured by the patch clamp
  • 25D is a diagram illustrating an example of a calcium current measured by the patch clamp method of Test Example 16.
  • FIG. 26 is a diagram illustrating an example of a result of action potentials measured by the patch clamp method reported so far.
  • FIG. 27 is a diagram showing another example of the result of the action potential measured by the patch clamp method reported so far.
  • the method for inducing differentiation of cardiomyocytes from pluripotent stem cells of the present invention includes at least an embryoid body formation step, a mesoderm induction step, and a cardiomyocyte induction step, and further includes other steps as necessary.
  • FIG. 4 An example of a method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention is shown in FIG.
  • Day 0 to 1 are the embryoid body formation process (embryoid body formation process start day 0 to day 1)
  • Day 1 to 3 are the mesoderm induction process (embryo-like process)
  • Day 3 to 3 are the first cardiomyocyte induction processes in the cardiomyocyte induction process (embryoid body formation process start 3 to 6)
  • Day 6 and subsequent steps are the second cardiomyocyte induction process in the cardiomyocyte induction process (after the 6th day of the embryoid body formation process start).
  • Y27632, CHIR99021, IWP2, SB431542, and estradiol (Estradiol) in FIG. 4 show an example of a differentiation induction regulator added to the medium in each step.
  • the embryoid body formation step is a step of forming embryoid bodies by suspension culture of pluripotent stem cells.
  • pluripotent stem cells There is no restriction
  • the method for preparing the pluripotent stem cells is not particularly limited, and a known method can be appropriately selected.
  • pluripotent stem cells maintained in an undifferentiated state by a feeder cell culture step described later are colonized. Those dissociated into a shape can be used in the embryoid body formation step.
  • embryoid body formation step medium The medium in the embryoid body formation step (hereinafter sometimes referred to as “embryoid body formation step medium” or “embryoid body formation step culture medium”) is added to the first differentiation induction medium as a ROCK inhibitor. Or a one-component differentiation induction medium with a ROCK inhibitor added, and further contains other components as necessary.
  • first medium may be referred to as a medium additive (hereinafter referred to as “first medium for differentiation-inducing medium”) as a basal medium. ), And further contains other components as necessary.
  • the basal medium in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose.
  • Dulbecco's modified Eagle medium / nutrient mixture F-12 ham hereinafter referred to as “DMEM / F12”
  • DMEM / F12 Dulbecco's modified Eagle medium / nutrient mixture F-12 ham
  • IMDM Iskov modified Dulbecco medium
  • DMEM high glucose
  • Dulbecco Examples thereof include a modified Eagle medium (low glucose) (hereinafter sometimes referred to as “DMEM (low glucose)”), an ⁇ MEM medium, and the like.
  • DMEM / F12 and IMDM are preferable, and DMEM / F12 is more preferable in terms of excellent differentiation induction efficiency into cardiomyocytes.
  • the DMEM may be high glucose or low glucose.
  • As the basal medium commercially available products may be used, or those prepared as appropriate may be used.
  • the first medium additive for differentiation induction medium contains at least insulin, transferrin, albumin, and 1-thioglycerol, and further contains other components as necessary.
  • the content of insulin in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 100 mg / L, and preferably 5 mg / L to 50 mg / L. More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the transferrin content in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 50 mg / L, and preferably 2 mg / L to 20 mg / L. More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of albumin in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 4,000 mg / L to 16,000 mg / L, and preferably 6,000 mg / L. L to 14,000 mg / L is more preferable, and 8,000 mg / L to 12,000 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • limiting in particular as said albumin According to the objective, it can select suitably, For example, human serum albumin, bovine serum albumin, recombinant human albumin, recombinant bovine albumin etc. are mentioned.
  • the 1-thioglycerol content in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 20 mg / L to 80 mg / L, and preferably 30 mg / L to 70 mg. / L is more preferable, and 40 mg / L to 60 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • Other components in the first medium for differentiation induction medium are not particularly limited and may be appropriately selected depending on the intended purpose.
  • the content of glycine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 200 mg / L, and is preferably 10 mg / L to 100 mg / L. More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-alanine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 20 mg / L, and 5 mg / L to 15 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-asparagine ⁇ H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 100 mg / L, and preferably 10 mg / L L to 50 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-aspartic acid in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 100 mg / L, and 10 mg / L to 50 mg. / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-glutamine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / L to 1,000 mg / L, preferably 100 mg / L to 500 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-glutamic acid in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 100 mg / L, and 10 mg / L to 50 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-histidine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 300 mg / L, and 10 mg / L to 200 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-isoleucine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / L to 4,000 mg / L, preferably 100 mg / L to 1,000 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-methionine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 250 mg / L, and 10 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-phenylalanine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 500 mg / L, and 10 mg / L to 400 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-proline in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 1,200 mg / L, and preferably 10 mg / L to 1,000 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-hydroxyproline in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 60 mg / L, and 5 mg / L to 40 mg. / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-serine in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 300 mg / L, 10 mg / L to 200 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-threonine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 10 mg / L to 650 mg / L, and preferably 100 mg / L to 500 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-tryptophan in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 mg / L to 150 mg / L, and 20 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-tyrosine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 3 mg / L to 200 mg / L, and 10 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-valine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 650 mg / L, and 10 mg / L to 500 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the thiamine content in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 25 mg / L, and preferably 2 mg / L to 15 mg / L. More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of reduced glutathione in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 25 mg / L, preferably 1.5 mg / L to 15 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of the magnesium salt of ascorbic acid-2-2PO 4 in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 250 mg / L. 10 mg / L to 100 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of AgNO 3 in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 0.0000008 mg / L to 0.008 mg / L, and 0.000008 mg / L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of AlCl 3 ⁇ 6H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000015 mg / L to 0.0015 mg / L. 0.00015 mg / L to 0.0015 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of Ba (C 2 H 3 O 2 ) 2 in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.00006 mg / L to 0.006 mg / L is preferable, and 0.0006 mg / L to 0.003 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of 3CdSO 4 .8H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00005 mg / L to 0.05 mg / L. 0.0005 mg / L to 0.04 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of CoCl 2 ⁇ 6H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00004 mg / L to 0.004 mg / L. 0.0004 mg / L to 0.003 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of Cr 2 (SO 4 ) 3 .H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.000000025 mg / L to 0.00. 0025 mg / L is preferable, and 0.0000025 mg / L to 0.001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of GeO 2 in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000009 mg / L to 0.0009 mg / L, preferably 0.00009 mg / L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of Na 2 SeO 3 in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0007 mg / L to 0.07 mg / L, More preferably, the content is 0.001 mg / L to 0.01 mg / L. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the KBr content in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000009 mg / L to 0.0009 mg / L, preferably 0.000009 mg / L L to 0.0005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of KI in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 0.00001 mg / L to 0.001 mg / L, preferably 0.00005 mg / L. L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of MnCl 2 .4H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000004 mg / L to 0.004 mg / L. 0.00004 mg / L to 0.001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of NaF in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00006 mg / L to 0.006 mg / L, preferably 0.0006 mg / L L to 0.005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of Na 2 SiO 3 ⁇ 9H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.001 mg / L to 1 mg / L. 0.01 mg / L to 0.5 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of NaVO 3 in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000005 mg / L to 0.005 mg / L, preferably 0.00005 mg / L to 0.004 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.00008 mg / L to 0.08 mg / L is preferable, and 0.0008 mg / L to 0.05 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of NiSO 4 .6H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000025 mg / L to 0.00025 mg / L. 0.000025 mg / L to 0.00025 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of RbCl in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000009 mg / L to 0.009 mg / L, preferably 0.000009 mg / L L to 0.005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of SnCl 2 .2H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000001 mg / L to 0.001 mg / L. 0.00001 mg / L to 0.0001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of ZrOCl 2 ⁇ 8H 2 O in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, 0.00007mg / L ⁇ 0.007mg / L is preferably 0.0007 mg / L to 0.006 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the first culture medium addition agent for differentiation induction medium may be added to the medium as one agent containing each component, or may be added to the medium as a separate agent for each component, or any plurality of components You may add to a culture medium as an agent containing.
  • the one-component differentiation induction medium (hereinafter sometimes referred to as “one-component culture medium”) is sometimes referred to as a medium additive (hereinafter referred to as “medium additive for one-component differentiation induction medium”) to IMDM. ), And further contains other components as necessary.
  • the basal medium in the one-component differentiation induction medium is IMDM.
  • the basal medium commercially available products may be used, or those prepared as appropriate may be used.
  • the medium additive for the one-part differentiation induction medium includes transferrin, albumin, polyvinyl alcohol, etanolamine hydrochloride, sodium selenite, hydrocortisone, DL- ⁇ -tocopherol acetate, N-acetyl- It contains at least L-cysteine, 1-thioglycerol, and ascorbic acid, and further contains other components as necessary.
  • the transferrin content in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 150 mg / L, and preferably 2 mg / L to 100 mg / L. More preferred is 3 mg / L to 50 mg / L. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of albumin in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably more than 500 mg / L and 8,000 mg / L or less, preferably 1,500 mg / L More than 7,000 mg / L is more preferable, and more than 3,000 mg / L and 6,000 mg / L or less is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • limiting in particular as said albumin According to the objective, it can select suitably, For example, human serum albumin, bovine serum albumin, recombinant human albumin, recombinant bovine albumin etc. are mentioned.
  • the content of polyvinyl alcohol in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably more than 500 mg / L and 4,000 mg / L or less, more than 600 mg / L. 2,000 mg / L is more preferable, and more than 800 mg / L and 1,500 mg / L or less is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the weight average molecular weight of the polyvinyl alcohol is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 30,000 to 70,000.
  • the combination of the polyvinyl alcohol content and the albumin content in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the albumin content is preferably more than 3,000 mg / L, and when the polyvinyl alcohol content is 4,000 mg / L, the albumin content is 500 mg / L. The above is preferable.
  • the content of etanolamine hydrochloride in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 mg / L to 18 mg / L, and 5 mg / L to 15 mg / L L is more preferable, and 7 mg / L to 12 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of sodium selenite in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.002 mg / L to 0.008 mg / L, 0.003 mg / L to 0.007 mg / L is more preferable, and 0.004 mg / L to 0.006 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of hydrocortisone in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.02 mg / L to 0.08 mg / L, preferably 0.03 mg / L L to 0.07 mg / L is more preferable, and 0.04 mg / L to 0.06 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of DL- ⁇ -tocopherol acetate in the one-component differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.001 mg / L to 0.008 mg / L. Preferably, 0.005 mg / L to 0.04 mg / L is more preferable, and 0.01 mg / L to 0.03 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of N-acetyl-L-cysteine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 500 mg / L to 3,000 mg / L, 1,000 mg / L to 2,500 mg / L is more preferable, and 1,500 mg / L to 2,000 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of 1-thioglycerol in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 20 mg / L to 80 mg / L, and preferably 30 mg / L to 70 mg. / L is more preferable, and 40 mg / L to 60 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of ascorbic acid in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 20 mg / L to 80 mg / L, preferably 30 mg / L to 70 mg / L. Is more preferable, and 40 mg / L to 60 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • components in the medium additive for the one-component differentiation induction medium are not particularly limited and may be appropriately selected depending on the intended purpose.
  • Magnesium salt AgNO 3 , AlCl 3 ⁇ 6H 2 O, Ba (C 2 H 3 O 2 ) 2 , 3CdSO 4 ⁇ 8H 2 O, CoCl 2 ⁇ 6H 2 O, Cr 2 (SO 4 ) 3 ⁇ H 2 O, GeO 2, Na 2 SeO 3, KBr, KI, MnCl 2 ⁇ 4H 2 O, NaF, Na 2 SiO 3 ⁇ 9H 2 O, Na O 3, (NH
  • the content of glycine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 200 mg / L, and preferably 10 mg / L to 100 mg / L More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-glutamine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / L to 1,000 mg / L, preferably 100 mg / L to 500 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-histidine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 300 mg / L, 10 mg / L to 200 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-isoleucine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / L to 4,000 mg / L, preferably 100 mg / L to 1,000 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-methionine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 250 mg / L, 10 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-phenylalanine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 500 mg / L, 10 mg / L to 400 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-proline in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 1,200 mg / L, and preferably 10 mg / L to 1,000 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-hydroxyproline in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 60 mg / L, and 5 mg / L to 40 mg. / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-serine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 300 mg / L, 10 mg / L to 200 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-threonine in the one-component differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 mg / L to 650 mg / L, preferably 100 mg / L to 500 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-tryptophan in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 mg / L to 150 mg / L, and 20 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-tyrosine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 3 mg / L to 200 mg / L, 10 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of L-valine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 650 mg / L, 10 mg / L to 500 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the thiamine content in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 25 mg / L, and preferably 2 mg / L to 15 mg / L. More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of reduced glutathione in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 25 mg / L, preferably 1.5 mg / L to 15 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of the magnesium salt of ascorbic acid-2-2PO 4 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 250 mg / L. 10 mg / L to 100 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of AgNO 3 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000008 mg / L to 0.008 mg / L, and 0.000008 mg / L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of AlCl 3 .6H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000015 mg / L to 0.0015 mg / L. 0.00015 mg / L to 0.0015 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of Ba (C 2 H 3 O 2 ) 2 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.00006 mg / L to 0.006 mg / L is preferable, and 0.0006 mg / L to 0.003 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of 3CdSO 4 ⁇ 8H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00005 mg / L to 0.05 mg / L. 0.0005 mg / L to 0.04 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of CoCl 2 ⁇ 6H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00004 mg / L to 0.004 mg / L. 0.0004 mg / L to 0.003 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of Cr 2 (SO 4 ) 3 .H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.000000025 mg / L to 0.00. 0025 mg / L is preferable, and 0.0000025 mg / L to 0.001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of GeO 2 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000009 mg / L to 0.0009 mg / L, preferably 0.00009 mg / L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of Na 2 SeO 3 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0007 mg / L to 0.07 mg / L, More preferably, the content is 0.001 mg / L to 0.01 mg / L. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the KBr content in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000009 mg / L to 0.0009 mg / L, preferably 0.000009 mg / L L to 0.0005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of KI in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 0.00001 mg / L to 0.001 mg / L, preferably 0.00005 mg / L. L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of MnCl 2 ⁇ 4H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000004 mg / L to 0.004 mg / L. 0.00004 mg / L to 0.001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of NaF in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00006 mg / L to 0.006 mg / L, preferably 0.0006 mg / L L to 0.005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of Na 2 SiO 3 ⁇ 9H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.001 mg / L to 1 mg / L. 0.01 mg / L to 0.5 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of NaVO 3 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000005 mg / L to 0.005 mg / L, preferably 0.00005 mg / L to 0.004 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.00008 mg / L to 0.08 mg / L is preferable, and 0.0008 mg / L to 0.05 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of NiSO 4 .6H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000025 mg / L to 0.00025 mg / L. 0.000025 mg / L to 0.00025 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of RbCl in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000009 mg / L to 0.009 mg / L, preferably 0.000009 mg / L L to 0.005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of SnCl 2 ⁇ 2H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000001 mg / L to 0.001 mg / L. 0.00001 mg / L to 0.0001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of ZrOCl 2 ⁇ 8H 2 O in the one-liquid type differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, 0.00007mg / L ⁇ 0.007mg / L is preferably 0.0007 mg / L to 0.006 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the medium additive for the one-component differentiation induction medium may be added to the medium as one agent containing each component, or may be added to the medium as a separate agent for each component, or any plurality of components You may add to a culture medium as an agent containing.
  • the ROCK inhibitor is one of differentiation induction regulators.
  • the differentiation induction regulator added to the embryoid body formation process medium (hereinafter sometimes referred to as “differentiation induction regulator for embryoid body formation process”) is not particularly limited as long as it contains a ROCK inhibitor. Can be appropriately selected according to the purpose.
  • Examples of the ROCK inhibitor include Y27632, Fastil Hydrochloride and the like. Among these, Y27632 is preferable because it is excellent in efficiency of inducing differentiation into cardiomyocytes.
  • the ROCK inhibitor may be used alone or in combination of two or more.
  • the ROCK inhibitor is known as a substance having an action of preventing cell death of human iPS cells.
  • the content of the differentiation induction regulator in the embryoid body formation step medium is not particularly limited and may be appropriately selected depending on the intended purpose.
  • Y27632 is used as a differentiation induction regulator in the medium for embryoid body formation step, it is preferably 2 ⁇ M to 10 ⁇ M, more preferably 3 ⁇ M to 8 ⁇ M, and particularly preferably 4 ⁇ M to 6 ⁇ M. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the differentiation-inducing regulator for the embryoid body formation step may or may not contain a differentiation-inducing regulator other than the ROCK inhibitor, but it is possible to obtain cardiomyocytes inexpensively and easily, It is preferable not to include a differentiation induction regulator other than the ROCK inhibitor.
  • the differentiation induction regulator other than the ROCK inhibitor in the differentiation induction regulator for the embryoid body formation step is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected according to the purpose.
  • BFGF BFGF
  • CHIR99021 CAS number: 252917-06-9
  • the content of bFGF when bFGF is added to the embryoid body formation step medium is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 10 ng / mL to 100 ng / mL. It is done.
  • the content of CHIR99021 in the case of adding CHIR99021 to the embryoid body formation step medium is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 5 nM to 5 ⁇ M.
  • the differentiation-inducing regulator for the embryoid body formation step a commercially available product or a chemically synthesized product may be used.
  • the differentiation-inducing regulator for embryoid body formation step may be added to the medium as one agent containing each component, may be added to the medium as a separate agent for each component, or any plurality of components You may add to a culture medium as an agent containing.
  • Preferred embodiments of the embryoid body forming step medium include the following embryoid body forming step mediums (1) and (2). Among these, the culture medium for embryoid body formation process (1) is more preferable.
  • Y27632 was added to the first differentiation induction medium supplemented with the first medium addition medium for differentiation induction medium so as to have the composition described in Tables 1-1 and 1-2 below. Medium added to 5 ⁇ M.
  • the suspension culture method is not particularly limited, and a known method can be appropriately selected.
  • the pluripotency can be selected using a culture dish that has not been subjected to adhesion processing or a culture dish that has undergone low adhesion processing.
  • a method of culturing stem cells can be mentioned.
  • Commercially available products can be used for the culture dish that has not been subjected to the adhesion process or the culture dish that has undergone a low adhesion process.
  • a well-known culture condition can be selected suitably.
  • the suspension culture may be performed under low oxygen conditions or may be performed under atmospheric conditions, but low oxygen conditions are preferable in terms of excellent differentiation efficiency.
  • Low oxygen conditions refer to oxygen concentration conditions below the normal oxygen concentration (21%).
  • the oxygen concentration in the low oxygen condition is not particularly limited as long as it is less than 21%, and can be appropriately selected according to the purpose, but it is preferably 0.1% or more and less than 21%, preferably 1% or more and 10%. The following is more preferable, and 4% or more and 6% or less are particularly preferable.
  • the period of the embryoid body formation step is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 24 to 48 hours.
  • the mesoderm induction step is a step of inducing the mesoderm by suspension culture of the embryoid body after the embryoid body formation step.
  • the medium in the mesoderm induction process (hereinafter sometimes referred to as “medium mesoderm induction process medium” or “mesodermal induction process culture medium”) contains a Wnt signal activator in the second differentiation induction medium. Either of the added medium and the one-part differentiation induction medium, a medium added with a Wnt signal activator, and further containing other components as necessary.
  • the second differentiation induction medium (hereinafter sometimes referred to as “second medium”) may be referred to as a medium additive (hereinafter referred to as “second medium for differentiation induction medium”) as a basal medium. ), And further contains other components as necessary.
  • the basal medium in the second differentiation induction medium is IMDM.
  • the basal medium commercially available products may be used, or those prepared as appropriate may be used.
  • the second medium additive for differentiation induction medium is albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL- ⁇ -tocopherol acetate, N-acetyl-L-cysteine. And at least 1-thioglycerol and ascorbic acid, and further contains other components as necessary.
  • the content of albumin in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably more than 500 mg / L and 8,000 mg / L or less, preferably 1,500 mg / L. More than 7,000 mg / L is more preferable, and more than 3,000 mg / L and 6,000 mg / L or less is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • limiting in particular as said albumin According to the objective, it can select suitably, For example, human serum albumin, bovine serum albumin, recombinant human albumin, recombinant bovine albumin etc. are mentioned.
  • the content of polyvinyl alcohol in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably more than 500 mg / L and less than 4,000 mg / L, more than 600 mg / L. 2,000 mg / L is more preferable, and more than 800 mg / L and 1,500 mg / L or less is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the weight average molecular weight of the polyvinyl alcohol is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 30,000 to 70,000.
  • the combination of the polyvinyl alcohol content and the albumin content in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the albumin content is preferably more than 3,000 mg / L, and when the polyvinyl alcohol content is 4,000 mg / L, the albumin content is 500 mg / L. The above is preferable.
  • the content of etanolamine hydrochloride in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 mg / L to 18 mg / L, and 5 mg / L to 15 mg / L L is more preferable, and 7 mg / L to 12 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of sodium selenite in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.002 mg / L to 0.008 mg / L, 0.003 mg / L to 0.007 mg / L is more preferable, and 0.004 mg / L to 0.006 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of hydrocortisone in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.02 mg / L to 0.08 mg / L, preferably 0.03 mg / L L to 0.07 mg / L is more preferable, and 0.04 mg / L to 0.06 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of DL- ⁇ -tocopherol acetate in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.001 mg / L to 0.008 mg / L. Preferably, 0.005 mg / L to 0.04 mg / L is more preferable, and 0.01 mg / L to 0.03 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of N-acetyl-L-cysteine in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 500 mg / L to 3,000 mg / L, 1,000 mg / L to 2,500 mg / L is more preferable, and 1,500 mg / L to 2,000 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of 1-thioglycerol in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 20 mg / L to 80 mg / L, and preferably 30 mg / L to 70 mg. / L is more preferable, and 40 mg / L to 60 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the content of ascorbic acid in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 20 mg / L to 80 mg / L, and preferably 30 mg / L to 70 mg / L. Is more preferable, and 40 mg / L to 60 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • components in the second culture medium supplement for differentiation induction medium are not particularly limited and may be appropriately selected depending on the intended purpose, but preferably include L-glutamine and transferrin.
  • the content of L-glutamine in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / L to 1,000 mg / L, preferably 100 mg / L to 500 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the transferrin content in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 150 mg / L, and preferably 2 mg / L to 100 mg / L. More preferred is 3 mg / L to 50 mg / L. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the second culture medium additive for differentiation induction medium may contain insulin as the other component, but preferably does not contain insulin.
  • the content of insulin when adding insulin to the second differentiation-inducing medium is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include 5 ng / mL to 15 ng / mL. .
  • the second medium for differentiation induction medium may be a commercially available product or a chemically synthesized product.
  • the second culture medium addition agent for differentiation induction medium may be added to the medium as one agent containing each component, may be added to the medium as a separate agent for each component, or any plurality of components You may add to a culture medium as an agent containing.
  • the one-part differentiation induction medium is the same as that described in the item of the one-part differentiation induction medium in the embryoid body formation step.
  • the differentiation induction regulator added to the medium for mesoderm induction process (hereinafter sometimes referred to as “differentiation induction regulator for mesoderm induction process”) is not particularly limited as long as it contains a Wnt signal activator. Can be appropriately selected according to the purpose. Examples of the Wnt signal activator include CHIR99021, BIO, Wnt agonist (CAS 853220-52-7), Wnt agonist II (SKL2001) and the like. Among these, CHIR99021 is preferable because it has low cytotoxicity and excellent differentiation induction efficiency. The Wnt signal activator may be used alone or in combination of two or more.
  • the content of the differentiation induction regulator in the medium for mesoderm induction process is not particularly limited and can be appropriately selected depending on the purpose.
  • CHIR99021 is used as the differentiation induction regulator in the medium for the mesoderm induction process, it is preferably 2 ⁇ M to 6 ⁇ M, and more preferably 3 ⁇ M to 5 ⁇ M. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the differentiation induction regulator for the mesoderm induction process may or may not contain a differentiation induction regulator other than the Wnt signal activator, but cardiomyocytes can be obtained inexpensively and easily. It is preferable that no differentiation induction regulator other than CHIR99021 is contained.
  • the Wnt signal activator preferably does not contain BIO, Wnt agonist, or Wnt agonist II.
  • the differentiation induction regulator other than the Wnt signal activator in the differentiation induction regulator for the mesoderm induction step is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected according to the purpose.
  • bFGF etc. are mentioned.
  • the content of bFGF in the case where bFGF is added to the medium for mesoderm induction process is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 10 ng / mL to 100 ng / mL. .
  • the differentiation induction regulator for the mesoderm induction process a commercially available product or a chemically synthesized product may be used.
  • the differentiation induction regulator for mesoderm induction process may be added to the medium as one agent containing each component, or may be added to the medium as a separate agent for each component, or any plurality of components may be added. You may add to a culture medium as an agent to contain.
  • Preferred embodiments of the medium for mesoderm induction process include the following mediums for mesoderm induction process (1) and (2). Among these, the medium (1) for mesoderm induction process is more preferable.
  • the suspension culture method and culture conditions are not particularly limited, and a known method can be appropriately selected.
  • the suspension culture method and the culture conditions may be the same as those described in the item of suspension culture in the embryoid body formation step. it can.
  • the duration of the mesoderm induction process is not particularly limited and may be appropriately selected depending on the purpose.
  • the embryoid body formation process starts on the first day to the third day, and the embryoid body formation process starts on the first day.
  • the cardiomyocyte inducing step is a step of inducing cardiomyocytes by suspension culture of the embryoid body after the mesoderm induction.
  • the cardiomyocyte induction step includes at least a first cardiomyocyte induction process and a second cardiomyocyte induction process, and further includes other processes as necessary.
  • first cardiomyocyte induction treatment medium the embryoid body after the mesoderm induction. It is the process which culture
  • first cardiomyocyte induction treatment medium The medium in the first cardiomyocyte induction treatment (hereinafter sometimes referred to as “first cardiomyocyte induction treatment medium” or “first cardiomyocyte induction treatment culture medium”) is used for the second differentiation induction.
  • Medium supplemented with Wnt signal suppressor, TGF- ⁇ signal inhibitor, and estrogen-like substance in medium, and one-part differentiation induction medium, Wnt signal suppressor, TGF- ⁇ signal inhibitor, and estrogen-like action It is any medium added with substances, and further contains other components as necessary.
  • the second differentiation induction medium is the same as that described in the item of the second differentiation induction medium in the mesoderm induction step.
  • One-part differentiation induction medium is the same as that described in the item of the one-part differentiation induction medium in the embryoid body formation step.
  • the differentiation-inducing regulator added to the first cardiomyocyte-inducing treatment medium is a Wnt signal inhibitor, TGF- It contains at least a ⁇ signal inhibitor and an estrogen-like substance, and further contains other components as necessary.
  • the content of the differentiation-inducing regulator in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose.
  • Wnt signal suppression substance there is no restriction
  • the Wnt signal inhibitor may be used alone or in combination of two or more.
  • the content of the Wnt signal inhibitor in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 ⁇ M to 10 ⁇ M, more preferably 2 ⁇ M to 9 ⁇ M. 4 ⁇ M to 7 ⁇ M is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the TGF- ⁇ signal inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include SB431542, SB505124, A-83-01 and the like. Among these, SB431542 is preferable because it is excellent in differentiation induction efficiency.
  • the TGF- ⁇ signal inhibitor may be used alone or in combination of two or more.
  • the content of the TGF- ⁇ signal inhibitor in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 ⁇ M to 10 ⁇ M, and 2 ⁇ M to 9 ⁇ M. More preferably, 3 ⁇ M to 8 ⁇ M is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the estrogen-like substance refers to hormones and low molecular compounds that exhibit estrogen action.
  • the estrogen-like substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include estradiol, estrone, estriol, and genistein. Among these, estradiol is preferable because it is excellent in differentiation induction efficiency.
  • the said estrogen-like active substance may be used individually by 1 type, and may use 2 or more types together.
  • the content of the estrogen-like substance in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 nM to 10,000 nM, 10 nM to 1, 000 nM is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the first differentiation-inducing regulator for cardiomyocyte induction treatment may or may not contain a differentiation-inducing regulator other than a Wnt signal inhibitor, a TGF- ⁇ signal inhibitor, and an estrogen-like agent.
  • VEGF can be used at a concentration of 10 ng / mL in place of the estrogen-like substance, but a Wnt signal inhibitor, TGF- ⁇ signal inhibition can be obtained in that cardiomyocytes can be obtained inexpensively and easily. It is preferable that a differentiation induction regulator other than an agent and an estrogen-like agent is not included.
  • the differentiation-inducing regulators other than the Wnt signal suppressor, TGF- ⁇ signal inhibitor, and estrogen-like agent in the differentiation-inducing regulator for the first cardiomyocyte induction treatment are not particularly limited as long as the effects of the present invention are not impaired.
  • the content of bFGF in the case of adding bFGF to the first cardiomyocyte induction treatment medium is not particularly limited and can be appropriately selected according to the purpose. For example, 10 ng / mL to 100 ng / mL, etc. Is mentioned.
  • the first differentiation induction regulator for cardiomyocyte induction treatment a commercially available product or a chemically synthesized product may be used.
  • the first differentiation-inducing regulator for cardiomyocyte induction treatment may be added to the medium as one agent containing each component, or may be added to the medium as a separate agent for each component. You may add to a culture medium as an agent containing these components.
  • Preferred embodiments of the first cardiomyocyte induction treatment medium include the following first cardiomyocyte induction treatment media (1) and (2). Among these, the first cardiomyocyte induction treatment medium (1) is more preferable.
  • suspension culture method and culture conditions are not particularly limited, and a known method can be appropriately selected.
  • the suspension culture method and the culture conditions may be the same as those described in the item of suspension culture in the embryoid body formation step. it can.
  • the period of the first cardiomyocyte induction treatment is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the embryoid body formation step is the 3rd to 6th day from the start of the embryoid body formation step.
  • the third day to the fifth day from the start, the fourth day to the sixth day from the start of the embryoid body formation process, and the like can be used.
  • Second cardiomyocyte induction process the embryoid body after the first cardiomyocyte induction treatment is referred to as a second cardiomyocyte induction treatment medium (hereinafter, “second cardiomyocyte induction treatment medium”). In some cases).
  • the medium in the second cardiomyocyte induction treatment (hereinafter sometimes referred to as “second cardiomyocyte induction treatment medium” or “second cardiomyocyte induction treatment culture medium”) is used for the second differentiation induction. It is either a medium obtained by adding an estrogen-like agent to a medium, or a medium obtained by adding an estrogen-like substance to a one-part differentiation induction medium, and further contains other components as necessary.
  • the second differentiation induction medium is the same as that described in the item of the second differentiation induction medium in the mesoderm induction step.
  • One-part differentiation induction medium is the same as that described in the item of the one-part differentiation induction medium in the embryoid body formation step.
  • the differentiation-inducing regulator added to the second cardiomyocyte-inducing treatment medium contains at least an estrogenic agent, If necessary, it further contains other components.
  • the content of the differentiation induction regulator in the second cardiomyocyte induction treatment medium is not particularly limited and can be appropriately selected depending on the purpose.
  • the estrogen-like substance and the content thereof are the same as those described in the item of the first cardiomyocyte induction treatment medium.
  • the second differentiation induction regulator for cardiomyocyte induction treatment may or may not contain a differentiation induction regulator other than an estrogen-like agent.
  • VEGF can be used at a concentration of 10 ng / mL instead of the estrogen-like substance, but differentiation-inducing regulators other than estrogen-like substances can be obtained at low cost and easily. It is preferable not to contain.
  • the differentiation induction regulator other than the estrogen-like agent in the second differentiation induction regulator for cardiomyocyte induction treatment is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose.
  • bFGF etc. are mentioned.
  • the content of bFGF in the case of adding bFGF to the second cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose. For example, 10 ng / mL to 100 ng / mL, etc. Is mentioned.
  • the second cardiomyocyte induction treatment differentiation induction regulator may be a commercially available product or a chemically synthesized product.
  • the second differentiation-inducing regulator for cardiomyocyte induction treatment may be added to the medium as one agent containing each component, or may be added to the medium as a separate agent for each component. You may add to a culture medium as an agent containing these components.
  • Preferred embodiments of the second cardiomyocyte induction treatment medium include the following second cardiomyocyte induction treatment media (1) and (2). Among these, the second cardiomyocyte induction treatment medium (1) is more preferable.
  • -Second medium for cardiomyocyte induction treatment (1)- A medium in which estradiol is added to 100 nM in the second differentiation induction medium to which the second differentiation medium medium additive is added so as to have the composition described in Table 3 above.
  • suspension culture method and culture conditions are not particularly limited, and a known method can be appropriately selected.
  • the suspension culture method and the culture conditions may be the same as those described in the item of suspension culture in the embryoid body formation step. it can.
  • the period of the second cardiomyocyte induction treatment is not particularly limited and can be appropriately selected according to the purpose.
  • the embryoid body formation process start day 6 and the embryoid body formation process start day 8 It can be after eyes.
  • the other treatment is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Examples thereof include further cardiomyocyte induction treatment and washing treatment.
  • the further cardiomyocyte induction process is a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process, and the embryo after the first cardiomyocyte induction process
  • a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment (hereinafter referred to as “first cardiomyocyte induction treatment and second cardiomyocyte induction treatment” And a culture medium for cardiomyocyte induction treatment during the treatment).
  • a Wnt signal inhibitory substance and an estrogen-like agent were added to the second differentiation induction medium. It is either a medium or a medium obtained by adding a Wnt signal inhibitory substance and an estrogen-like agent to a one-part differentiation induction medium, and further contains other components as necessary.
  • Second differentiation induction medium is the same as that described in the item of the second differentiation induction medium in the mesoderm induction step.
  • One-part differentiation induction medium is the same as that described in the item of the one-part differentiation induction medium in the embryoid body formation step.
  • the cardiomyocyte induction treatment differentiation differentiation regulator for cardiomyocyte induction treatment may include at least a Wnt signal suppressor and an estrogen-like agent, and may further contain other components as necessary. Including.
  • the content of the differentiation-inducing regulator in the cardiomyocyte induction treatment medium between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is not particularly limited and is appropriately selected depending on the purpose. be able to.
  • the Wnt signal suppressing substance and the content thereof are the same as those described in the item of the first medium for cardiomyocyte induction treatment.
  • the estrogen-like substance and the content thereof are the same as those described in the item of the first cardiomyocyte induction treatment medium.
  • a differentiation-inducing regulator for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment may or may not be included.
  • VEGF can be used at a concentration of 10 ng / mL instead of the estrogen-like substance, but a Wnt signal inhibitor and estrogen-like substance can be obtained at low cost and easily. It is preferable not to contain any other differentiation-inducing regulator.
  • a differentiation induction regulator other than a Wnt signal inhibitory substance and an estrogen-like agent in a differentiation induction regulator for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment As long as the effects of the present invention are not impaired, there is no particular limitation, and it can be appropriately selected according to the purpose. Examples thereof include bFGF.
  • the content of bFGF in the case where bFGF is added to the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is not particularly limited and depends on the purpose. For example, it can be 10 ng / mL to 100 ng / mL.
  • the differentiation induction regulator for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment a commercially available product or a chemically synthesized product may be used. .
  • the differentiation-inducing regulator for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment may be added to the medium as one agent containing each component, or for each component May be added to the medium as separate agents, or may be added to the medium as agents containing any of a plurality of components.
  • the following first cardiomyocyte induction treatment and second cardiomyocyte induction treatment are described below.
  • Medium for cardiomyocyte induction treatment between (1) and (2) are described below.
  • the culture medium for cardiomyocyte induction treatment (1) between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is more preferable.
  • -Medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment (1)- A medium obtained by adding 5 ⁇ M of IWP2 and 100 nM of estradiol to the second differentiation-inducing medium to which the second culture medium additive for differentiation-inducing medium is added so as to have the composition described in Table 3 above.
  • suspension culture-- The suspension culture method and culture conditions are not particularly limited, and a known method can be appropriately selected.
  • the suspension culture method and the culture conditions may be the same as those described in the item of suspension culture in the embryoid body formation step. it can.
  • the period of the cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process is not particularly limited and can be appropriately selected according to the purpose. It may be the 6th to 8th day from the start of the body formation process, the 5th to 8th day from the start of the embryoid body formation process.
  • the washing process is a process of washing the embryoid body after at least one of the first cardiomyocyte induction process, the second cardiomyocyte induction process, and the further cardiomyocyte induction process, which will be described later. It can carry out similarly to the washing
  • the other steps are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Examples thereof include a feeder cell culture step and a washing step.
  • the feeder cell culturing step is a step of culturing pluripotent stem cells and feeder cells together to maintain the pluripotent stem cells in an undifferentiated state.
  • the feeder cell culturing step is a step of culturing pluripotent stem cells and feeder cells together to maintain the pluripotent stem cells in an undifferentiated state.
  • limiting in particular as said feeder cell A well-known thing can be selected suitably.
  • the washing step is a step of washing the embryoid body after at least one of the aforementioned embryoid body forming step, mesoderm induction step, and cardiomyocyte induction step.
  • the washing method is not particularly limited and can be appropriately selected depending on the purpose.
  • the cultured embryoid bodies are collected in a container together with the culture solution and allowed to stand to precipitate the embryoid bodies. After the treatment, the supernatant is removed, and then a culture solution or a buffer solution is added, and the mixture is allowed to stand again to precipitate the embryoid body, and then the supernatant is removed.
  • cleaning Although it can select suitably according to the objective, It is preferable to perform in multiple times.
  • the medium combination in each step is not particularly limited and can be appropriately selected depending on the purpose.
  • the medium in the embryoid body formation step is a first differentiation-inducing medium, and a ROCK inhibitor is added to the medium.
  • the medium in the mesoderm induction step is a medium obtained by adding a Wnt signal activator to the second differentiation induction medium, and the medium in the first cardiomyocyte induction treatment is the second differentiation induction.
  • the medium in the embryoid body formation step is a medium in which a ROCK inhibitor is added to the one-part differentiation induction medium, and the medium in the mesoderm induction step is one.
  • a medium obtained by adding a Wnt signal activator to a formula differentiation-inducing medium, and the medium in the first cardiomyocyte induction treatment includes a one-part differentiation induction medium, a Wnt signal inhibitor, a TGF- ⁇ signal inhibitor, and
  • An embodiment in which an estrogen-like agent is added, and the medium in the second cardiomyocyte induction treatment is preferably a medium in which an estrogen-like agent is added to a one-part differentiation induction medium, in the embryoid body formation step
  • the medium is a medium obtained by adding a ROCK inhibitor to the first differentiation induction medium
  • the medium in the mesoderm induction step is a medium obtained by adding a Wnt signal activator to the second differentiation induction medium
  • the medium for the cardiomyocyte induction treatment of 1 is a medium obtained by adding
  • cardiomyocytes are obtained from pluripotent stem cells by the above method can be confirmed by whether or not the embryoid body is beating.
  • the patch clamp method etc. are mentioned.
  • an action potential having a large and clear plateau phase action potential amplitude of 140 mV or more
  • a large peak potassium current 300 pA or more
  • a large peak calcium current 1 nA or more
  • a large peak sodium current 6.5 nA or more
  • the medium additive of the present invention is a medium additive added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells, and contains at least one of albumin and 1-thioglycerol, and further if necessary Contains other ingredients.
  • the medium additive can be suitably used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
  • the other components are not particularly limited and may be appropriately selected depending on the purpose.
  • the other components may be used alone or in combination of two or more.
  • An embodiment comprising at least one selected from the group consisting of tocopherol acetate, N-acetyl-L-cysteine, and ascorbic acid, glycine, L-alanine, L-asparagine / H 2 O, L-aspartic acid, L- Glutamine, L-glutamic acid, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine , Thiamine, reduced glutathione, ascorbic acid-2-2P Magnesium salt of O 4 , AgNO 3 , AlCl 3 .6H
  • the mode of the medium additive is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the purpose. At least one of the following medium additives (1) to (3) This embodiment is preferred.
  • the medium additive (1) contains at least insulin, transferrin, albumin, and 1-thioglycerol, and further contains other components as necessary.
  • the other components in the medium additive (1) are not particularly limited and may be appropriately selected depending on the intended purpose.
  • the culture medium additive (1) can be suitably used as a first culture medium addition medium for differentiation induction medium in the first differentiation induction medium of the above-described method for inducing differentiation of cardiomyocytes from pluripotent stem cells.
  • the content and the like in can also be the same as those in the first medium-inducing medium for differentiation-inducing medium of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
  • the medium additive (2) comprises albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL- ⁇ -tocopherol acetate, N-acetyl-L-cysteine, 1- It contains at least thioglycerol and ascorbic acid, and further contains other components as necessary.
  • Other components in the medium additive (2) are not particularly limited and may be appropriately selected depending on the intended purpose, but preferably include L-glutamine and transferrin.
  • the medium additive (2) may contain insulin as the other component, but preferably does not contain insulin.
  • the medium additive (2) can be suitably used as the second medium for medium for inducing differentiation in the second medium for inducing differentiation of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
  • the content and the like in can also be the same as those of the second medium-inducing medium for differentiation-inducing medium of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells.
  • the medium additive (3) includes transferrin, albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL- ⁇ -tocopherol acetate, N-acetyl-L-cysteine, , 1-thioglycerol and ascorbic acid, and further contains other components as necessary.
  • Other components in the medium additive (3) are not particularly limited and may be appropriately selected depending on the intended purpose.
  • the medium additive (3) can be suitably used as a medium additive for a one-part differentiation induction medium in a one-part differentiation induction medium for the method of inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
  • the content and the like in can also be the same as the medium additive for a one-part differentiation induction medium for the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
  • the medium additive may be a commercially available product or a chemically synthesized product.
  • the medium additive may be one agent containing each component, may be a separate agent for each component, or may be a combination of agents containing any plural components.
  • the differentiation-inducing regulator of the present invention is a differentiation-inducing regulator added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells, which is a ROCK inhibitor, a Wnt signal activator, a Wnt signal suppressor, TGF -It contains at least one member selected from the group consisting of ⁇ signal inhibitor and estrogen-like substance, and further contains other components as necessary.
  • the differentiation-inducing regulator can be suitably used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
  • ROCK inhibitor There is no restriction
  • the ROCK inhibitor may be used alone or in combination of two or more.
  • the Wnt signal activator is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include CHIR99021, BIO, Wnt agonist (CAS 853220-52-7), Wnt agonist II (SKL2001) and the like. Can be mentioned. Among these, CHIR99021 is preferable because it is excellent in differentiation induction efficiency.
  • the Wnt signal activator may be used alone or in combination of two or more.
  • Wnt signal suppressor> There is no restriction
  • the Wnt signal inhibitor may be used alone or in combination of two or more.
  • TGF- ⁇ signal inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include SB431542, SB505124, A-83-01 and the like. Among these, SB431542 is preferable because it is excellent in differentiation induction efficiency.
  • the TGF- ⁇ signal inhibitor may be used alone or in combination of two or more.
  • the estrogen-like substance refers to hormones and low molecular compounds that exhibit estrogen action.
  • the estrogen-like substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include estradiol, estrone, estriol, and genistein. Among these, estradiol is preferable because it is excellent in differentiation induction efficiency.
  • the said estrogen-like active substance may be used individually by 1 type, and may use 2 or more types together.
  • the other components are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include bFGF and VEGF.
  • the mode of the differentiation induction regulator is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. At least one of the following differentiation induction regulators (1) to (5) Either embodiment is preferred.
  • the differentiation-inducing regulator (1) contains at least a ROCK inhibitor, and further contains other components as necessary. Although the differentiation-inducing regulator (1) may contain a differentiation-inducing regulator other than the ROCK inhibitor, it is preferably not contained.
  • the differentiation-inducing regulator (1) can be suitably used as a differentiation-inducing regulator for the embryoid body formation step in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above, and the content in the medium is also described above. It can be the same as the differentiation-inducing regulator for the embryoid body formation step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells.
  • the differentiation-inducing regulator (2) contains at least a Wnt signal activator, and further contains other components as necessary.
  • the differentiation-inducing regulator (2) may contain a differentiation-inducing regulator other than the Wnt signal activator, but preferably does not contain it.
  • the Wnt signal activator preferably does not contain BIO, Wnt agonist, or Wnt agonist II.
  • the differentiation-inducing regulator (2) can be suitably used as a differentiation-inducing regulator for the mesoderm-inducing process for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above, and the content in the medium is also described above.
  • the method of inducing differentiation of cardiomyocytes from pluripotent stem cells can be the same as the differentiation induction regulator for the mesoderm induction process.
  • the differentiation induction regulator (3) contains at least a Wnt signal inhibitor, a TGF- ⁇ signal inhibitor, and an estrogen-like substance, and further contains other components as necessary.
  • the differentiation induction regulator (3) may contain, but preferably does not contain, a differentiation induction regulator other than a Wnt signal inhibitor, a TGF- ⁇ signal inhibitor, and an estrogen-like agent.
  • the differentiation induction regulator (3) can be suitably used as the first differentiation induction regulator for cardiomyocyte induction treatment in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. Can also be the same as the first differentiation-inducing regulator for cardiomyocyte induction treatment in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
  • the differentiation-inducing regulator (4) contains at least an estrogen-like substance, and further contains other components as necessary.
  • the differentiation-inducing regulator (4) may contain a differentiation-inducing regulator other than an estrogen-like agent, but preferably does not contain it.
  • the differentiation-inducing regulator (4) can be suitably used as the second differentiation-inducing regulator for cardiomyocyte induction treatment in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. Can also be the same as the second differentiation-inducing regulator for cardiomyocyte induction in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
  • the differentiation induction regulator (5) contains at least a Wnt signal inhibitor and an estrogen-like substance, and further contains other components as necessary.
  • the differentiation-inducing regulator (5) may contain a differentiation-inducing regulator other than a Wnt signal suppressor and an estrogen-like agent, but preferably does not contain it.
  • the differentiation induction regulator (5) is used for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
  • the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment of the method of inducing differentiation of cardiomyocytes from the pluripotent stem cells described above which can be suitably used as a differentiation induction regulator and whose content in the medium is also described above It can be the same as the differentiation-inducing regulator for cardiomyocyte induction treatment.
  • the differentiation induction regulator may be a commercially available product or a chemically synthesized product.
  • the differentiation-inducing regulator may be one agent containing each component, may be a separate agent for each component, or may be a combination of agents containing any plural components.
  • the medium of the present invention is a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells, and includes at least the medium additive of the present invention and the differentiation-inducing regulator of the present invention, and if necessary, other Contains the ingredients.
  • the medium can be suitably used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
  • the basal medium used for the medium is at least one selected from the group consisting of DMEM / F12, DMEM, IMDM, RPMI1640 medium, and ⁇ MEM medium.
  • the DMEM may be high glucose or low glucose.
  • the form of the medium is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. However, at least one of the following mediums (1) to (10) is preferable. .
  • the medium (1) is at least one selected from the group consisting of DMEM / F12, DMEM, IMDM, RPMI1640 medium, and ⁇ MEM medium as a basal medium, and the medium additive (1 ), And the differentiation-inducing regulator (1) as the differentiation-inducing regulator, and further containing other components as necessary.
  • DMEM / F12 is preferable.
  • the medium (1) can be suitably used as a medium in which a ROCK inhibitor is added to the first differentiation induction medium in the embryoid body formation step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. .
  • the said culture medium (1) for embryoid body formation processes is mentioned.
  • the medium (2) uses IMDM as a basal medium, includes the medium additive (3) as the medium additive, and includes the differentiation induction regulator (1) as the differentiation induction regulator. Depending on the above, other components are further included.
  • the medium (2) can be suitably used as a medium in which a ROCK inhibitor is added to the one-part differentiation induction medium in the embryoid body formation step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. .
  • the said culture medium (2) for embryoid body formation processes is mentioned.
  • the medium (3) uses IMDM as a basal medium, includes the medium additive (2) as the medium additive, and includes the differentiation induction regulator (2) as the differentiation induction regulator. Depending on the above, other components are further included.
  • the medium (3) is preferably used as a medium in which a Wnt signal activator is added to the second differentiation induction medium in the mesoderm induction step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. it can.
  • the said culture medium (1) for mesoderm induction processes is mentioned.
  • the medium (4) uses IMDM as a basal medium, includes the medium additive (3) as the medium additive, and includes the differentiation induction regulator (2) as the differentiation induction regulator. Depending on the above, other components are further included.
  • the medium (4) is preferably used as a medium in which a Wnt signal activator is added to the one-component differentiation induction medium in the mesoderm induction step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. it can.
  • the said culture medium (2) for mesoderm induction processes is mentioned.
  • the medium (5) uses IMDM as a basal medium, includes the medium additive (2) as the medium additive, and includes the differentiation induction regulator (3) as the differentiation induction regulator. Depending on the above, other components are further included.
  • the medium (5) includes a Wnt signal inhibitor, a TGF- ⁇ signal inhibitor, a second differentiation induction medium in the first cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above, And an estrogen-like substance-added medium.
  • the first cardiomyocyte induction treatment medium (1) may be mentioned.
  • the medium (6) uses IMDM as a basal medium, includes the medium additive (3) as the medium additive, and includes the differentiation induction regulator (3) as the differentiation induction regulator. Depending on the above, other components are further included.
  • the medium (6) includes a Wnt signal suppressor, a TGF- ⁇ signal inhibitor, a one-part differentiation induction medium in the first cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above, And an estrogen-like substance-added medium.
  • the said culture medium (2) for said 1st cardiomyocyte induction is mentioned.
  • the medium (7) uses IMDM as a basal medium, includes the medium additive (2) as the medium additive, and includes the differentiation induction regulator (4) as the differentiation induction regulator. Depending on the above, other components are further included.
  • the medium (7) is suitably used as a medium in which an estrogen-like agent is added to the second differentiation inducing medium in the second cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. be able to.
  • the second culture medium for cardiomyocyte induction treatment (1) may be mentioned.
  • the medium (8) uses IMDM as a basal medium, includes the medium additive (3) as the medium additive, and includes the differentiation induction regulator (4) as the differentiation induction regulator. Depending on the above, other components are further included.
  • the medium (8) is preferably used as a medium in which an estrogen-like agent is added to the one-part differentiation induction medium in the second cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. be able to.
  • the first culture medium for cardiomyocyte induction treatment (2) may be mentioned.
  • the medium (9) uses IMDM as a basal medium, includes the medium additive (2) as the medium additive, and includes the differentiation induction regulator (5) as the differentiation induction regulator. Depending on the above, other components are further included.
  • the medium (9) is a second cardiomyocyte induction process between the first cardiomyocyte induction process of the above-described method for inducing differentiation of cardiomyocytes from the pluripotent stem cells and the second cardiomyocyte induction process. Can be suitably used as a medium obtained by adding a Wnt signal inhibitory substance and an estrogen-like agent to the differentiation induction medium.
  • a preferred embodiment of the medium (9) includes a medium for cardiomyocyte induction treatment (1) between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment.
  • the medium (10) uses IMDM as a basal medium, includes the medium additive (3) as the medium additive, and includes the differentiation induction regulator (5) as the differentiation induction regulator. Depending on the above, other components are further included.
  • the medium (10) is a solution in the cardiomyocyte induction process between the first cardiomyocyte induction process of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above and the second cardiomyocyte induction process. It can be suitably used as a medium obtained by adding a Wnt signal inhibitory substance and an estrogen-like substance to a formula differentiation-inducing medium.
  • a preferable embodiment of the culture medium (10) includes a culture medium for cardiomyocyte induction treatment (2) between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment.
  • the medium preparation kit of the present invention is a medium preparation kit for inducing differentiation of cardiomyocytes from pluripotent stem cells, the medium additive of the present invention, the differentiation induction regulator of the present invention, a basal medium, And at least further other configurations as necessary.
  • the medium preparation kit can be suitably used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
  • the basal medium in the medium preparation kit is at least one selected from the group consisting of DMEM / F12, DMEM, IMDM, RPMI1640 medium, and ⁇ MEM medium.
  • the DMEM may be high glucose or low glucose.
  • the form of the medium preparation kit is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. At least one of the following medium preparation kits (1) to (10) can be used. Either embodiment is preferred.
  • the medium preparation kit (1) comprises the medium additive (1), the differentiation-inducing regulator (1), and DMEM / F12, DMEM, IMDM, RPMI1640 medium, and ⁇ MEM medium as a basal medium. At least one selected from the above, and further includes other configurations as necessary. Among the basal media, DMEM / F12 is preferable.
  • the medium preparation kit (1) is a medium preparation kit obtained by adding a ROCK inhibitor to the first differentiation induction medium in the embryoid body formation step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. Can be suitably used.
  • the embryoid body formation process culture medium (1) may be mentioned.
  • the medium preparation kit (2) includes the medium additive (3), the differentiation induction regulator (1), and IMDM as a basal medium, and further includes other components as necessary.
  • the medium preparation kit (2) is a medium preparation kit in which a ROCK inhibitor is added to the one-part differentiation induction medium in the embryoid body formation step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. Can be suitably used.
  • the above-mentioned medium for embryoid body formation step (2) can be mentioned.
  • the medium preparation kit (3) includes the medium additive (2), the differentiation induction regulator (2), and IMDM as a basal medium, and further includes other components as necessary.
  • the medium preparation kit (3) is for preparing a medium in which a Wnt signal activator is added to the second differentiation induction medium in the mesoderm induction step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a kit.
  • the medium for mesoderm induction process (1) can be mentioned.
  • the medium preparation kit (4) includes the medium additive (3), the differentiation induction regulator (2), and IMDM as a basal medium, and further includes other components as necessary.
  • the medium preparation kit (4) is for preparation of a medium in which a Wnt signal activator is added to a one-part differentiation induction medium in the mesoderm induction step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a kit.
  • the medium for mesoderm induction process (2) can be mentioned.
  • the medium preparation kit (5) includes the medium additive (2), the differentiation induction regulator (3), and IMDM as a basal medium, and further includes other components as necessary.
  • the medium preparation kit (5) includes a Wnt signal inhibitor, a TGF- ⁇ signal, in the second differentiation induction medium in the first cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a kit for preparing a medium containing an inhibitor and an estrogen-like agent.
  • a preferred embodiment of the medium prepared by the medium preparation kit (5) includes the first cardiomyocyte induction treatment medium (1).
  • the medium preparation kit (6) includes the medium additive (3), the differentiation induction regulator (3), and IMDM as a basal medium, and further includes other components as necessary.
  • the medium preparation kit (6) includes a Wnt signal suppressor, a TGF- ⁇ signal, in a one-part differentiation induction medium in the first cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a kit for preparing a medium containing an inhibitor and an estrogen-like agent.
  • a preferred embodiment of the medium prepared by the medium preparation kit (6) is the first cardiomyocyte induction treatment medium (2).
  • the medium preparation kit (7) includes the medium additive (2), the differentiation induction regulator (4), and IMDM as a basal medium, and further includes other components as necessary.
  • the medium preparation kit (7) comprises a medium obtained by adding an estrogen-like agent to the second differentiation inducing medium in the second cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a preparation kit.
  • a preferred embodiment of the medium prepared by the medium preparation kit (7) is the second cardiomyocyte induction treatment medium (1).
  • the medium preparation kit (8) includes the medium additive (3), the differentiation induction regulator (4), and IMDM as a basal medium, and further includes other components as necessary.
  • the medium preparation kit (8) comprises a medium obtained by adding an estrogen-like agent to a one-part differentiation induction medium in the second cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a preparation kit.
  • a preferred embodiment of the medium prepared by the medium preparation kit (8) includes the first cardiomyocyte induction treatment medium (2).
  • the medium preparation kit (9) includes the medium additive (2), the differentiation induction regulator (5), and IMDM as a basal medium, and further includes other components as necessary.
  • the medium preparation kit (9) is a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
  • a preferred embodiment of the medium prepared by the medium preparation kit (9) is a medium for cardiomyocyte induction treatment (1) between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment. It is done.
  • the medium preparation kit (10) includes the medium additive (3), the differentiation induction regulator (5), and IMDM as a basal medium, and further includes other components as necessary.
  • the medium preparation kit (10) is a cardiomyocyte induction process between the first cardiomyocyte induction process of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells and the second cardiomyocyte induction process.
  • a preferred embodiment of the medium prepared by the medium preparation kit (10) is a cardiomyocyte induction treatment medium (2) between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment. It is done.
  • the kit for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention includes at least one of the medium of the present invention and the medium preparation kit of the present invention, and further includes other components as necessary.
  • the kit for inducing differentiation of cardiomyocytes from the pluripotent stem cells can be suitably used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
  • the medium and the medium preparation kit may contain either one or both.
  • the medium is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the medium includes at least one of the mediums (1) to (10) described in the item of the medium of the present invention described above.
  • the medium (1), (3), (5), and (7) are more preferably included, and the medium (1), (3), (5), (7), and (9) are included. It is particularly preferable to include it.
  • the medium preparation kit is not particularly limited and may be appropriately selected depending on the intended purpose. However, the medium preparation kits (1) to (10) described in the item of the medium preparation kit of the present invention described above. ), More preferably the medium preparation kits (1), (3), (5), and (7), and the medium preparation kits (1), (3 ), (5), (7) and (9) are particularly preferred.
  • the other configuration is not particularly limited and can be appropriately selected according to the purpose.
  • Examples thereof include a culture dish and instructions for teaching a method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention. Can be mentioned. Among these, it is preferable to include instructions that teach a method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
  • ⁇ Pluripotent stem cells Two human iPS cells (clone 1 and clone 2) established at the University of Tokyo Hospital were used as pluripotent stem cells.
  • ⁇ Embryoid body formation process After the human iPS cells maintained in an undifferentiated state according to a conventional method are dissociated into colonies and suspended in the following medium (hereinafter, sometimes referred to as “culture medium for embryoid body formation process”)
  • the suspension culture (culture conditions: 37 ° C., 5% CO 2 , 5% O 2 ) was started on the culture dish (Corning ultra-low adhesion processed surface (manufactured by Corning)) subjected to the low adhesion process (Day 0)
  • the human iPS cell aggregate embryombryoid body (EB) was formed.
  • DMEM / F12 manufactured by Nacalai Tesque Co., Ltd.
  • Y27632 manufactured by Sigma, 5 ⁇ M
  • Table 4 Table 4 below. Each component was added so that the concentrations described in -1 to Table 4-2 were obtained, and used as a culture solution for embryoid body formation process.
  • ⁇ Washing process> The embryoid bodies were collected in a centrifuge tube together with the culture solution, and allowed to stand to precipitate the embryoid bodies, and then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
  • culture medium for mesoderm induction process After the washing step, the following medium (hereinafter sometimes referred to as “culture medium for mesoderm induction process”) was added, and suspension culture was resumed in the culture dish (embryoid body formation process starting day 1 to 3 Day).
  • culture medium for mesoderm induction process StemPro (registered trademark) 34 (manufactured by Invitrogen) is used as the basal medium, and CHIR99021 (manufactured by Millipore, 1 ⁇ M, 3 ⁇ M, 5 ⁇ M, or 7 ⁇ M) is used as the differentiation induction regulator in the basal medium.
  • ⁇ Washing process> The embryoid body after the mesoderm induction was collected in a centrifuge tube together with the culture solution, allowed to stand to precipitate the embryoid body, and then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
  • first culture medium for cardiomyocyte induction treatment ⁇ Cardiomyocyte induction process >> After the washing step, the following medium (hereinafter also referred to as “first culture medium for cardiomyocyte induction treatment”) was added, and suspension culture was resumed in the culture dish (embryoid body formation process start day 3 Eyes to 6th).
  • first culture medium for cardiomyocyte induction treatment StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, and IWP2 (manufactured by Sigma, 5 ⁇ M), SB431542 (manufactured by Sigma, 5 ⁇ M) and VEGF (R & D) were used as the differentiation induction regulators in the basal medium.
  • ⁇ Cleaning process The embryoid body after the first cardiomyocyte induction treatment was collected in a centrifuge tube together with the culture solution, allowed to stand to precipitate the embryoid body, and then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
  • a culture solution for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is added, and the culture is performed. Suspension culture was resumed in the dish (embryoid body formation process from day 6 to day 8).
  • -Culture medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment- StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, and IWP2 (manufactured by Sigma, 5 ⁇ M) and VEGF (manufactured by R & D, 10 ng / mL) as differentiation regulators.
  • L-glutamine manufactured by Invitrogen, 2 mM
  • transferrin manufactured by Sigma, 150 ⁇ g / mL
  • ascorbic acid manufactured by Sigma, 50 ⁇ g / mL
  • 1-thioglycerol manufactured by Sigma, 50 ⁇ g) / ML
  • ⁇ Cleaning process The embryoid body after the cardiomyocyte inducing process between the first cardiomyocyte inducing process and the second cardiomyocyte inducing process is collected in a centrifuge tube together with the culture solution and allowed to stand to precipitate the embryoid body And then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
  • Second cardiomyocyte induction process After the washing treatment, the following medium (hereinafter sometimes referred to as “second culture medium for cardiomyocyte induction treatment”) was added, and suspension culture was resumed in the culture dish (embryoid body formation process start day 8 Eyes to 16th day).
  • second culture medium for cardiomyocyte induction treatment StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, and VEGF (manufactured by R & D, 10 ng / mL) and bFGF (manufactured by Wako Pure Chemical Industries, Ltd.) as the differentiation induction regulator.
  • CHIR represents CHIR99021
  • the left side (black) in each item represents the result of clone 1
  • the right side (gray) represents the result of clone 2.
  • ⁇ Mesodermal induction process Test Example 1 except that the Wnt signal activator in Test Example 1 was CHIR99021 (concentration: 1 ⁇ M, 3 ⁇ M, 3.5 ⁇ M, 4 ⁇ M, 4.5 ⁇ M, 5 ⁇ M, 5.5 ⁇ M, 6 ⁇ M, or 7 ⁇ M). In the same manner, a mesoderm induction process was performed.
  • Second cardiomyocyte induction process In the same manner as in Test Example 1, a second cardiomyocyte induction treatment was performed.
  • FIG. 6 shows the results of clone 1
  • FIG. 7 shows the results of clone 2. From the results of FIG. 6 and FIG. 7, it was found that a beating embryoid body was obtained when the concentration of CHIR99021 was in the range of 3 ⁇ M to 6 ⁇ M. Further, from the results of FIG. 6, it was found that the optimum concentration of CHIR99021 in clone 1 was 3.91 ⁇ M, and from the results of FIG. 7, the optimum concentration of CHIR99021 in clone 2 was 4.31 ⁇ M.
  • ⁇ Mesodermal induction process> A mesoderm induction step was performed in the same manner as in Test Example 1 except that the Wnt signal activator in Test Example 1 was changed to CHIR99021 (concentration: 4 ⁇ M).
  • the differentiation-inducing regulator in the first culture solution for cardiomyocyte induction treatment was the following Wnt signal inhibitory substance, SB431542 (SIGMA, 5 ⁇ M) as a TGF- ⁇ signal inhibitor, and VEGF (R & D).
  • the first cardiomyocyte induction treatment was performed in the same manner as in Test Example 1 except that the product was changed to 10 ng / mL.
  • DMSO manufactured by Sigma
  • the differentiation-inducing regulator in the culture solution for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is used as the culture for the first cardiomyocyte induction treatment.
  • the first cardiomyocyte induction treatment and the second cardiomyocyte induction were carried out in the same manner as in Test Example 1 except that the Wnt signal inhibitory substance and VEGF (manufactured by R & D, 10 ng / mL) were used. Cardiomyocyte induction treatment between treatments was performed.
  • a negative control a culture solution to which DMSO (manufactured by Sigma) was added instead of the Wnt signal suppressing substance was also prepared.
  • Second cardiomyocyte induction process In the same manner as in Test Example 1, a second cardiomyocyte induction treatment was performed.
  • negative con represents a negative control.
  • the left side represents the result of clone 1
  • the right side represents the result of clone 2.
  • the first cardiomyocyte induction was performed in the same manner as in Test Example 3 except that the Wnt signal inhibitory substance was IWP2 (concentration: 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, or 10 ⁇ M) in Test Example 3.
  • a cardiomyocyte induction process was performed between the treatment and the second cardiomyocyte induction process.
  • Second cardiomyocyte induction process In the same manner as in Test Example 1, a second cardiomyocyte induction treatment was performed.
  • FIG. 9 shows the results of clone 1
  • FIG. 10 shows the results of clone 2. From the results shown in FIGS. 9 and 10, it was found that a beating embryoid body can be obtained at least in the range where the concentration of IWP2 is 1 ⁇ M or more and 10 ⁇ M or less. Further, from the results of FIG. 9, it was found that the optimal concentration of IWP2 in clone 1 was 6.22 ⁇ M, and from the results of FIG. 10, the optimal concentration of IWP2 in clone 2 was 5.11 ⁇ M.
  • pluripotent stem cells As pluripotent stem cells, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
  • the mesoderm induction step was performed in the same manner as in Test Example 1 except that the following medium for mesoderm induction step was used as the medium for mesoderm induction step.
  • -Medium for mesoderm induction process- IMDM manufactured by Nacalai Tesque Co., Ltd.
  • CHIR99021 manufactured by Millipore, 4 ⁇ M
  • Each component was added so that it might become the density
  • -First culture solution for cardiomyocyte induction treatment The mesoderm induction except that the differentiation induction regulator in the culture solution for the mesodermal induction process of Test Example 5 was replaced with IWP2 (Sigma, 5 ⁇ M), SB431542 (Sigma, 5 ⁇ M), and the following candidate substances:
  • the first culture solution for cardiomyocyte induction treatment was used in the same manner as the process culture solution.
  • a negative control (( ⁇ )) a culture solution not containing the following candidate substances was also prepared.
  • a cardiomyocyte between the following first cardiomyocyte induction process and the second cardiomyocyte induction process as a medium for cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process In the same manner as in the first cardiomyocyte induction treatment of Test Example 5, except that the culture solution for induction treatment was used and the period of suspension culture was changed to the sixth to eighth days from the start of the embryoid body formation step. A cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed.
  • -Culture medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment As in the culture medium for the mesoderm induction process, except that the differentiation induction regulator in the culture medium for the mesoderm induction process of Test Example 5 was replaced with IWP2 (manufactured by Sigma, 5 ⁇ M) and the following candidate substances:
  • IWP2 manufactured by Sigma, 5 ⁇ M
  • the culture solution for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was used.
  • a negative control (( ⁇ )) a culture solution not containing the following candidate substances was also prepared.
  • Second cardiomyocyte induction process A second cardiomyocyte induction treatment was performed in the same manner as in Test Example 1 except that the following second cardiomyocyte induction treatment medium was used as the second cardiomyocyte induction treatment medium.
  • -Second culture solution for cardiomyocyte induction treatment Except that the differentiation-inducing regulator in the culture medium for mesoderm induction process of Test Example 5 was replaced with the following candidate substances, the second culture medium for cardiomyocyte induction treatment was the same as the culture medium for mesoderm induction process, did.
  • a negative control (( ⁇ )) a culture solution not containing the following candidate substances was also prepared.
  • RA retinoic acid.
  • E2 represents estradiol. From the results shown in FIGS. 11 and 12, it was confirmed that differentiation efficiency exceeding VEGF can be obtained by adding estradiol at a concentration of 1 nM to 10 ⁇ M.
  • the embryoid body formation step was performed in the same manner as in Test Example 1 except that the following culture fluids for the embryoid body formation step (1) to (6) were used as the culture fluid for the embryoid body formation step. .
  • Basal medium ⁇ ⁇ ⁇ 5mL KnockOut Serum Replacement (manufactured by Invitrogen, hereinafter, sometimes referred to as “KSR”) 1 mL MEM Non-Essential Amino Acids (manufactured by Nacalai Tesque, Inc., hereinafter sometimes referred to as “MEM NEAA”) 60 ⁇ L 1-thioglycerol (manufactured by Sigma) 50 mg / L Y27632 (manufactured by Sigma) 5 ⁇ M bFGF (manufactured by Wako Pure Chemical Industries, Ltd.) ...
  • the basal medium is (1) DMEM (high glucose) (manufactured by Nacalai Tesque), (2) DMEM (low glucose) (manufactured by Nacalai Tesque), (3) DMEM / F12 (manufactured by Nacalai Tesque) (4) IMDM (manufactured by Nacalai Tesque) or (5) ⁇ MEM medium (manufactured by Nacalai Tesque) was used.
  • DMEM high glucose
  • DMEM low glucose
  • DMEM / F12 manufactured by Nacalai Tesque
  • IMDM manufactured by Nacalai Tesque
  • ⁇ MEM medium manufactured by Nacalai Tesque
  • the mesoderm induction step was performed in the same manner as in Test Example 1 except that the following medium for mesoderm induction step was used as the medium for mesoderm induction step.
  • -Medium for mesoderm induction process StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, CHIR99021 (manufactured by Millipore, 4 ⁇ M) as the differentiation induction regulator, and L-glutamine (manufactured by Invitrogen) as the medium additive.
  • transferrin Sigma, 150 ⁇ g / mL
  • ascorbic acid Sigma, 50 ⁇ g / mL
  • 1-thioglycerol Sigma, 50 ⁇ g / mL
  • the first cardiomyocyte induction treatment was performed in the same manner as in Test Example 1 except that the following first cardiomyocyte induction treatment culture medium was used as the first cardiomyocyte induction treatment medium.
  • -First culture solution for cardiomyocyte induction treatment StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, and IWP2 (manufactured by Sigma, 5 ⁇ M), SB431542 (manufactured by Sigma, 5 ⁇ M), and VEGF (as a differentiation induction regulator) were used as the basal medium.
  • a cardiomyocyte between the following first cardiomyocyte induction process and the second cardiomyocyte induction process as a medium for cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed in the same manner as in the first cardiomyocyte induction treatment of Test Example 6 except that the culture medium for induction treatment was used and the period of suspension culture was changed to the sixth to eighth days from the start of the embryoid body formation process.
  • the cardiomyocyte induction process between the cardiomyocyte induction process and the second cardiomyocyte induction process was performed.
  • -Culture medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment- StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, and IWP2 (manufactured by Sigma, 5 ⁇ M) and VEGF (manufactured by R & D, 10 ng / mL) as differentiation regulators.
  • L-glutamine manufactured by Invitrogen, 2 mM
  • transferrin manufactured by Sigma, 150 ⁇ g / mL
  • ascorbic acid manufactured by Sigma, 50 ⁇ g / mL
  • 1-thioglycerol manufactured by Sigma, 50 ⁇ g) / ML
  • Second cardiomyocyte induction process A second cardiomyocyte induction treatment was performed in the same manner as in Test Example 1 except that the following second cardiomyocyte induction treatment medium was used as the second cardiomyocyte induction treatment medium.
  • -Second culture solution for cardiomyocyte induction treatment - StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, VEGF (manufactured by R & D, 10 ng / mL) as the differentiation induction regulator, and L-glutamine (Invitrogen) as the medium additive.
  • the culture medium for embryoid body formation process As the culture medium for embryoid body formation process, the culture medium for embryoid body formation process of Test Example 6 (3) (using DMEM / F12 as the basal medium), CHIR99021 (manufactured by Millipore) and bFGF (The embryoid body forming step was performed in the same manner as in Test Example 6 except that the culture solution for embryoid body forming step having the following concentration was added to Wako Pure Chemical Industries, Ltd.). As a control, the embryoid body formation step was similarly performed for the culture fluid for embryoid body formation step (6) of Test Example 6 (using ReproFF2, hereinafter sometimes referred to as “ReproFF2”). Went.
  • ReproFF2 ReproFF2
  • FGF 0 ng / mL
  • FGF 50 50 ng / mL
  • FGF 100 100 ng / mL
  • CHIR99021 0.005 ⁇ M
  • CHIR 0.25 0.25 ⁇ M
  • CHIR 5 5 ⁇ M
  • Second cardiomyocyte induction process In the same manner as in Test Example 6, a second cardiomyocyte induction treatment was performed.
  • Embryoid body formation process The embryoid body formation step was performed in the same manner as in Test Example 6 except that the following embryoid body formation step culture medium A or B was used as the embryoid body formation step culture medium.
  • -Culture fluid A for embryoid body formation process The culture solution for embryoid body formation process in Test Example 1.
  • -Culture solution B for embryoid body formation process The culture solution obtained by removing bFGF in the culture solution (3) for embryoid body formation process of Test Example 6 ((using DMEM / F12 as a basal medium)).
  • Second cardiomyocyte induction process In the same manner as in Test Example 6, a second cardiomyocyte induction treatment was performed.
  • the embryoid body formation step was performed in the same manner as in Test Example 5 except that the culture fluid B for embryoid body formation step in Test Example 8 was used as the culture solution for embryoid body formation step.
  • ⁇ Mesodermal induction process> As a medium for mesoderm induction process, polyvinyl alcohol (hereinafter sometimes referred to as “PVA”) and bovine serum albumin (hereinafter referred to as “BSA”) in the culture medium for mesoderm induction process of Test Example 5 may be referred to.
  • PVA polyvinyl alcohol
  • BSA bovine serum albumin
  • the mesoderm induction step was performed in the same manner as in Test Example 5 except that the culture solution having the following concentration was used.
  • SP34 StemPro (registered trademark) 34 (manufactured by Invitrogen), differentiation induction regulator, CHIR99021 (manufactured by Millipore, 4 ⁇ M), medium additive, Added L-glutamine (Invitrogen, 2 mM), transferrin (Sigma, 150 ⁇ g / mL), ascorbic acid (Sigma, 50 ⁇ g / mL), and 1-thioglycerol (Sigma, 50 ⁇ g / mL)
  • SP34 StemPro 34
  • CHIR99021 manufactured by Millipore, 4 ⁇ M
  • medium additive Added L-glutamine
  • Invitrogen 2 mM
  • transferrin Sigma, 150 ⁇ g / mL
  • ascorbic acid Sigma, 50 ⁇ g / mL
  • 1-thioglycerol Sigma, 50 ⁇ g / mL
  • PVA 4,000 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA4000 / BSA5000”) (2) PVA 4,000 mg / L, BSA 500 mg / L (hereinafter sometimes referred to as “PVA4000 / BSA500”) (3) PVA 4,000 mg / L, BSA 0 mg / L (hereinafter sometimes referred to as “PVA4000 / BSA0”) (4) PVA 1,000 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA5000”) (5) PVA 1,000 mg / L, BSA 500 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA500”) (6) PVA 1,000 mg / L, BSA 0 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA0”) (7) PVA 0 mg / L, BSA
  • ⁇ Cardiomyocyte induction process> ⁇ First cardiomyocyte induction process As the first cardiomyocyte induction treatment medium, CHIR99021 in the culture solution for the mesoderm induction process of Test Example 9 was used as IWP2 (Sigma, 5 ⁇ M), SB431542 (Sigma, 5 ⁇ M), and VEGF (R & D). The first cardiomyocyte induction treatment medium was used in the same manner except that insulin (Sigma, 10 ng / mL) was added instead of 10 ng / mL, and the period of suspension culture was the embryoid body formation step. The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 5 except that the changes were made on the third to fifth days from the start.
  • StemPro (registered trademark) 34 manufactured by Invitrogen
  • differentiation induction regulators as IWP2 manufactured by Sigma, 5 ⁇ M
  • SB431542 manufactured by Sigma, 5 ⁇ M
  • VEGF manufactured by R & D, 10 ng) / ML
  • L-glutamine manufactured by Invitrogen, 2 mM
  • transferrin manufactured by Sigma, 150 ⁇ g / mL
  • ascorbic acid manufactured by Sigma, 50 ⁇ g / mL
  • 1-thioglycerol Sigma
  • StemPro (registered trademark) 34 manufactured by Invitrogen
  • IWP2 manufactured by Sigma, 5 ⁇ M
  • VEGF manufactured by R & D, 10 ng / mL
  • Added L-glutamine Invitrogen, 2 mM
  • transferrin Sigma, 150 ⁇ g / mL
  • ascorbic acid Sigma, 50 ⁇ g / mL
  • 1-thioglycerol Sigma, 50 ⁇ g / mL
  • Second cardiomyocyte induction process As the second culture medium for cardiomyocyte induction treatment, CHIR99021 in the culture solution for the mesoderm induction process of Test Example 9 was replaced with VEGF (R & D, 10 ng / mL), and insulin (Sigma, 10 ng / mL). The second cardiomyocyte induction treatment was performed in the same manner as in Test Example 5 except that the second medium for inducing cardiomyocyte induction treatment was used except that (2) was added.
  • StemPro (registered trademark) 34 manufactured by Invitrogen
  • VEGF manufactured by R & D, 10 ng / mL
  • L-glutamine manufactured by Invitrogen, 2 mM
  • transferrin Sigma, 150 ⁇ g / mL
  • ascorbic acid Sigma, 50 ⁇ g / mL
  • 1-thioglycerol Sigma, 50 ⁇ g / mL
  • ⁇ Mesodermal induction process> As a medium for mesoderm induction process, polyvinyl alcohol (hereinafter sometimes referred to as “PVA”) and bovine serum albumin (hereinafter referred to as “BSA”) in the culture medium for mesoderm induction process of Test Example 5 may be referred to.
  • PVA polyvinyl alcohol
  • BSA bovine serum albumin
  • the mesoderm induction step was performed in the same manner as in Test Example 9 except that the culture solution having the following concentration was used.
  • SP34 StemPro (registered trademark) 34 (manufactured by Invitrogen), differentiation induction regulator, CHIR99021 (manufactured by Millipore, 4 ⁇ M), medium additive, Added L-glutamine (Invitrogen, 2 mM), transferrin (Sigma, 150 ⁇ g / mL), ascorbic acid (Sigma, 50 ⁇ g / mL), and 1-thioglycerol (Sigma, 50 ⁇ g / mL)
  • SP34 StemPro 34
  • CHIR99021 manufactured by Millipore, 4 ⁇ M
  • medium additive Added L-glutamine
  • Invitrogen 2 mM
  • transferrin Sigma, 150 ⁇ g / mL
  • ascorbic acid Sigma, 50 ⁇ g / mL
  • 1-thioglycerol Sigma, 50 ⁇ g / mL
  • PVA 500 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA500 / BSA5000”)
  • PVA 500 mg / L, BSA 1,500 mg / L (hereinafter sometimes referred to as “PVA500 / BSA1500”)
  • PVA 500 mg / L, BSA 500 mg / L (hereinafter sometimes referred to as “PVA500 / BSA500”)
  • PVA 500 mg / L, BSA 500 mg / L (hereinafter sometimes referred to as “PVA500 / BSA500”)
  • PVA500 mg / L, BSA 0 mg / L (hereinafter sometimes referred to as “PVA500 / BSA0”)
  • the first cardiomyocyte induction treatment medium was prepared on the basis of the culture medium for the mesodermal induction process of Test Example 9, and the culture medium for the mesoderm induction process of Test Example 10 was used.
  • the first cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that the first cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, the first cardiomyocyte induction treatment was performed for the control.
  • a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction process and the second cardiomyocyte induction process is prepared based on the culture medium for the mesodermal induction process of Test Example 9. Except that the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was prepared based on the medium for mesodermal induction process of Test Example 10
  • a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed. Further, in the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed for the control.
  • Second cardiomyocyte induction process the second cardiomyocyte induction treatment medium was prepared based on the culture medium for the mesodermal induction process of the test example 9, and the culture medium for the mesodermal induction process of the test example 10 was used.
  • a second cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that a second cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, a second cardiomyocyte induction treatment was performed for the control.
  • ⁇ Mesodermal induction process> As a medium for mesoderm induction process, polyvinyl alcohol (hereinafter sometimes referred to as “PVA”) and bovine serum albumin (hereinafter referred to as “BSA”) in the culture medium for mesoderm induction process of Test Example 5 may be referred to.
  • PVA polyvinyl alcohol
  • BSA bovine serum albumin
  • the mesoderm induction step was performed in the same manner as in Test Example 9 except that the culture solution having the following concentration was used.
  • SP34 StemPro (registered trademark) 34 (manufactured by Invitrogen), differentiation induction regulator, CHIR99021 (manufactured by Millipore, 4 ⁇ M), medium additive, Added L-glutamine (Invitrogen, 2 mM), transferrin (Sigma, 150 ⁇ g / mL), ascorbic acid (Sigma, 50 ⁇ g / mL), and 1-thioglycerol (Sigma, 50 ⁇ g / mL)
  • SP34 StemPro 34
  • CHIR99021 manufactured by Millipore, 4 ⁇ M
  • medium additive Added L-glutamine
  • Invitrogen 2 mM
  • transferrin Sigma, 150 ⁇ g / mL
  • ascorbic acid Sigma, 50 ⁇ g / mL
  • 1-thioglycerol Sigma, 50 ⁇ g / mL
  • PVA 1,500 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA1500 / BSA5000”) (13) PVA 1,500 mg / L, BSA 4,000 mg / L (hereinafter sometimes referred to as “PVA1500 / BSA4000”) (14) PVA 1,500 mg / L, BSA 3,000 mg / L (hereinafter sometimes referred to as “PVA1500 / BSA3000”)
  • PVA 1,000 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA5000”) (16) PVA 1,000 mg / L, BSA 4,000 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA4000”) (17) PVA 1,000 mg / L, BSA 3,000 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA3000”) (18) PVA 1000 mg / L, BSA 3,000 mg / L (hereinafter sometimes
  • the first cardiomyocyte induction treatment medium was prepared based on the culture medium for the mesodermal induction process of the test example 9, and the culture medium for the mesodermal induction process of the test example 11 was used.
  • the first cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that the first cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, the first cardiomyocyte induction treatment was performed for the control.
  • a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction process and the second cardiomyocyte induction process is prepared based on the culture medium for the mesodermal induction process of Test Example 9. Except that the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was prepared based on the culture solution for mesodermal induction process of Test Example 11 In the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed. Further, in the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed for the control.
  • Second cardiomyocyte induction process >> In Test Example 9, the second cardiomyocyte induction treatment medium was prepared based on the culture medium for mesoderm induction process of Test Example 9, and the culture medium for mesoderm induction process of Test Example 11 was used. A second cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that a second cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, a second cardiomyocyte induction treatment was performed for the control.
  • ⁇ Mesodermal induction process> As a medium for mesoderm induction process, polyvinyl alcohol (hereinafter sometimes referred to as “PVA”) and bovine serum albumin (hereinafter referred to as “BSA”) in the culture medium for mesoderm induction process of Test Example 5 may be referred to.
  • PVA polyvinyl alcohol
  • BSA bovine serum albumin
  • the mesoderm induction step was performed in the same manner as in Test Example 9 except that the culture solution having the following concentration was used.
  • StemPro registered trademark
  • CHIR99021 manufactured by Millipore, 4 ⁇ M
  • L-glutamine Invitrogen
  • SP34 StemPro (registered trademark) 34 (manufactured by Invitrogen), CHIR99021 (manufactured by Millipore, 4 ⁇ M), and L-glutamine (Invitrogen) as a medium additive. 2 mM), transferrin (Sigma, 150 ⁇ g / mL), ascorbic acid (Sigma, 50 ⁇ g / mL), and 1-thioglycerol (Sigma, 50 ⁇ g / mL) for mesoderm induction process Similarly, the mesoderm induction process was performed for the culture solution.
  • PVA 500 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA500 / BSA5000”)
  • PVA 0 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA0 / BSA5000”)
  • PVA0 mg / L, BSA 4,000 mg / L (hereinafter sometimes referred to as “PVA0 / BSA4000”)
  • PVA 0 mg / L, BSA 3,000 mg / L (hereinafter sometimes referred to as “PVA0 / BSA3000”)
  • the first cardiomyocyte induction treatment medium was prepared based on the culture medium for the mesodermal induction process of the test example 9, and the culture medium for the mesodermal induction process of the test example 12 was used.
  • the first cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that the first cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, the first cardiomyocyte induction treatment was performed for the control.
  • a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction process and the second cardiomyocyte induction process is prepared based on the culture medium for the mesodermal induction process of Test Example 9. Except that the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was prepared based on the culture solution for the mesoderm induction process of Test Example 12 In the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed. Further, in the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed for the control.
  • Second cardiomyocyte induction process >> In Test Example 9, the second cardiomyocyte induction treatment medium was prepared on the basis of the culture medium for the mesodermal induction process of Test Example 9, and the culture medium for the mesoderm induction process of Test Example 12 was used. A second cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that a second cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, a second cardiomyocyte induction treatment was performed for the control.
  • ⁇ Mesodermal induction process> The same as in Test Example 9, except that the medium for transferrin in the culture medium for mesoderm induction process in Test Example 5 was 5 mg / L or 0 mg / L as the medium for mesoderm induction process. Then, a mesoderm induction process was performed. Further, as a control (hereinafter sometimes referred to as “SP34”), StemPro (registered trademark) 34 (manufactured by Invitrogen), CHIR99021 (manufactured by Millipore, 4 ⁇ M), and L-glutamine (Invitrogen) as a medium additive.
  • SP34 StemPro (registered trademark) 34 (manufactured by Invitrogen), CHIR99021 (manufactured by Millipore, 4 ⁇ M), and L-glutamine (Invitrogen) as a medium additive.
  • mesoderm induction process was performed for the culture solution.
  • the first cardiomyocyte induction treatment medium was prepared on the basis of the culture medium for the mesodermal induction process of Test Example 9, and the culture medium for the mesodermal induction process of Test Example 13 was used.
  • the same cardiomyocyte induction treatment medium was prepared, and the same as in Test Example 9 except that each medium was prepared with and without the addition of insulin (10 ng / mL).
  • the first cardiomyocyte induction treatment was performed. Further, in the same manner as in Test Example 9, the first cardiomyocyte induction treatment was performed for the control.
  • a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction process and the second cardiomyocyte induction process is prepared based on the culture medium for the mesodermal induction process of Test Example 9.
  • a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is prepared based on the culture solution for the mesodermal induction process of Test Example 13,
  • the first cardiomyocyte induction treatment and the second myocardial cell induction treatment were performed in the same manner as in Test Example 9 except that the respective media were prepared with and without insulin (10 ng / mL). Cardiomyocyte induction treatment between cell induction treatments was performed. Further, in the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed for the control.
  • Second cardiomyocyte induction process the second cardiomyocyte induction treatment medium was prepared on the basis of the culture medium for mesoderm induction process of Test Example 9, and the culture medium for mesoderm induction process of Test Example 13 was used.
  • a second cardiomyocyte induction treatment medium was prepared based on the same procedure as in Test Example 9 except that each medium was prepared with and without the addition of insulin (10 ng / mL). Then, the second cardiomyocyte induction treatment was performed. Further, in the same manner as in Test Example 9, a second cardiomyocyte induction treatment was performed for the control.
  • transferrin and insulin are indicated by + when they are contained in the culture solution and by-when they are not contained.
  • the results of FIG. 21 show that the differentiation efficiency is higher than that of the control (StemPro34 conventionally used) even without transferrin, but the differentiation efficiency is superior when transferrin is included.
  • the differentiation efficiency exceeding control was shown even if insulin was contained in the culture medium used at the cardiomyocyte induction
  • Embryoid body formation process The embryo was treated in the same manner as in Test Example 9 except that the oxygen condition during suspension culture was (1) hypoxic condition (oxygen concentration 5%) or (2) non-hypoxic condition (oxygen concentration 21%). A body forming step was performed.
  • ⁇ Mesodermal induction process As a medium for mesoderm induction process, a culture medium to which insulin (10 ng / mL) was added or not added to the culture medium for mesoderm induction process of Test Example 5 was used. The mesoderm induction process was performed in the same manner as in Test Example 9 except that the oxygen condition was (1) low oxygen condition (oxygen concentration 5%) or (2) non-low oxygen condition (oxygen concentration 21%). went.
  • control hereinafter sometimes referred to as “SP34”
  • SP34 StemPro (registered trademark) 34 (manufactured by Invitrogen), CHIR99021 (manufactured by Millipore, 4 ⁇ M), and L-glutamine (Invitrogen) as a medium additive. 2 mM), transferrin (Sigma, 150 ⁇ g / mL), ascorbic acid (Sigma, 50 ⁇ g / mL), and 1-thioglycerol (Sigma, 50 ⁇ g / mL) for mesoderm induction process Similarly, the mesoderm induction process was performed for the culture solution.
  • the oxygen condition during suspension culture in the control is a hypoxic condition.
  • ⁇ Cardiomyocyte induction process> ⁇ First cardiomyocyte induction process As the first medium for inducing cardiomyocyte treatment, a medium similar to that in the first cardiomyocyte inducing treatment medium in Test Example 9 was used except that insulin was not added. The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that (1) hypoxic conditions (oxygen concentration 5%) or (2) non-hypoxic conditions (oxygen concentration 21%) were used. went. Further, in the same manner as in Test Example 9, the first cardiomyocyte induction treatment was performed for the control. The oxygen condition during suspension culture in the control is a hypoxic condition.
  • the oxygen condition at the time of suspension culture is (1) low oxygen condition (oxygen concentration 5%), or ( 2)
  • a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed for the control.
  • the oxygen condition during suspension culture in the control is a hypoxic condition.
  • Second cardiomyocyte induction process The medium for the second cardiomyocyte induction treatment was the same as that for the second cardiomyocyte induction treatment in Test Example 9 except that no insulin was added.
  • the second cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that (1) hypoxic condition (oxygen concentration 5%), or (2) non-hypoxic condition (oxygen concentration 21%). went. Further, in the same manner as in Test Example 9, a second cardiomyocyte induction treatment was performed for the control.
  • the oxygen condition during suspension culture in the control is a hypoxic condition.
  • the case of hypoxic condition is indicated by +
  • the case of non-hypoxic condition is indicated by-
  • the case where insulin is contained in the culture medium in the mesoderm induction step is indicated by +
  • the case where it is not included is indicated by-.
  • the culture solution in the mesoderm induction step does not contain insulin, and thus shows a differentiation efficiency higher than that of the control (StemPro34 used conventionally).
  • the conventional differentiation-inducing method was performed under hypoxic conditions, it was found that by using the medium of the present invention, differentiation efficiency superior to that of the control was exhibited even under non-hypoxic conditions.
  • ReproFF2 manufactured by Reprocell, Inc., hereinafter sometimes referred to as “ReproFF2”.
  • IMDM manufactured by Nacalai Tesque Co., Ltd.
  • each component is added to the basal medium as a medium additive to the concentrations shown in Table 6-1 to Table 6-2 below.
  • a prepared medium (hereinafter sometimes referred to as “one-component medium”) was prepared.
  • DMEM / F12 manufactured by Nacalai Tesque Co., Ltd.
  • each component is added to the basal medium as the medium additive to the concentrations shown in Table 7-1 to Table 7-2 below. (Hereinafter sometimes referred to as “first medium”) was prepared.
  • IMDM manufactured by Nacalai Tesque Co., Ltd.
  • No. 2 medium a medium (hereinafter referred to as “No. 2 medium), sometimes referred to as “two media”.
  • ⁇ Embryoid body formation process A medium obtained by adding Y27632 (manufactured by Sigma, 5 ⁇ M) as a differentiation induction regulator to the medium prepared above (StemPro34, ReproFF2, one-part medium, or first medium)
  • the embryoid body formation process was performed like Test Example 5 except having performed.
  • ⁇ Mesodermal induction process A medium prepared by adding CHIR99021 (Millipore, 4 ⁇ M) as a differentiation-inducing regulator to the medium prepared above (StemPro34, one-part medium, or second medium) was used as a medium for mesoderm induction process. In the same manner as in Test Example 5, a mesoderm induction step was performed.
  • Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process A medium prepared by adding IWP2 (manufactured by Sigma, 5 ⁇ M) and estradiol (manufactured by Sigma, 100 nM) as a differentiation induction regulator to the medium prepared above (StemPro34, one-pack medium, or second medium)
  • the first cardiomyocyte induction treatment, the second cardiomyocyte induction treatment, and the second cardiomyocyte induction treatment in the same manner as in Test Example 5, except that the culture solution for the cardiomyocyte induction treatment is used. Cardiomyocyte induction treatment was performed between the cardiomyocyte induction treatment.
  • Second cardiomyocyte induction process A medium obtained by adding estradiol (manufactured by Sigma, 100 nM) as a differentiation-inducing regulator to the medium prepared above (StemPro34, one-component medium, or second medium) is a second cardiomyocyte-inducing culture medium. Except that, the second cardiomyocyte induction treatment was performed in the same manner as in Test Example 5.
  • Table 9 below shows media and differentiation-inducing regulators in each step of Test Example 15.
  • a medium obtained by adding BMP4 (manufactured by R & D, 0.5 ng / mL) or Y27632 (manufactured by Sigma, 5 ⁇ M) as a differentiation-inducing regulator to the first medium prepared above is a culture solution for embryoid body formation process.
  • An embryoid body formation step was performed in the same manner as in Test Example 5 except that.
  • Activin A R & D, 6 ng / mL
  • BMP4 R & D, 10 ng / mL
  • bFGF FGF2, R & D, 5 ng / mL
  • a medium supplemented with CHIR99021 is used as a culture solution for the mesoderm induction process, and the period of suspension culture is defined as the embryoid body formation process from the first day to the third day
  • the mesoderm induction process was performed in the same manner as in Test Example 5 except that the first to fourth days were used.
  • the first cardiomyocyte induction treatment was performed in the same manner as in Test Example 5 except that the period of 3 was 6 days or 4 days to 6 days from the start of the embryoid body formation process.
  • ⁇ Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process In the second medium prepared above, as a differentiation induction regulator, (1) Dkk1 (R & D, 150 ng / mL), SB431542 (Sigma, 5 ⁇ M), and VEGF (R & D, 10 ng / mL), Or (2) cardiomyocyte induction between the first cardiomyocyte induction treatment and the medium containing IWP2 (Sigma, 5 ⁇ M) and estradiol (Sigma, 100 nM) added A cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed in the same manner as in Test Example 5 except that the culture solution for treatment was used.
  • Dkk1 R & D, 150 ng / mL
  • SB431542 Sigma, 5 ⁇ M
  • VEGF R & D, 10 ng / mL
  • Second cardiomyocyte induction process A medium obtained by adding estradiol (manufactured by Sigma, 100 nM) as a differentiation-inducing regulator to the medium prepared above (StemPro34, one-component medium, or second medium) is a second cardiomyocyte-inducing culture medium. Except that, the second cardiomyocyte induction treatment was performed in the same manner as in Test Example 5.
  • VEGF manufactured by R & D, 10 ng / mL
  • bFGF FGF2, R & D, 5 ng / mL
  • estradiol Sigma
  • Table 10 below shows the differentiation induction regulator and the medium in each step of Test Example 16.
  • the composition of the extracellular fluid was NaCl 150 mmol / L, KCl 4 mmol / L, CaCl 2 1.2 mmol / L, MgCl 2 1 mmol / L, D (+)-Glucose 10 mmol / L, HEPES 10 mmol / L (using NaOH adjusted to pH 7.4).
  • the composition of the electrode internal solution used was KCl 140 mmol / L, MgCl 2 1 mmol / L, EGTA 5 mmol / L, MgATP 5 mmol / L, HEPES 10 mmol / L (adjusted to pH 7.2 using KOH).
  • Sodium current A depolarization stimulus of 20 milliseconds from a holding potential of ⁇ 90 mV to ⁇ 20 mV was applied at 1 second intervals.
  • Calcium current A depolarization stimulus of 100 milliseconds was applied at intervals of 10 seconds from a holding potential of ⁇ 40 mV to ⁇ 0 mV.
  • Potassium current After 50 milliseconds of depolarization from a holding potential of -90 mV to -40 mV, a stimulus of 20 mV, 2 seconds of depolarization, and further -40 mV, repolarization to 0.5 seconds was given at 15 second intervals.
  • FIG. 25A shows the result of measuring action potential
  • FIG. 25B shows the result of measuring sodium current
  • FIG. 25C shows the result of measuring potassium current
  • FIG. 25D shows the result of measuring calcium current. Show. From the results of FIGS. 25A to 27, 95% of cardiomyocytes induced to differentiate by the method of the present invention have a large action potential with an action potential amplitude of 140 mV or more, and iPS cell-derived myocardium reported in the past. An action potential having a large and clear plateau phase as compared with the cells was confirmed.
  • the cardiomyocytes induced to differentiate by the method of the present invention exhibited a peak potassium current of 300 pA or more, a peak calcium current of 1 nA or more, and a peak sodium current of 6.5 nA or more. Therefore, it was shown that the cardiomyocytes induced to differentiate by the method of the present invention are high-quality cardiomyocytes.
  • the Wnt signal activator and Wnt signal suppressor alone have very low efficiency in inducing differentiation into cardiomyocytes. Therefore, the addition of a TGF- ⁇ signal inhibitor and estradiol can improve the adhesion culture system. It is presumed that it was necessary to differentiate into cardiomyocytes only in the suspension culture system without intervention.
  • cardiomyocytes can be induced to differentiate without going through adhesion culture after the start of suspension culture. Cells can be produced. Further, in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention, a stable and high-quality cardiomyocyte is obtained because a component that is stably available and has little difference between lots is used as a differentiation induction regulator. Can be manufactured. In addition, since the medium used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention is composed of inexpensive components, high-quality cardiomyocytes can be produced at low cost.
  • the medium used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention does not contain a toxic substance (for example, 2-mercaptoethanol) or a lipid component (for example, Human-ExCyte) whose component is not clear. Excellent safety.
  • a toxic substance for example, 2-mercaptoethanol
  • a lipid component for example, Human-ExCyte
  • cardiomyocytes can be produced regardless of differences between clones.
  • iPS cell single-cell culture coating of extracellular matrix on a culture dish, medium exchange of cells that are easily detached, medium exchange at strict timing, High-quality cardiomyocytes can be produced without using special techniques such as resuspension of differentiated myocardium.
  • Examples of the aspect of the present invention include the following. ⁇ 1> Embryoid body formation step of suspension culture of pluripotent stem cells to form embryoid bodies, Mesoderm induction step of culturing the embryoid body in suspension and inducing mesoderm, A suspension culture of the embryoid body after the mesoderm induction, and a cardiomyocyte induction step of inducing cardiomyocytes,
  • the cardiomyocyte induction step includes a first cardiomyocyte induction process and a second cardiomyocyte induction process;
  • the medium in the embryoid body formation step is any one of a medium in which a ROCK inhibitor is added to a first differentiation induction medium, and a medium in which a ROCK inhibitor is added to a one-part differentiation induction medium.
  • the medium in the mesoderm induction step is any one of a medium obtained by adding a Wnt signal activator to a second differentiation induction medium and a medium obtained by adding a Wnt signal activator to a one-part differentiation induction medium.
  • the medium in the first cardiomyocyte induction treatment is a medium obtained by adding a Wnt signal inhibitor, a TGF- ⁇ signal inhibitor, and an estrogen-like agent to a second differentiation induction medium, and a one-part differentiation induction medium.
  • the medium in the second cardiomyocyte induction treatment is either a medium in which an estrogen-like agent is added to the second differentiation-inducing medium, or a medium in which an estrogen-like agent is added to a one-part differentiation induction medium.
  • the first differentiation induction medium is a medium obtained by adding insulin, transferrin, albumin, and 1-thioglycerol to a basal medium;
  • the second differentiation-inducing medium is an Iskov-modified Dulbecco medium that contains albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL- ⁇ -tocopherol acetate, N-acetyl-L-cysteine, 1-thiol.
  • the one-part differentiation induction medium is transferred to Iscov modified Dulbecco medium with transferrin, albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL- ⁇ -tocopherol acetate, N-acetyl-L-cysteine, 1
  • Iscov modified Dulbecco medium with transferrin, albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL- ⁇ -tocopherol acetate, N-acetyl-L-cysteine, 1
  • a method for inducing differentiation of cardiomyocytes from pluripotent stem cells characterized in that the medium is a medium supplemented with thioglycerol and ascorbic acid.
  • the medium in the embryoid body formation step is a medium obtained by adding a ROCK inhibitor to the first differentiation induction medium
  • the medium in the mesoderm induction step is a medium obtained by adding a Wnt signal activator to the second differentiation induction medium
  • the medium in the first cardiomyocyte induction treatment is a medium obtained by adding a Wnt signal inhibitor, a TGF- ⁇ signal inhibitor, and an estrogen-like agent to the second differentiation induction medium
  • the medium in the embryoid body formation step is a medium in which a ROCK inhibitor is added to a one-part differentiation induction medium
  • the medium in the mesoderm induction step is a medium obtained by adding a Wnt signal activator to a one-part differentiation induction medium
  • the medium in the first cardiomyocyte induction treatment is a medium in which a Wnt signal inhibitor, a TGF- ⁇ signal inhibitor, and an estrogen-like agent are added to a one-part differentiation induction medium
  • ⁇ 4> Medium obtained by adding a Wnt signal inhibitory substance and an estrogen-like substance to the second differentiation induction medium between the first cardiomyocyte induction process and the second cardiomyocyte induction process, and one solution
  • the first differentiation-inducing medium further contains glycine, L-alanine, L-asparagine / H 2 O, L-aspartic acid, L-glutamine, L-glutamic acid, L-histidine, L-isoleucine, L- Methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, ascorbic acid 2-2PO 4 magnesium Salt, AgNO 3 , AlCl 3 ⁇ 6H 2 O, Ba (C 2 H 3 O 2 ) 2 , 3CdSO 4 ⁇ 8H 2 O, CoCl 2 ⁇ 6H 2 O, Cr 2 (SO 4 ) 3 ⁇ H 2 O, GeO 2 , Na 2 SeO 3 , KBr, KI, MnCl 2 .4H 2 O, NaF, Na 2 SiO
  • a medium additive added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells A medium additive comprising at least one of albumin and 1-thioglycerol.
  • a medium additive comprising at least one of albumin and 1-thioglycerol.
  • a differentiation-inducing regulator added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
  • a differentiation-inducing regulator comprising at least one selected from the group consisting of a ROCK inhibitor, a Wnt signal activator, a Wnt signal suppressor, a TGF- ⁇ signal inhibitor, and an estrogen-like agent .
  • the medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
  • the medium additive according to any one of ⁇ 6> to ⁇ 8>, Including the differentiation-inducing regulator according to ⁇ 9> above,
  • the basal medium is at least one selected from the group consisting of Dulbecco's modified Eagle medium / nutrient mixture F-12 ham, Dulbecco's modified Eagle medium, Iskov's modified Dulbecco medium, RPMI 1640 medium, and ⁇ MEM medium. It is.
  • a medium preparation kit for inducing differentiation of cardiomyocytes from pluripotent stem cells The medium additive according to any one of ⁇ 6> to ⁇ 8>, The differentiation-inducing regulator according to ⁇ 9>, A medium comprising at least one basal medium selected from the group consisting of Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 Ham, Dulbecco's Modified Eagle Medium, Iskov's Modified Dulbecco Medium, RPMI 1640 Medium, and ⁇ MEM Medium This is a preparation kit.
  • a kit for inducing differentiation of cardiomyocytes from pluripotent stem cells comprising at least one of the medium according to ⁇ 10> and the medium preparation kit according to ⁇ 11>. is there.

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Abstract

A method for inducing the differentiation of pluripotent stem cells into cardiomyocytes, said method characterized by comprising an embryoid body formation step, a mesodermal induction step, and a cardiomyocyte induction step, wherein: the cardiomyocyte induction step comprises a first cardiomyocyte induction process and a second cardiomyocyte induction process; the culture medium used in the embryoid body formation step is a first differentiation-induction culture medium to which a ROCK inhibitor has been added, or the like; the culture medium used in the mesodermal induction step is a second differentiation-induction culture medium to which a Wnt signal-activating substance has been added, or the like; the culture medium used in the first cardiomyocyte induction process is a second differentiation-induction culture medium to which a Wnt signal-inhibiting substance etc. has been added, or the like; the culture medium used in the second cardiomyocyte induction process is a second differentiation-induction culture medium to which an estrogenic agonist has been added, or the like; the first differentiation-induction culture medium is a basal medium to which insulin etc. has been added; and the second differentiation-induction culture medium is an Iscove's modified Dulbecco's medium to which albumin etc. has been added.

Description

多能性幹細胞から心筋細胞を分化誘導する方法、並びに該方法に好適な培地添加剤、分化誘導調節剤、培地、培地作製用キット、及び多能性幹細胞から心筋細胞を分化誘導するためのキットMethod for inducing differentiation of cardiomyocytes from pluripotent stem cells, medium additive suitable for the method, differentiation induction regulator, medium, kit for preparing medium, and kit for inducing differentiation of cardiomyocytes from pluripotent stem cells
　本発明は、多能性幹細胞から心筋細胞を分化誘導する方法、並びに該方法に好適な多能性幹細胞から心筋細胞を分化誘導するための培地に添加する培地添加剤、多能性幹細胞から心筋細胞を分化誘導するための培地に添加する分化誘導調節剤、多能性幹細胞から心筋細胞を分化誘導するための培地、多能性幹細胞から心筋細胞を分化誘導するための培地作製用キット、及び多能性幹細胞から心筋細胞を分化誘導するためのキットに関する。
本願は、２０１４年９月２日に、日本に出願された特願２０１４－１７８２８５号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to a method for inducing differentiation of cardiomyocytes from pluripotent stem cells, a medium additive added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells suitable for the method, and from pluripotent stem cells to cardiac muscle A differentiation-inducing regulator added to a medium for inducing cell differentiation, a medium for inducing cardiomyocyte differentiation from pluripotent stem cells, a medium preparation kit for inducing cardiomyocyte differentiation from pluripotent stem cells, and The present invention relates to a kit for inducing differentiation of cardiomyocytes from pluripotent stem cells.
This application claims priority based on Japanese Patent Application No. 2014-178285 filed in Japan on September 2, 2014, the contents of which are incorporated herein by reference.
　近年、多能性幹細胞の１つである人工多能性幹細胞（以下、「ｉＰＳ細胞」と称することがある）の研究が進められている。例えば、ヒトｉＰＳ細胞から分化誘導した心筋細胞は、再生医療、創薬研究、薬物安全性試験などの様々な用途への応用が期待されている。 In recent years, research on induced pluripotent stem cells (hereinafter sometimes referred to as “iPS cells”), which is one of pluripotent stem cells, has been underway. For example, cardiomyocytes differentiated from human iPS cells are expected to be applied to various uses such as regenerative medicine, drug discovery research, and drug safety tests.
　再生医療では、例えば、自己のｉＰＳ細胞やバンキングされたｉＰＳ細胞から分化誘導された心筋細胞を心臓に移植することにより、失われた心臓の機能を回復させることが期待されている。また、創薬研究では、例えば、健常者のｉＰＳ細胞や難病患者から作製された疾患特異的ｉＰＳ細胞から分化誘導された心筋細胞を解析やスクリーニングに利用し、循環器疾患や難病に対する新薬を開発することが期待されている。また、薬物安全性試験では、開発中もしくは開発された薬の心毒性のスクリーニングのために心筋細胞を用いたりすることが期待されている。 In regenerative medicine, for example, it is expected to restore lost heart function by transplanting myocardial cells differentiated from self iPS cells or banked iPS cells into the heart. In drug discovery research, for example, the development of new drugs for cardiovascular diseases and intractable diseases using iPS cells of healthy individuals and cardiomyocytes differentiated from disease-specific iPS cells prepared from patients with intractable diseases is used for analysis and screening. Is expected to be. In drug safety studies, cardiomyocytes are expected to be used for screening for cardiotoxicity during development or for developed drugs.
　再生医療、創薬研究、薬物安全性試験などの様々な用途に心筋細胞が産業利用されるためには、多能性幹細胞から、大量に、安定して心筋細胞を得る必要があり、そのためには心筋細胞を分化誘導する方法が、技術的に容易で少ない手順から構成されており、かつ安価な方法でなければならない。また、前記心筋細胞は、大きく明瞭なプラトー相を有する活動電位を示し、大きなピークカリウム電流（３００ｐＡ以上）、ピークカルシウム電流（１ｎＡ以上）、ピークナトリウム電流（６．５ｎＡ以上）を安定して示す、高品質なものであることが好ましい。 In order for cardiomyocytes to be industrially used for various applications such as regenerative medicine, drug discovery research, and drug safety testing, it is necessary to stably obtain cardiomyocytes in large quantities from pluripotent stem cells. The method for inducing differentiation of cardiomyocytes is technically easy and is composed of a small number of procedures and must be an inexpensive method. Further, the cardiomyocytes show an action potential having a large and clear plateau phase, and stably show a large peak potassium current (300 pA or more), a peak calcium current (1 nA or more), and a peak sodium current (6.5 nA or more). High quality is preferable.
　これまでに、胚様体形成法で心筋細胞を分化誘導する方法が提案されている（例えば、非特許文献１参照）。
　前記提案では、フィーダー細胞上で培養されているｉＰＳ細胞を剥離した後、一旦フィーダー細胞が無い状態で平面培養し、再度剥離した後に浮遊培養を行っている（図１参照）。また、前記提案では、浮遊培養時の培地として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、培地添加剤として、Ｌ－グルタミンを２ｍＭ、トランスフェリンを１５０μｇ／ｍＬ、アスコルビン酸を５０μｇ／ｍＬ、１－チオグリセロールを５０μｇ／ｍＬ加えた培地を用い、分化誘導調節剤として、浮遊培養開始時から１日目までは骨形成タンパク質４（以下、「ＢＭＰ４」と称することがある）を０．５ｎｇ／ｍＬ添加し、１日目から４日目まではＢＭＰ４を１０ｎｇ／ｍＬ、塩基性線維芽細胞成長因子（以下、「ｂＦＧＦ」と称することがある）を５ｎｇ／ｍＬ、及びアクチビンＡを６ｎｇ／ｍＬ添加し、４日目から８日目までは血管内皮増殖因子（以下、「ＶＥＧＦ」と称することがある）を１０ｎｇ／ｍＬ、及び分泌タンパク質Ｄｉｃｋｋｏｐｆ１（以下、「Ｄｋｋ－１」と称することがある）を１５０ｎｇ／ｍＬを添加し、８日目以降はＶＥＧＦを１０ｎｇ／ｍＬ、Ｄｋｋ－１を１５０ｎｇ／ｍＬ、及びｂＦＧＦを５ｎｇ／ｍＬ添加することで心筋細胞への分化を誘導している。
　前記提案によれば、浮遊培養（三次元培養）により心筋細胞へ分化誘導することができるので、大量に心筋細胞を得ることができると考えられる。
　しかしながら、前記提案では、高価で製造業者間による品質の差やロット間差の大きいサイトカイン（アクチビンＡ、ＢＭＰ４、Ｄｋｋ－１、ｂＦＧＦ、及びＶＥＧＦ）が用いられており、また、ＳｔｅｍＰｒｏ（登録商標）３４には、毒物である２－メルカプトエタノール、成分が明らかでない脂質成分であるＨｕｍａｎ－ＥｘＣｙｔｅ、及び高価でロット間差が大きいヒト血清アルブミンが用いられているため、安全性に優れ、安定して、安価に心筋細胞を得ることができないという問題がある。 So far, a method for inducing differentiation of cardiomyocytes by an embryoid body formation method has been proposed (see, for example, Non-Patent Document 1).
In the above proposal, after detaching the iPS cells cultured on the feeder cells, the cells are once cultured in a flat state in the absence of the feeder cells, and after detaching again, the suspension culture is performed (see FIG. 1). In the above proposal, StemPro (registered trademark) 34 (manufactured by Invitrogen) is used as a medium for suspension culture, L-glutamine is 2 mM, transferrin is 150 μg / mL, ascorbic acid is 50 μg / mL, Using a medium supplemented with 1-thioglycerol at 50 μg / mL, 0.5 ng of bone morphogenetic protein 4 (hereinafter sometimes referred to as “BMP4”) is used as a differentiation induction regulator from the beginning of suspension culture until the first day. From day 1 to day 4, BMP4 was 10 ng / mL, basic fibroblast growth factor (hereinafter sometimes referred to as “bFGF”) was 5 ng / mL, and activin A was 6 ng / mL. mL was added, and from day 4 to day 8, vascular endothelial growth factor (hereinafter sometimes referred to as “VEGF”) was 10 ng / mL, and 150 ng / mL of protein Dickkopf1 (hereinafter sometimes referred to as “Dkk-1”) is added, and after 8 days, VEGF is 10 ng / mL, Dkk-1 is 150 ng / mL, and bFGF is 5 ng / mL. Addition induces cardiomyocyte differentiation.
According to the above proposal, it is considered that cardiomyocytes can be obtained in a large amount because differentiation into cardiomyocytes can be induced by suspension culture (three-dimensional culture).
However, in the above proposal, cytokines (activin A, BMP4, Dkk-1, bFGF, and VEGF) that are expensive and have large quality differences among manufacturers and lot-to-lot differences are used, and StemPro (registered trademark) is used. No. 34 uses 2-mercaptoethanol, which is a toxic substance, Human-ExCyte, which is a lipid component whose component is not clear, and human serum albumin that is expensive and has a large lot-to-lot difference. There is a problem that cardiomyocytes cannot be obtained at low cost.
　また、サイトカインを使わずに心筋細胞を分化誘導する方法も提案されている（例えば、非特許文献２参照）。
　前記提案では、フィーダーレス培養後、単細胞に単離し、その後、高密度で播種し、接着培養を行っている（図２参照）。
　前記提案によれば、高価でロット間差の大きいサイトカインではなく、ＣＨＩＲ９９０２１やＩＷＰ２などの低分子化合物を用いているため、安定に、安価に心筋細胞を得ることができると考えられる。
　しかしながら、前記提案は、接着培養により行われるものであり、大量に心筋細胞を得ることが困難であるという問題があり、また、フィーダーレス培養に馴化することが可能な細胞でなければ分化させることが困難であるという問題や、培養液を交換するタイミングが非常に厳密でかつ培養液を交換する際に細胞が剥がれやすいなど技術的に困難であるという問題がある。 A method for inducing differentiation of cardiomyocytes without using cytokines has also been proposed (see, for example, Non-Patent Document 2).
In the above proposal, after feeder-less culture, the cells are isolated into single cells and then seeded at a high density to perform adhesion culture (see FIG. 2).
According to the above proposal, it is considered that cardiomyocytes can be obtained stably and inexpensively because low molecular weight compounds such as CHIR99021 and IWP2 are used instead of expensive cytokines with large lot-to-lot differences.
However, the above proposal is performed by adhesion culture, and there is a problem that it is difficult to obtain a large amount of cardiomyocytes, and it is differentiated unless the cells can be adapted to feeder-less culture. There is a problem that it is difficult, and the timing of exchanging the culture medium is very strict, and there is a problem that it is technically difficult, for example, when the culture medium is replaced, the cells are easily detached.
　また、接着培養と、浮遊培養とを併用し、心筋細胞を分化誘導する方法も提案されている（例えば、非特許文献３参照）。
　前記提案では、フィーダー細胞培養後、浮遊培養を行い、その後、接着培養を介して、更に浮遊培養を行っている（図３参照）。
　前記提案によれば、高価でロット間差の大きいサイトカインではなく、ＣＨＩＲ９９０２１、ＢＩＯ、ＫＹ０２１１１などの低分子化合物を用いているため、安定に、安価に心筋細胞を得ることができると考えられる。
　しかしながら、前記提案は、接着培養と、浮遊培養とを併用するため、技術的に困難な複数の手順から構成されており、非常に煩雑であるという問題がある。 In addition, a method of inducing differentiation of cardiomyocytes by using both adhesion culture and suspension culture has been proposed (see Non-Patent Document 3, for example).
In the above proposal, suspension culture is performed after feeder cell culture, and then further suspension culture is performed via adhesion culture (see FIG. 3).
According to the above proposal, since low molecular compounds such as CHIR99021, BIO, and KY02111 are used instead of expensive cytokines with large lot-to-lot differences, it is considered that cardiomyocytes can be obtained stably and inexpensively.
However, since the above-mentioned proposal uses both adhesion culture and suspension culture, it consists of a plurality of technically difficult procedures and has a problem that it is very complicated.
　したがって、高品質な心筋細胞を、大量に、安定して、安価に、かつ簡便に製造することが可能な心筋細胞の分化誘導方法の開発が強く求められているのが現状である。 Accordingly, there is a strong demand for the development of a method for inducing differentiation of cardiomyocytes that can produce high-quality cardiomyocytes in large quantities, stably, inexpensively and simply.
　本発明は、従来における前記諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、高品質な心筋細胞を、大量に、安定して、安価に、かつ簡便に製造することが可能な多能性幹細胞から心筋細胞を分化誘導する方法、並びに該方法に好適な培地添加剤、分化誘導調節剤、培地、培地作製用キット、及び多能性幹細胞から心筋細胞を分化誘導するためのキットを提供することを目的とする。 This invention makes it a subject to solve the said various problems in the past and to achieve the following objectives. That is, the present invention is a method for inducing differentiation of cardiomyocytes from pluripotent stem cells capable of producing high-quality cardiomyocytes in large quantities, stably, inexpensively and simply, and suitable for the method. An object of the present invention is to provide a medium additive, a differentiation induction regulator, a medium, a medium preparation kit, and a kit for inducing differentiation of cardiomyocytes from pluripotent stem cells.
　前記課題を解決するための手段としては、以下の通りである。即ち、
　＜１＞　多能性幹細胞を浮遊培養し、胚様体を形成する胚様体形成工程と、
　前記胚様体を浮遊培養し、中胚葉を誘導する中胚葉誘導工程と、
　前記中胚葉誘導後の胚様体を浮遊培養し、心筋細胞を誘導する心筋細胞誘導工程とを含み、
　前記心筋細胞誘導工程が、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理とを含み、
　前記胚様体形成工程における培地が、第１の分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地、及び一液式分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地のいずれかであり、
　前記中胚葉誘導工程における培地が、第２の分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地のいずれかであり、
　前記第１の心筋細胞誘導処理における培地が、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、トランスフォーミング増殖因子β（以下、「ＴＧＦ－β」と称することがある）シグナル阻害剤、及びエストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地のいずれかであり、
　前記第２の心筋細胞誘導処理における培地が、第２の分化誘導培地に、エストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、エストロゲン様作用物質を加えた培地のいずれかであり、
　前記第１の分化誘導培地が、基礎培地に、インスリン、トランスフェリン、アルブミン、及び１－チオグリセロールを加えた培地であり、
　前記第２の分化誘導培地が、イスコフ改変ダルベッコ培地に、アルブミン、ポリビニルアルコール、エタノラミン塩酸塩、亜セレン酸ナトリウム、ヒドロコルチゾン、ＤＬ－α－トコフェロール酢酸エステル、Ｎ－アセチル－Ｌ－システイン、１－チオグリセロール、及びアスコルビン酸を加えた培地であり、
　前記一液式分化誘導培地が、イスコフ改変ダルベッコ培地に、トランスフェリン、アルブミン、ポリビニルアルコール、エタノラミン塩酸塩、亜セレン酸ナトリウム、ヒドロコルチゾン、ＤＬ－α－トコフェロール酢酸エステル、Ｎ－アセチル－Ｌ－システイン、１－チオグリセロール、及びアスコルビン酸を加えた培地であることを特徴とする多能性幹細胞から心筋細胞を分化誘導する方法である。
　＜２＞　多能性幹細胞から心筋細胞を分化誘導するための培地に添加する培地添加剤であって、
　アルブミン及び１－チオグリセロールの少なくともいずれかを含むことを特徴とする培地添加剤である。
　＜３＞　多能性幹細胞から心筋細胞を分化誘導するための培地に添加する分化誘導調節剤であって、
　ＲＯＣＫ阻害剤、Ｗｎｔシグナル活性化物質、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質からなる群から選択される少なくとも１種を含むことを特徴とする分化誘導調節剤である。
　＜４＞　多能性幹細胞から心筋細胞を分化誘導するための培地であって、
　前記＜２＞に記載の培地添加剤と、
　前記＜３＞に記載の分化誘導調節剤とを含み、
　基礎培地が、ダルベッコ改変イーグル培地／栄養混合物Ｆ－１２ハム、ダルベッコ改変イーグル培地、イスコフ改変ダルベッコ培地、ＲＰＭＩ１６４０培地、及びαＭＥＭ培地からなる群から選択される少なくとも１種であることを特徴とする培地である。
　＜５＞　多能性幹細胞から心筋細胞を分化誘導するための培地作製用キットであって、
　前記＜２＞に記載の培地添加剤と、
　前記＜３＞に記載の分化誘導調節剤と、
　ダルベッコ改変イーグル培地／栄養混合物Ｆ－１２ハム、ダルベッコ改変イーグル培地、イスコフ改変ダルベッコ培地、ＲＰＭＩ１６４０培地、及びαＭＥＭ培地からなる群から選択される少なくとも１種の基礎培地とを含むことを特徴とする培地作製用キットである。
　＜６＞　前記＜４＞に記載の培地、及び前記＜５＞に記載の培地作製用キットの少なくともいずれかを含むことを特徴とする多能性幹細胞から心筋細胞を分化誘導するためのキットである。 Means for solving the problems are as follows. That is,
<1> Embryoid body formation step of suspension culture of pluripotent stem cells to form embryoid bodies,
Mesoderm induction step of culturing the embryoid body in suspension and inducing mesoderm,
A suspension culture of the embryoid body after the mesoderm induction, and a cardiomyocyte induction step of inducing cardiomyocytes,
The cardiomyocyte induction step includes a first cardiomyocyte induction process and a second cardiomyocyte induction process;
The medium in the embryoid body formation step is any one of a medium in which a ROCK inhibitor is added to a first differentiation induction medium, and a medium in which a ROCK inhibitor is added to a one-part differentiation induction medium.
The medium in the mesoderm induction step is any one of a medium obtained by adding a Wnt signal activator to a second differentiation induction medium and a medium obtained by adding a Wnt signal activator to a one-part differentiation induction medium. ,
In the first cardiomyocyte induction treatment, the second differentiation induction medium includes a Wnt signal inhibitor, a transforming growth factor β (hereinafter sometimes referred to as “TGF-β”) signal inhibitor, and an estrogen. Any one of a medium added with a like-like substance and a medium added with a Wnt signal inhibitory substance, a TGF-β signal inhibitor, and an estrogen-like acting substance in a one-part differentiation induction medium,
The medium in the second cardiomyocyte induction treatment is either a medium in which an estrogen-like agent is added to the second differentiation-inducing medium, or a medium in which an estrogen-like agent is added to a one-part differentiation induction medium. Yes,
The first differentiation induction medium is a medium obtained by adding insulin, transferrin, albumin, and 1-thioglycerol to a basal medium;
The second differentiation-inducing medium is an Iskov-modified Dulbecco medium that contains albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine, 1-thiol. A medium to which glycerol and ascorbic acid are added,
The one-part differentiation induction medium is transferred to Iscov modified Dulbecco medium with transferrin, albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine, 1 A method for inducing differentiation of cardiomyocytes from pluripotent stem cells, characterized in that the medium is a medium supplemented with thioglycerol and ascorbic acid.
<2> A medium additive added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
A medium additive comprising at least one of albumin and 1-thioglycerol.
<3> A differentiation-inducing regulator added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
A differentiation-inducing regulator comprising at least one selected from the group consisting of a ROCK inhibitor, a Wnt signal activator, a Wnt signal suppressor, a TGF-β signal inhibitor, and an estrogen-like agent .
<4> A medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
The culture medium additive according to <2>,
Including the differentiation-inducing regulator according to <3>,
The basal medium is at least one selected from the group consisting of Dulbecco's modified Eagle medium / nutrient mixture F-12 ham, Dulbecco's modified Eagle medium, Iskov's modified Dulbecco medium, RPMI 1640 medium, and αMEM medium. It is.
<5> A medium preparation kit for inducing differentiation of cardiomyocytes from pluripotent stem cells,
The culture medium additive according to <2>,
The differentiation-inducing regulator according to <3>,
A medium comprising at least one basal medium selected from the group consisting of Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 Ham, Dulbecco's Modified Eagle Medium, Iskov's Modified Dulbecco Medium, RPMI 1640 Medium, and αMEM Medium This is a preparation kit.
<6> A kit for inducing differentiation of cardiomyocytes from pluripotent stem cells, comprising at least one of the medium according to <4> and the medium preparation kit according to <5>. is there.
　本発明によれば、従来における前記諸問題を解決し、前記目的を達成することができ、高品質な心筋細胞を、大量に、安定して、安価に、かつ簡便に製造することが可能な多能性幹細胞から心筋細胞を分化誘導する方法、並びに該方法に好適な培地添加剤、分化誘導調節剤、培地、培地作製用キット、及び多能性幹細胞から心筋細胞を分化誘導するためのキットを提供することができる。 According to the present invention, it is possible to solve the conventional problems and achieve the object, and to manufacture high-quality cardiomyocytes in a large amount stably, inexpensively and simply. Method for inducing differentiation of cardiomyocytes from pluripotent stem cells, medium additive suitable for the method, differentiation induction regulator, medium, kit for preparing medium, and kit for inducing differentiation of cardiomyocytes from pluripotent stem cells Can be provided.
図１は、従来の多能性幹細胞から心筋細胞を分化誘導する方法の一例を説明する図である。FIG. 1 is a diagram for explaining an example of a conventional method for inducing differentiation of cardiomyocytes from pluripotent stem cells. 図２は、従来の多能性幹細胞から心筋細胞を分化誘導する方法の他の一例を説明する図である。FIG. 2 is a diagram for explaining another example of a conventional method for inducing differentiation of cardiomyocytes from pluripotent stem cells. 図３は、従来の多能性幹細胞から心筋細胞を分化誘導する方法の他の一例を説明する図である。FIG. 3 is a diagram for explaining another example of a conventional method for inducing differentiation of cardiomyocytes from pluripotent stem cells. 図４は、本発明の多能性幹細胞から心筋細胞を分化誘導する方法の一例を説明する図である。FIG. 4 is a diagram for explaining an example of a method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention. 図５は、試験例１の結果を示す図である。FIG. 5 is a diagram showing the results of Test Example 1. 図６は、試験例２におけるクローン１の結果を示す図である。FIG. 6 is a diagram showing the results of clone 1 in Test Example 2. 図７は、試験例２におけるクローン２の結果を示す図である。FIG. 7 is a diagram showing the results of clone 2 in Test Example 2. 図８は、試験例３の結果を示す図である。FIG. 8 is a diagram showing the results of Test Example 3. 図９は、試験例４におけるクローン１の結果を示す図である。FIG. 9 is a diagram showing the results of clone 1 in Test Example 4. 図１０は、試験例４におけるクローン２の結果を示す図である。FIG. 10 is a diagram showing the results of clone 2 in Test Example 4. 図１１は、試験例５の結果を示す図－１である。FIG. 11 is a diagram -1 showing the results of Test Example 5. 図１２は、試験例５の結果を示す図－２である。FIG. 12 is a diagram 2 showing the results of Test Example 5. 図１３は、試験例６の結果を示す図である。FIG. 13 is a diagram showing the results of Test Example 6. 図１４は、試験例７におけるｂＦＧＦを添加した場合の結果を示す図である。FIG. 14 is a diagram showing the results when bFGF was added in Test Example 7. 図１５は、試験例７におけるＣＨＩＲ９９０２１を添加した場合の結果を示す図である。FIG. 15 is a diagram showing the results when CHIR99021 in Test Example 7 is added. 図１６は、試験例８の結果を示す図である。FIG. 16 is a diagram showing the results of Test Example 8. 図１７は、試験例９の結果を示す図である。FIG. 17 is a diagram showing the results of Test Example 9. 図１８は、試験例１０の結果を示す図である。FIG. 18 is a diagram showing the results of Test Example 10. 図１９は、試験例１１の結果を示す図である。FIG. 19 is a diagram showing the results of Test Example 11. 図２０は、試験例１２の結果を示す図である。FIG. 20 is a diagram showing the results of Test Example 12. 図２１は、試験例１３の結果を示す図である。FIG. 21 is a diagram showing the results of Test Example 13. 図２２は、試験例１４の結果を示す図である。FIG. 22 is a diagram showing the results of Test Example 14. 図２３は、試験例１５の結果を示す図である。FIG. 23 is a diagram showing the results of Test Example 15. 図２４は、試験例１６の拍動している胚様体の割合を測定した結果を示す図である。FIG. 24 is a diagram showing the results of measuring the ratio of beating embryoid bodies in Test Example 16. 図２５Ａは、試験例１６のパッチクランプ法で測定した活動電位の一例を示す図である。25A is a diagram illustrating an example of action potentials measured by the patch clamp method of Test Example 16. FIG. 図２５Ｂは、試験例１６のパッチクランプ法で測定したナトリウム電流の一例を示す図である。FIG. 25B is a diagram showing an example of a sodium current measured by the patch clamp method of Test Example 16. 図２５Ｃは、試験例１６のパッチクランプ法で測定したカリウム電流の一例を示す図である。FIG. 25C is a diagram illustrating an example of a potassium current measured by the patch clamp method of Test Example 16. 図２５Ｄは、試験例１６のパッチクランプ法で測定したカルシウム電流の一例を示す図である。FIG. 25D is a diagram illustrating an example of a calcium current measured by the patch clamp method of Test Example 16. 図２６は、これまでに報告されているパッチクランプ法で測定した活動電位の結果の一例を示す図である。FIG. 26 is a diagram illustrating an example of a result of action potentials measured by the patch clamp method reported so far. 図２７は、これまでに報告されているパッチクランプ法で測定した活動電位の結果の他の一例を示す図である。FIG. 27 is a diagram showing another example of the result of the action potential measured by the patch clamp method reported so far.
　以下、本発明の好ましい例を説明するが、本発明はこれらの例に限定されることはない。本発明の趣旨を逸脱しない範囲で、構成の付加、省略、置換、およびその他の変更が可能である。 Hereinafter, preferred examples of the present invention will be described, but the present invention is not limited to these examples. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit of the present invention.
（多能性幹細胞から心筋細胞を分化誘導する方法）
　本発明の多能性幹細胞から心筋細胞を分化誘導する方法は、胚様体形成工程と、中胚葉誘導工程と、心筋細胞誘導工程とを少なくとも含み、必要に応じて更にその他の工程を含む。 (Method for inducing cardiomyocyte differentiation from pluripotent stem cells)
The method for inducing differentiation of cardiomyocytes from pluripotent stem cells of the present invention includes at least an embryoid body formation step, a mesoderm induction step, and a cardiomyocyte induction step, and further includes other steps as necessary.
　本発明の多能性幹細胞から心筋細胞を分化誘導する方法の一例を図４に示す。
　図４中、Ｄａｙ　０～１は、前記胚様体形成工程であり（胚様体形成工程開始０日目～１日目）、Ｄａｙ　１～３は、前記中胚葉誘導工程であり（胚様体形成工程開始１日目～３日目）、Ｄａｙ　３～６は、前記心筋細胞誘導工程における第１の心筋細胞誘導処理であり（胚様体形成工程開始３日目～６日目）、Ｄａｙ　６以降は、前記心筋細胞誘導工程における第２の心筋細胞誘導処理である（胚様体形成工程開始６日目以降）。
　また、図４中、Ｙ２７６３２、ＣＨＩＲ９９０２１、ＩＷＰ２、ＳＢ４３１５４２、及びエストラジオール（Ｅｓｔｒａｄｉｏｌ）は、前記各工程の培地に添加する分化誘導調節剤の一例を示す。 An example of a method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention is shown in FIG.
In FIG. 4, Day 0 to 1 are the embryoid body formation process (embryoid body formation process start day 0 to day 1), and Day 1 to 3 are the mesoderm induction process (embryo-like process) Day 3 to 3), Day 3 to 6 are the first cardiomyocyte induction processes in the cardiomyocyte induction process (embryoid body formation process start 3 to 6), Day 6 and subsequent steps are the second cardiomyocyte induction process in the cardiomyocyte induction process (after the 6th day of the embryoid body formation process start).
Moreover, Y27632, CHIR99021, IWP2, SB431542, and estradiol (Estradiol) in FIG. 4 show an example of a differentiation induction regulator added to the medium in each step.
＜胚様体形成工程＞
　前記胚様体形成工程は、多能性幹細胞を浮遊培養し、胚様体を形成する工程である。 <Embryoid body formation process>
The embryoid body formation step is a step of forming embryoid bodies by suspension culture of pluripotent stem cells.
＜＜多能性幹細胞＞＞
　前記多能性幹細胞としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ｉＰＳ細胞、ＥＳ細胞などが挙げられる。これらの中でも、ｉＰＳ細胞が好ましい。
　前記多能性幹細胞の種としては、特に制限はなく、目的に応じて適宜選択することができる。 << pluripotent stem cells >>
There is no restriction | limiting in particular as said pluripotent stem cell, According to the objective, it can select suitably, For example, an iPS cell, ES cell, etc. are mentioned. Among these, iPS cells are preferable.
There is no restriction | limiting in particular as a seed | species of the said pluripotent stem cell, According to the objective, it can select suitably.
　前記多能性幹細胞の調製方法としては、特に制限はなく、公知の方法を適宜選択することができ、例えば、後述するフィーダー細胞培養工程により未分化状態で維持されている多能性幹細胞をコロニー状に解離したものを前記胚様体形成工程に用いることができる。 The method for preparing the pluripotent stem cells is not particularly limited, and a known method can be appropriately selected. For example, pluripotent stem cells maintained in an undifferentiated state by a feeder cell culture step described later are colonized. Those dissociated into a shape can be used in the embryoid body formation step.
＜＜培地＞＞
　前記胚様体形成工程における培地（以下、「胚様体形成工程用培地」、「胚様体形成工程用培養液」と称することがある）は、第１の分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地、及び一液式分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地のいずれかであり、必要に応じて更にその他の成分を含む。 << Medium >>
The medium in the embryoid body formation step (hereinafter sometimes referred to as “embryoid body formation step medium” or “embryoid body formation step culture medium”) is added to the first differentiation induction medium as a ROCK inhibitor. Or a one-component differentiation induction medium with a ROCK inhibitor added, and further contains other components as necessary.
－第１の分化誘導培地－
　前記第１の分化誘導培地（以下、「第一培地」と称することがある）は、基礎培地に、培地添加剤（以下、「第１の分化誘導培地用培地添加剤」と称することがある）を加えた培地であり、必要に応じて更にその他の成分を含む。 -First differentiation-inducing medium-
The first differentiation-inducing medium (hereinafter sometimes referred to as “first medium”) may be referred to as a medium additive (hereinafter referred to as “first medium for differentiation-inducing medium”) as a basal medium. ), And further contains other components as necessary.
－－基礎培地－－
　前記第１の分化誘導培地における基礎培地としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ダルベッコ改変イーグル培地／栄養混合物Ｆ－１２ハム（以下、「ＤＭＥＭ／Ｆ１２」と称することがある）、イスコフ改変ダルベッコ培地（以下、「ＩＭＤＭ」と称することがある）、ダルベッコ改変イーグル培地（高グルコース）（以下、「ＤＭＥＭ（高グルコース）」と称することがある）、ダルベッコ改変イーグル培地（低グルコース）（以下、「ＤＭＥＭ（低グルコース）」と称することがある）、αＭＥＭ培地などが挙げられる。これらの中でも、心筋細胞への分化誘導効率に優れる点で、ＤＭＥＭ／Ｆ１２、ＩＭＤＭが好ましく、ＤＭＥＭ／Ｆ１２がより好ましい。
　前記ＤＭＥＭは、高グルコースであってもよいし、低グルコースであってもよい。
　前記基礎培地は、市販品を用いてもよいし、適宜調製したものを用いてもよい。 --Basic medium--
The basal medium in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. For example, Dulbecco's modified Eagle medium / nutrient mixture F-12 ham (hereinafter referred to as “DMEM / F12”) ), Iskov modified Dulbecco medium (hereinafter sometimes referred to as “IMDM”), Dulbecco modified Eagle medium (high glucose) (hereinafter sometimes referred to as “DMEM (high glucose)”), Dulbecco Examples thereof include a modified Eagle medium (low glucose) (hereinafter sometimes referred to as “DMEM (low glucose)”), an αMEM medium, and the like. Among these, DMEM / F12 and IMDM are preferable, and DMEM / F12 is more preferable in terms of excellent differentiation induction efficiency into cardiomyocytes.
The DMEM may be high glucose or low glucose.
As the basal medium, commercially available products may be used, or those prepared as appropriate may be used.
－－第１の分化誘導培地用培地添加剤－－
　前記第１の分化誘導培地用培地添加剤は、インスリンと、トランスフェリンと、アルブミンと、１－チオグリセロールとを少なくとも含み、必要に応じて更にその他の成分を含む。 --First medium additive for differentiation induction medium--
The first medium additive for differentiation induction medium contains at least insulin, transferrin, albumin, and 1-thioglycerol, and further contains other components as necessary.
　前記第１の分化誘導培地におけるインスリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～１００ｍｇ／Ｌが好ましく、５ｍｇ／Ｌ～５０ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of insulin in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 100 mg / L, and preferably 5 mg / L to 50 mg / L. More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるトランスフェリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～５０ｍｇ／Ｌが好ましく、２ｍｇ／Ｌ～２０ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The transferrin content in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 50 mg / L, and preferably 2 mg / L to 20 mg / L. More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるアルブミンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、４，０００ｍｇ／Ｌ～１６，０００ｍｇ／Ｌが好ましく、６，０００ｍｇ／Ｌ～１４，０００ｍｇ／Ｌがより好ましく、８，０００ｍｇ／Ｌ～１２，０００ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。
　前記アルブミンとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト血清アルブミン、牛血清アルブミン、リコンビナントヒトアルブミン、リコンビナント牛アルブミンなどが挙げられる。 The content of albumin in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 4,000 mg / L to 16,000 mg / L, and preferably 6,000 mg / L. L to 14,000 mg / L is more preferable, and 8,000 mg / L to 12,000 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
There is no restriction | limiting in particular as said albumin, According to the objective, it can select suitably, For example, human serum albumin, bovine serum albumin, recombinant human albumin, recombinant bovine albumin etc. are mentioned.
　前記第１の分化誘導培地における１－チオグリセロールの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、２０ｍｇ／Ｌ～８０ｍｇ／Ｌが好ましく、３０ｍｇ／Ｌ～７０ｍｇ／Ｌがより好ましく、４０ｍｇ／Ｌ～６０ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The 1-thioglycerol content in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 20 mg / L to 80 mg / L, and preferably 30 mg / L to 70 mg. / L is more preferable, and 40 mg / L to 60 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地用培地添加剤におけるその他の成分としては、特に制限はなく、目的に応じて適宜選択することができるが、グリシン、Ｌ－アラニン、Ｌ－アスパラギン・Ｈ２Ｏ、Ｌ－アスパラギン酸、Ｌ－グルタミン、Ｌ－グルタミン酸、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏを含むことが好ましい。 Other components in the first medium for differentiation induction medium are not particularly limited and may be appropriately selected depending on the intended purpose. However, glycine, L-alanine, L-asparagine / H 2 O, L -Aspartic acid, L-glutamine, L-glutamic acid, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L - tyrosine, L- valine, thiamine, reduced glutathione, magnesium salt of ascorbic acid -2-2PO 4, AgNO 3, AlCl 3 · 6H 2 O, Ba (C 2 H 3 O 2) 2, 3CdSO 4 · 8H 2 O, CoCl 2 · 6H 2 O , Cr 2 (SO 4) 3 · H 2 O, GeO 2, Na 2 SeO 3, Br, KI, MnCl 2 · 4H 2 O, NaF, Na 2 SiO 3 · 9H 2 O, NaVO 3, (NH 4) 6 Mo 7 O 24 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl, SnCl 2 · 2H 2 O, and preferably includes a ZrOCl 2 · 8H 2 O.
　前記第１の分化誘導培地におけるグリシンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～２００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of glycine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 200 mg / L, and is preferably 10 mg / L to 100 mg / L. More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－アラニンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～２０ｍｇ／Ｌが好ましく、５ｍｇ／Ｌ～１５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-alanine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 20 mg / L, and 5 mg / L to 15 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－アスパラギン・Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～１００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～５０ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-asparagine · H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 100 mg / L, and preferably 10 mg / L L to 50 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－アスパラギン酸の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～１００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～５０ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-aspartic acid in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 100 mg / L, and 10 mg / L to 50 mg. / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－グルタミンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５０ｍｇ／Ｌ～１，０００ｍｇ／Ｌが好ましく、１００ｍｇ／Ｌ～５００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-glutamine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / L to 1,000 mg / L, preferably 100 mg / L to 500 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－グルタミン酸の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～１００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～５０ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-glutamic acid in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 100 mg / L, and 10 mg / L to 50 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－ヒスチジンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～３００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～２００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-histidine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 300 mg / L, and 10 mg / L to 200 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－イソロイシンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５０ｍｇ／Ｌ～４，０００ｍｇ／Ｌが好ましく、１００ｍｇ／Ｌ～１，０００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-isoleucine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / L to 4,000 mg / L, preferably 100 mg / L to 1,000 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－メチオニンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～２５０ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-methionine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 250 mg / L, and 10 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－フェニルアラニンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～５００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～４００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-phenylalanine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 500 mg / L, and 10 mg / L to 400 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－プロリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～１，２００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～１，０００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-proline in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 1,200 mg / L, and preferably 10 mg / L to 1,000 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－ヒドロキシプロリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～６０ｍｇ／Ｌが好ましく、５ｍｇ／Ｌ～４０ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-hydroxyproline in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 60 mg / L, and 5 mg / L to 40 mg. / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－セリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～３００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～２００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-serine in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 300 mg / L, 10 mg / L to 200 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－スレオニンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１０ｍｇ／Ｌ～６５０ｍｇ／Ｌが好ましく、１００ｍｇ／Ｌ～５００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-threonine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 10 mg / L to 650 mg / L, and preferably 100 mg / L to 500 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－トリプトファンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、２ｍｇ／Ｌ～１５０ｍｇ／Ｌが好ましく、２０ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-tryptophan in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 mg / L to 150 mg / L, and 20 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－チロシンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、３ｍｇ／Ｌ～２００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-tyrosine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 3 mg / L to 200 mg / L, and 10 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＬ－バリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～６５０ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～５００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-valine in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 650 mg / L, and 10 mg / L to 500 mg / L. L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるチアミンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～２５ｍｇ／Ｌが好ましく、２ｍｇ／Ｌ～１５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The thiamine content in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 25 mg / L, and preferably 2 mg / L to 15 mg / L. More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地における還元型グルタチオンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～２５ｍｇ／Ｌが好ましく、１．５ｍｇ／Ｌ～１５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of reduced glutathione in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 25 mg / L, preferably 1.5 mg / L to 15 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるアスコルビン酸－２－２ＰＯ４のマグネシウム塩の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～２５０ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of the magnesium salt of ascorbic acid-2-2PO 4 in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 250 mg / L. 10 mg / L to 100 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＡｇＮＯ３の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００００８ｍｇ／Ｌ～０．００８ｍｇ／Ｌが好ましく、０．０００００８ｍｇ／Ｌ～０．０００８ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of AgNO 3 in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 0.0000008 mg / L to 0.008 mg / L, and 0.000008 mg / L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＡｌＣｌ３・６Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００１５ｍｇ／Ｌ～０．００１５ｍｇ／Ｌが好ましく、０．０００１５ｍｇ／Ｌ～０．００１５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of AlCl 3 · 6H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000015 mg / L to 0.0015 mg / L. 0.00015 mg / L to 0.0015 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＢａ（Ｃ２Ｈ３Ｏ２）２の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００６ｍｇ／Ｌ～０．００６ｍｇ／Ｌが好ましく、０．０００６ｍｇ／Ｌ～０．００３ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of Ba (C 2 H 3 O 2 ) 2 in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.00006 mg / L to 0.006 mg / L is preferable, and 0.0006 mg / L to 0.003 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地における３ＣｄＳＯ４・８Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００５ｍｇ／Ｌ～０．０５ｍｇ／Ｌが好ましく、０．０００５ｍｇ／Ｌ～０．０４ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of 3CdSO 4 .8H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00005 mg / L to 0.05 mg / L. 0.0005 mg / L to 0.04 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＣｏＣｌ２・６Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００４ｍｇ／Ｌ～０．００４ｍｇ／Ｌが好ましく、０．０００４ｍｇ／Ｌ～０．００３ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of CoCl 2 · 6H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00004 mg / L to 0.004 mg / L. 0.0004 mg / L to 0.003 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＣｒ２（ＳＯ４）３・Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００００２５ｍｇ／Ｌ～０．００２５ｍｇ／Ｌが好ましく、０．０００００２５ｍｇ／Ｌ～０．００１ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of Cr 2 (SO 4 ) 3 .H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.000000025 mg / L to 0.00. 0025 mg / L is preferable, and 0.0000025 mg / L to 0.001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＧｅＯ２の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００００９ｍｇ／Ｌ～０．０００９ｍｇ／Ｌが好ましく、０．００００９ｍｇ／Ｌ～０．０００８ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of GeO 2 in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000009 mg / L to 0.0009 mg / L, preferably 0.00009 mg / L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＮａ２ＳｅＯ３の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００７ｍｇ／Ｌ～０．０７ｍｇ／Ｌが好ましく、０．００１ｍｇ／Ｌ～０．０１ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of Na 2 SeO 3 in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0007 mg / L to 0.07 mg / L, More preferably, the content is 0.001 mg / L to 0.01 mg / L. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＫＢｒの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００００９ｍｇ／Ｌ～０．０００９ｍｇ／Ｌが好ましく、０．０００００９ｍｇ／Ｌ～０．０００５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The KBr content in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000009 mg / L to 0.0009 mg / L, preferably 0.000009 mg / L L to 0.0005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＫＩの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００１ｍｇ／Ｌ～０．００１ｍｇ／Ｌが好ましく、０．００００５ｍｇ／Ｌ～０．０００８ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of KI in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 0.00001 mg / L to 0.001 mg / L, preferably 0.00005 mg / L. L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＭｎＣｌ２・４Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００００４ｍｇ／Ｌ～０．００４ｍｇ／Ｌが好ましく、０．００００４ｍｇ／Ｌ～０．００１ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of MnCl 2 .4H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000004 mg / L to 0.004 mg / L. 0.00004 mg / L to 0.001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＮａＦの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００６ｍｇ／Ｌ～０．００６ｍｇ／Ｌが好ましく、０．０００６ｍｇ／Ｌ～０．００５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of NaF in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00006 mg / L to 0.006 mg / L, preferably 0.0006 mg / L L to 0.005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＮａ２ＳｉＯ３・９Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００１ｍｇ／Ｌ～１ｍｇ／Ｌが好ましく、０．０１ｍｇ／Ｌ～０．５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of Na 2 SiO 3 · 9H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.001 mg / L to 1 mg / L. 0.01 mg / L to 0.5 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＮａＶＯ３の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００００５ｍｇ／Ｌ～０．００５ｍｇ／Ｌが好ましく、０．００００５ｍｇ／Ｌ～０．００４ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of NaVO 3 in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000005 mg / L to 0.005 mg / L, preferably 0.00005 mg / L to 0.004 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地における（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００８ｍｇ／Ｌ～０．０８ｍｇ／Ｌが好ましく、０．０００８ｍｇ／Ｌ～０．０５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of (NH 4 ) 6 Mo 7 O 24 · 4H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.00008 mg / L to 0.08 mg / L is preferable, and 0.0008 mg / L to 0.05 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＮｉＳＯ４・６Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００００２５ｍｇ／Ｌ～０．０００２５ｍｇ／Ｌが好ましく、０．００００２５ｍｇ／Ｌ～０．０００２５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of NiSO 4 .6H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000025 mg / L to 0.00025 mg / L. 0.000025 mg / L to 0.00025 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＲｂＣｌの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００００９ｍｇ／Ｌ～０．００９ｍｇ／Ｌが好ましく、０．０００００９ｍｇ／Ｌ～０．００５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of RbCl in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000009 mg / L to 0.009 mg / L, preferably 0.000009 mg / L L to 0.005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＳｎＣｌ２・２Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００００１ｍｇ／Ｌ～０．００１ｍｇ／Ｌが好ましく、０．００００１ｍｇ／Ｌ～０．０００１ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of SnCl 2 .2H 2 O in the first differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000001 mg / L to 0.001 mg / L. 0.00001 mg / L to 0.0001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地におけるＺｒＯＣｌ２・８Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００７ｍｇ／Ｌ～０．００７ｍｇ／Ｌが好ましく、０．０００７ｍｇ／Ｌ～０．００６ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of ZrOCl 2 · 8H 2 O in the first differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, 0.00007mg / L ~ 0.007mg / L is preferably 0.0007 mg / L to 0.006 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の分化誘導培地用培地添加剤は、市販品を用いてもよいし、化学合成したものを用いてもよい。 Commercially available products or chemically synthesized products may be used as the first culture medium additive for differentiation induction medium.
　前記第１の分化誘導培地用培地添加剤は、各成分を含む１剤として培地に添加してもよいし、成分ごとに別々の剤として培地に添加してもよいし、任意の複数の成分を含む剤として培地に添加してもよい。 The first culture medium addition agent for differentiation induction medium may be added to the medium as one agent containing each component, or may be added to the medium as a separate agent for each component, or any plurality of components You may add to a culture medium as an agent containing.
－一液式分化誘導培地－
　前記一液式分化誘導培地（以下、「一液式培地」と称することがある）は、ＩＭＤＭに、培地添加剤（以下、「一液式分化誘導培地用培地添加剤」と称することがある）を加えた培地であり、必要に応じて更にその他の成分を含む。 -One-part differentiation induction medium-
The one-component differentiation induction medium (hereinafter sometimes referred to as “one-component culture medium”) is sometimes referred to as a medium additive (hereinafter referred to as “medium additive for one-component differentiation induction medium”) to IMDM. ), And further contains other components as necessary.
－－基礎培地－－
　前記一液式分化誘導培地における基礎培地は、ＩＭＤＭである。
　前記基礎培地は、市販品を用いてもよいし、適宜調製したものを用いてもよい。 --Basic medium--
The basal medium in the one-component differentiation induction medium is IMDM.
As the basal medium, commercially available products may be used, or those prepared as appropriate may be used.
－－一液式分化誘導培地用培地添加剤－－
　前記一液式分化誘導培地用培地添加剤は、トランスフェリンと、アルブミンと、ポリビニルアルコールと、エタノラミン塩酸塩と、亜セレン酸ナトリウムと、ヒドロコルチゾンと、ＤＬ－α－トコフェロール酢酸エステルと、Ｎ－アセチル－Ｌ－システインと、１－チオグリセロールと、アスコルビン酸とを少なくとも含み、必要に応じて更にその他の成分を含む。 --- Medium additive for one-part differentiation induction medium-
The medium additive for the one-part differentiation induction medium includes transferrin, albumin, polyvinyl alcohol, etanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl- It contains at least L-cysteine, 1-thioglycerol, and ascorbic acid, and further contains other components as necessary.
　前記一液式分化誘導培地におけるトランスフェリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～１５０ｍｇ／Ｌが好ましく、２ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましく、３ｍｇ／Ｌ～５０ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The transferrin content in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 150 mg / L, and preferably 2 mg / L to 100 mg / L. More preferred is 3 mg / L to 50 mg / L. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるアルブミンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５００ｍｇ／Ｌ超８，０００ｍｇ／Ｌ以下が好ましく、１，５００ｍｇ／Ｌ超７，０００ｍｇ／Ｌ以下がより好ましく、３，０００ｍｇ／Ｌ超６，０００ｍｇ／Ｌ以下が特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。
　前記アルブミンとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト血清アルブミン、牛血清アルブミン、リコンビナントヒトアルブミン、リコンビナント牛アルブミンなどが挙げられる。 The content of albumin in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably more than 500 mg / L and 8,000 mg / L or less, preferably 1,500 mg / L More than 7,000 mg / L is more preferable, and more than 3,000 mg / L and 6,000 mg / L or less is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
There is no restriction | limiting in particular as said albumin, According to the objective, it can select suitably, For example, human serum albumin, bovine serum albumin, recombinant human albumin, recombinant bovine albumin etc. are mentioned.
　前記一液式分化誘導培地におけるポリビニルアルコールの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５００ｍｇ／Ｌ超４，０００ｍｇ／Ｌ以下が好ましく、６００ｍｇ／Ｌ超２,０００ｍｇ／Ｌがより好ましく、８００ｍｇ／Ｌ超１,５００ｍｇ／Ｌ以下が特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。
　前記ポリビニルアルコールの重量平均分子量としては、特に制限はなく、目的に応じて適宜選択することができるが、３０，０００～７０，０００が好ましい。 The content of polyvinyl alcohol in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably more than 500 mg / L and 4,000 mg / L or less, more than 600 mg / L. 2,000 mg / L is more preferable, and more than 800 mg / L and 1,500 mg / L or less is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
The weight average molecular weight of the polyvinyl alcohol is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 30,000 to 70,000.
　前記一液式分化誘導培地におけるポリビニルアルコールの含有量と、アルブミンの含有量との組合せとしては、特に制限はなく、目的に応じて適宜選択することができるが、ポリビニルアルコールの含有量が５００ｍｇ／Ｌ～１，５００ｍｇ／Ｌの場合は、アルブミンの含有量は、３，０００ｍｇ／Ｌ超が好ましく、ポリビニルアルコールの含有量が４，０００ｍｇ／Ｌの場合は、アルブミンの含有量は、５００ｍｇ／Ｌ以上が好ましい。 The combination of the polyvinyl alcohol content and the albumin content in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. In the case of L to 1,500 mg / L, the albumin content is preferably more than 3,000 mg / L, and when the polyvinyl alcohol content is 4,000 mg / L, the albumin content is 500 mg / L. The above is preferable.
　前記一液式分化誘導培地におけるエタノラミン塩酸塩の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、２ｍｇ／Ｌ～１８ｍｇ／Ｌが好ましく、５ｍｇ／Ｌ～１５ｍｇ／Ｌがより好ましく、７ｍｇ／Ｌ～１２ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of etanolamine hydrochloride in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 mg / L to 18 mg / L, and 5 mg / L to 15 mg / L L is more preferable, and 7 mg / L to 12 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地における亜セレン酸ナトリウムの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００２ｍｇ／Ｌ～０．００８ｍｇ／Ｌが好ましく、０．００３ｍｇ／Ｌ～０．００７ｍｇ／Ｌがより好ましく、０．００４ｍｇ／Ｌ～０．００６ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of sodium selenite in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.002 mg / L to 0.008 mg / L, 0.003 mg / L to 0.007 mg / L is more preferable, and 0.004 mg / L to 0.006 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるヒドロコルチゾンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０２ｍｇ／Ｌ～０．０８ｍｇ／Ｌが好ましく、０．０３ｍｇ／Ｌ～０．０７ｍｇ／Ｌがより好ましく、０．０４ｍｇ／Ｌ～０．０６ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of hydrocortisone in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.02 mg / L to 0.08 mg / L, preferably 0.03 mg / L L to 0.07 mg / L is more preferable, and 0.04 mg / L to 0.06 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＤＬ－α－トコフェロール酢酸エステルの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００１ｍｇ／Ｌ～０．００８ｍｇ／Ｌが好ましく、０．００５ｍｇ／Ｌ～０．０４ｍｇ／Ｌがより好ましく、０．０１ｍｇ／Ｌ～０．０３ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of DL-α-tocopherol acetate in the one-component differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.001 mg / L to 0.008 mg / L. Preferably, 0.005 mg / L to 0.04 mg / L is more preferable, and 0.01 mg / L to 0.03 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＮ－アセチル－Ｌ－システインの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５００ｍｇ／Ｌ～３，０００ｍｇ／Ｌが好ましく、１，０００ｍｇ／Ｌ～２，５００ｍｇ／Ｌがより好ましく、１，５００ｍｇ／Ｌ～２，０００ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of N-acetyl-L-cysteine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 500 mg / L to 3,000 mg / L, 1,000 mg / L to 2,500 mg / L is more preferable, and 1,500 mg / L to 2,000 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地における１－チオグリセロールの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、２０ｍｇ／Ｌ～８０ｍｇ／Ｌが好ましく、３０ｍｇ／Ｌ～７０ｍｇ／Ｌがより好ましく、４０ｍｇ／Ｌ～６０ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of 1-thioglycerol in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 20 mg / L to 80 mg / L, and preferably 30 mg / L to 70 mg. / L is more preferable, and 40 mg / L to 60 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるアスコルビン酸の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、２０ｍｇ／Ｌ～８０ｍｇ／Ｌが好ましく、３０ｍｇ／Ｌ～７０ｍｇ／Ｌがより好ましく、４０ｍｇ／Ｌ～６０ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of ascorbic acid in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 20 mg / L to 80 mg / L, preferably 30 mg / L to 70 mg / L. Is more preferable, and 40 mg / L to 60 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地用培地添加剤におけるその他の成分としては、特に制限はなく、目的に応じて適宜選択することができるが、グリシン、Ｌ－グルタミン、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏを含むことが好ましい。 Other components in the medium additive for the one-component differentiation induction medium are not particularly limited and may be appropriately selected depending on the intended purpose. However, glycine, L-glutamine, L-histidine, L-isoleucine, L -Methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, ascorbic acid 2-2PO 4 Magnesium salt, AgNO 3 , AlCl 3 · 6H 2 O, Ba (C 2 H 3 O 2 ) 2 , 3CdSO 4 · 8H 2 O, CoCl 2 · 6H 2 O, Cr 2 (SO 4 ) 3 · H 2 O, GeO 2, Na 2 SeO 3, KBr, KI, MnCl 2 · 4H 2 O, NaF, Na 2 SiO 3 · 9H 2 O, Na O 3, (NH 4) 6 Mo 7 O 24 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl, SnCl 2 · 2H 2 O, and may include ZrOCl 2 · 8H 2 O preferred.
　前記一液式分化誘導培地におけるグリシンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～２００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of glycine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 200 mg / L, and preferably 10 mg / L to 100 mg / L More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－グルタミンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５０ｍｇ／Ｌ～１，０００ｍｇ／Ｌが好ましく、１００ｍｇ／Ｌ～５００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-glutamine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / L to 1,000 mg / L, preferably 100 mg / L to 500 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－ヒスチジンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～３００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～２００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-histidine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 300 mg / L, 10 mg / L to 200 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－イソロイシンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５０ｍｇ／Ｌ～４，０００ｍｇ／Ｌが好ましく、１００ｍｇ／Ｌ～１，０００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-isoleucine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / L to 4,000 mg / L, preferably 100 mg / L to 1,000 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－メチオニンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～２５０ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-methionine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 250 mg / L, 10 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－フェニルアラニンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～５００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～４００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-phenylalanine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 500 mg / L, 10 mg / L to 400 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－プロリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～１，２００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～１，０００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-proline in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 1,200 mg / L, and preferably 10 mg / L to 1,000 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－ヒドロキシプロリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～６０ｍｇ／Ｌが好ましく、５ｍｇ／Ｌ～４０ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-hydroxyproline in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 60 mg / L, and 5 mg / L to 40 mg. / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－セリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～３００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～２００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-serine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 300 mg / L, 10 mg / L to 200 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－スレオニンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１０ｍｇ／Ｌ～６５０ｍｇ／Ｌが好ましく、１００ｍｇ／Ｌ～５００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-threonine in the one-component differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 mg / L to 650 mg / L, preferably 100 mg / L to 500 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－トリプトファンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、２ｍｇ／Ｌ～１５０ｍｇ／Ｌが好ましく、２０ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-tryptophan in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 mg / L to 150 mg / L, and 20 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－チロシンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、３ｍｇ／Ｌ～２００ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-tyrosine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 3 mg / L to 200 mg / L, 10 mg / L to 100 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＬ－バリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５ｍｇ／Ｌ～６５０ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～５００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-valine in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 mg / L to 650 mg / L, 10 mg / L to 500 mg / L L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるチアミンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～２５ｍｇ／Ｌが好ましく、２ｍｇ／Ｌ～１５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The thiamine content in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 25 mg / L, and preferably 2 mg / L to 15 mg / L. More preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地における還元型グルタチオンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～２５ｍｇ／Ｌが好ましく、１．５ｍｇ／Ｌ～１５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of reduced glutathione in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 25 mg / L, preferably 1.5 mg / L to 15 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるアスコルビン酸－２－２ＰＯ４のマグネシウム塩の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～２５０ｍｇ／Ｌが好ましく、１０ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of the magnesium salt of ascorbic acid-2-2PO 4 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mg / L to 250 mg / L. 10 mg / L to 100 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＡｇＮＯ３の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００００８ｍｇ／Ｌ～０．００８ｍｇ／Ｌが好ましく、０．０００００８ｍｇ／Ｌ～０．０００８ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of AgNO 3 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000008 mg / L to 0.008 mg / L, and 0.000008 mg / L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＡｌＣｌ３・６Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００１５ｍｇ／Ｌ～０．００１５ｍｇ／Ｌが好ましく、０．０００１５ｍｇ／Ｌ～０．００１５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of AlCl 3 .6H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000015 mg / L to 0.0015 mg / L. 0.00015 mg / L to 0.0015 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＢａ（Ｃ２Ｈ３Ｏ２）２の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００６ｍｇ／Ｌ～０．００６ｍｇ／Ｌが好ましく、０．０００６ｍｇ／Ｌ～０．００３ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of Ba (C 2 H 3 O 2 ) 2 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.00006 mg / L to 0.006 mg / L is preferable, and 0.0006 mg / L to 0.003 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地における３ＣｄＳＯ４・８Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００５ｍｇ／Ｌ～０．０５ｍｇ／Ｌが好ましく、０．０００５ｍｇ／Ｌ～０．０４ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of 3CdSO 4 · 8H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00005 mg / L to 0.05 mg / L. 0.0005 mg / L to 0.04 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＣｏＣｌ２・６Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００４ｍｇ／Ｌ～０．００４ｍｇ／Ｌが好ましく、０．０００４ｍｇ／Ｌ～０．００３ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of CoCl 2 · 6H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00004 mg / L to 0.004 mg / L. 0.0004 mg / L to 0.003 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＣｒ２（ＳＯ４）３・Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００００２５ｍｇ／Ｌ～０．００２５ｍｇ／Ｌが好ましく、０．０００００２５ｍｇ／Ｌ～０．００１ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of Cr 2 (SO 4 ) 3 .H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.000000025 mg / L to 0.00. 0025 mg / L is preferable, and 0.0000025 mg / L to 0.001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＧｅＯ２の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００００９ｍｇ／Ｌ～０．０００９ｍｇ／Ｌが好ましく、０．００００９ｍｇ／Ｌ～０．０００８ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of GeO 2 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000009 mg / L to 0.0009 mg / L, preferably 0.00009 mg / L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＮａ２ＳｅＯ３の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００７ｍｇ／Ｌ～０．０７ｍｇ／Ｌが好ましく、０．００１ｍｇ／Ｌ～０．０１ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of Na 2 SeO 3 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0007 mg / L to 0.07 mg / L, More preferably, the content is 0.001 mg / L to 0.01 mg / L. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＫＢｒの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００００９ｍｇ／Ｌ～０．０００９ｍｇ／Ｌが好ましく、０．０００００９ｍｇ／Ｌ～０．０００５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The KBr content in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000009 mg / L to 0.0009 mg / L, preferably 0.000009 mg / L L to 0.0005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＫＩの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００１ｍｇ／Ｌ～０．００１ｍｇ／Ｌが好ましく、０．００００５ｍｇ／Ｌ～０．０００８ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of KI in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 0.00001 mg / L to 0.001 mg / L, preferably 0.00005 mg / L. L to 0.0008 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＭｎＣｌ２・４Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００００４ｍｇ／Ｌ～０．００４ｍｇ／Ｌが好ましく、０．００００４ｍｇ／Ｌ～０．００１ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of MnCl 2 · 4H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000004 mg / L to 0.004 mg / L. 0.00004 mg / L to 0.001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＮａＦの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００６ｍｇ／Ｌ～０．００６ｍｇ／Ｌが好ましく、０．０００６ｍｇ／Ｌ～０．００５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of NaF in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.00006 mg / L to 0.006 mg / L, preferably 0.0006 mg / L L to 0.005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＮａ２ＳｉＯ３・９Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００１ｍｇ／Ｌ～１ｍｇ／Ｌが好ましく、０．０１ｍｇ／Ｌ～０．５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of Na 2 SiO 3 · 9H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.001 mg / L to 1 mg / L. 0.01 mg / L to 0.5 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＮａＶＯ３の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００００５ｍｇ／Ｌ～０．００５ｍｇ／Ｌが好ましく、０．００００５ｍｇ／Ｌ～０．００４ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of NaVO 3 in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000005 mg / L to 0.005 mg / L, preferably 0.00005 mg / L to 0.004 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地における（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００８ｍｇ／Ｌ～０．０８ｍｇ／Ｌが好ましく、０．０００８ｍｇ／Ｌ～０．０５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of (NH 4 ) 6 Mo 7 O 24 · 4H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.00008 mg / L to 0.08 mg / L is preferable, and 0.0008 mg / L to 0.05 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＮｉＳＯ４・６Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００００２５ｍｇ／Ｌ～０．０００２５ｍｇ／Ｌが好ましく、０．００００２５ｍｇ／Ｌ～０．０００２５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of NiSO 4 .6H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000025 mg / L to 0.00025 mg / L. 0.000025 mg / L to 0.00025 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＲｂＣｌの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００００９ｍｇ／Ｌ～０．００９ｍｇ／Ｌが好ましく、０．０００００９ｍｇ／Ｌ～０．００５ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of RbCl in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.0000009 mg / L to 0.009 mg / L, preferably 0.000009 mg / L L to 0.005 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＳｎＣｌ２・２Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０００００１ｍｇ／Ｌ～０．００１ｍｇ／Ｌが好ましく、０．００００１ｍｇ／Ｌ～０．０００１ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of SnCl 2 · 2H 2 O in the one-part differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.000001 mg / L to 0.001 mg / L. 0.00001 mg / L to 0.0001 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地におけるＺｒＯＣｌ２・８Ｈ２Ｏの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００００７ｍｇ／Ｌ～０．００７ｍｇ／Ｌが好ましく、０．０００７ｍｇ／Ｌ～０．００６ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of ZrOCl 2 · 8H 2 O in the one-liquid type differentiation-inducing medium is not particularly limited and may be appropriately selected depending on the intended purpose, 0.00007mg / L ~ 0.007mg / L is preferably 0.0007 mg / L to 0.006 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記一液式分化誘導培地用培地添加剤は、市販品を用いてもよいし、化学合成したものを用いてもよい。 Commercially available products or chemically synthesized products may be used as the medium additive for the one-component differentiation induction medium.
　前記一液式分化誘導培地用培地添加剤は、各成分を含む１剤として培地に添加してもよいし、成分ごとに別々の剤として培地に添加してもよいし、任意の複数の成分を含む剤として培地に添加してもよい。 The medium additive for the one-component differentiation induction medium may be added to the medium as one agent containing each component, or may be added to the medium as a separate agent for each component, or any plurality of components You may add to a culture medium as an agent containing.
－分化誘導調節剤－
　前記ＲＯＣＫ阻害剤は、分化誘導調節剤の１つである。
　前記胚様体形成工程用培地に添加する分化誘導調節剤（以下、「胚様体形成工程用分化誘導調節剤」と称することがある）としては、ＲＯＣＫ阻害剤を含む限り、特に制限はなく、目的に応じて適宜選択することができる。
　前記ＲＯＣＫ阻害剤としては、例えば、Ｙ２７６３２、Ｆａｓｕｄｉｌ　Ｈｙｄｒｏｃｈｌｏｒｉｄｅなどが挙げられる。これらの中でも、心筋細胞への分化誘導効率に優れる点で、Ｙ２７６３２が好ましい。
　前記ＲＯＣＫ阻害剤は、１種単独で使用してもよいし、２種以上を併用してもよい。
　前記ＲＯＣＫ阻害剤は、ヒトｉＰＳ細胞の細胞死を防ぐ作用がある物質として知られている。 -Differentiation induction regulator-
The ROCK inhibitor is one of differentiation induction regulators.
The differentiation induction regulator added to the embryoid body formation process medium (hereinafter sometimes referred to as “differentiation induction regulator for embryoid body formation process”) is not particularly limited as long as it contains a ROCK inhibitor. Can be appropriately selected according to the purpose.
Examples of the ROCK inhibitor include Y27632, Fastil Hydrochloride and the like. Among these, Y27632 is preferable because it is excellent in efficiency of inducing differentiation into cardiomyocytes.
The ROCK inhibitor may be used alone or in combination of two or more.
The ROCK inhibitor is known as a substance having an action of preventing cell death of human iPS cells.
　前記胚様体形成工程用培地における分化誘導調節剤の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。例えば、前記胚様体形成工程用培地における分化誘導調節剤としてＹ２７６３２を用いる場合には、２μＭ～１０μＭが好ましく、３μＭ～８μＭがより好ましく、４μＭ～６μＭが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of the differentiation induction regulator in the embryoid body formation step medium is not particularly limited and may be appropriately selected depending on the intended purpose. For example, when Y27632 is used as a differentiation induction regulator in the medium for embryoid body formation step, it is preferably 2 μM to 10 μM, more preferably 3 μM to 8 μM, and particularly preferably 4 μM to 6 μM. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記胚様体形成工程用分化誘導調節剤は、ＲＯＣＫ阻害剤以外の分化誘導調節剤を含んでもよいし、含まなくてもよいが、心筋細胞を安価、かつ簡便に得ることができる点で、ＲＯＣＫ阻害剤以外の分化誘導調節剤を含まないことが好ましい。 The differentiation-inducing regulator for the embryoid body formation step may or may not contain a differentiation-inducing regulator other than the ROCK inhibitor, but it is possible to obtain cardiomyocytes inexpensively and easily, It is preferable not to include a differentiation induction regulator other than the ROCK inhibitor.
　前記胚様体形成工程用分化誘導調節剤におけるＲＯＣＫ阻害剤以外の分化誘導調節剤としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、ｂＦＧＦ、ＣＨＩＲ９９０２１（ＣＡＳ番号：２５２９１７－０６－９）などが挙げられる。
　前記胚様体形成工程用培地にｂＦＧＦを添加する場合におけるｂＦＧＦの含有量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、１０ｎｇ／ｍＬ～１００ｎｇ／ｍＬなどが挙げられる。
　前記胚様体形成工程用培地にＣＨＩＲ９９０２１を添加する場合におけるＣＨＩＲ９９０２１の含有量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、５ｎＭ～５μＭなどが挙げられる。 The differentiation induction regulator other than the ROCK inhibitor in the differentiation induction regulator for the embryoid body formation step is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected according to the purpose. , BFGF, CHIR99021 (CAS number: 252917-06-9) and the like.
The content of bFGF when bFGF is added to the embryoid body formation step medium is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 10 ng / mL to 100 ng / mL. It is done.
The content of CHIR99021 in the case of adding CHIR99021 to the embryoid body formation step medium is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 5 nM to 5 μM.
　前記胚様体形成工程用分化誘導調節剤は、市販品を用いてもよいし、化学合成したものを用いてもよい。 As the differentiation-inducing regulator for the embryoid body formation step, a commercially available product or a chemically synthesized product may be used.
　前記胚様体形成工程用分化誘導調節剤は、各成分を含む１剤として培地に添加してもよいし、成分ごとに別々の剤として培地に添加してもよいし、任意の複数の成分を含む剤として培地に添加してもよい。 The differentiation-inducing regulator for embryoid body formation step may be added to the medium as one agent containing each component, may be added to the medium as a separate agent for each component, or any plurality of components You may add to a culture medium as an agent containing.
　前記胚様体形成工程用培地の好ましい態様としては、下記胚様体形成工程用培地（１）、（２）が挙げられる。これらの中でも、胚様体形成工程用培地（１）がより好ましい。 Preferred embodiments of the embryoid body forming step medium include the following embryoid body forming step mediums (1) and (2). Among these, the culture medium for embryoid body formation process (1) is more preferable.
－胚様体形成工程用培地（１）－
　基礎培地としてＤＭＥＭ／Ｆ１２を用い、下記表１－１及び１－２に記載の組成となるように、第１の分化誘導培地用培地添加剤を添加した第１の分化誘導培地に、Ｙ２７６３２を５μＭとなるように添加した培地。 -Medium for embryoid body formation process (1)-
Using DMEM / F12 as the basal medium, Y27632 was added to the first differentiation induction medium supplemented with the first medium addition medium for differentiation induction medium so as to have the composition described in Tables 1-1 and 1-2 below. Medium added to 5 μM.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
－胚様体形成工程用培地（２）－
　下記表２－１及び２－２に記載の組成となるように、一液式分化誘導培地用培地添加剤を添加した一液式分化誘導培地に、Ｙ２７６３２を５μＭとなるように添加した培地。
Figure JPOXMLDOC01-appb-T000003
 -Medium for embryoid body formation process (2)-
A medium obtained by adding Y27632 to 5 μM to a one-part differentiation induction medium supplemented with a medium additive for a one-part differentiation induction medium so as to have the composition described in Tables 2-1 and 2-2 below.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
＜＜浮遊培養＞＞
　前記浮遊培養の方法としては、特に制限はなく、公知の方法を適宜選択することができ、例えば、接着加工を行っていない培養皿や低接着加工を行った培養皿を用い、前記多能性幹細胞を培養する方法が挙げられる。
　前記接着加工を行っていない培養皿や低接着加工を行った培養皿は、市販品を用いることができる。 << Floating culture >>
The suspension culture method is not particularly limited, and a known method can be appropriately selected. For example, the pluripotency can be selected using a culture dish that has not been subjected to adhesion processing or a culture dish that has undergone low adhesion processing. A method of culturing stem cells can be mentioned.
Commercially available products can be used for the culture dish that has not been subjected to the adhesion process or the culture dish that has undergone a low adhesion process.
　前記浮遊培養の培養条件としては、特に制限はなく、公知の培養条件を適宜選択することができる。
　前記浮遊培養は、低酸素条件で行ってもよいし、大気条件で行ってもよいが、分化効率に優れる点で、低酸素条件が好ましい。
　低酸素条件とは、常酸素濃度（２１％）を下回る酸素濃度条件をいう。
　前記低酸素条件における酸素濃度としては、２１％未満であれば、特に制限はなく、目的に応じて適宜選択することができるが、０．１％以上２１％未満が好ましく、１％以上１０％以下がより好ましく、４％以上６％以下が特に好ましい。 There is no restriction | limiting in particular as a culture condition of the said suspension culture, A well-known culture condition can be selected suitably.
The suspension culture may be performed under low oxygen conditions or may be performed under atmospheric conditions, but low oxygen conditions are preferable in terms of excellent differentiation efficiency.
Low oxygen conditions refer to oxygen concentration conditions below the normal oxygen concentration (21%).
The oxygen concentration in the low oxygen condition is not particularly limited as long as it is less than 21%, and can be appropriately selected according to the purpose, but it is preferably 0.1% or more and less than 21%, preferably 1% or more and 10%. The following is more preferable, and 4% or more and 6% or less are particularly preferable.
　前記胚様体形成工程の期間としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、２４時間～４８時間などが挙げられる。 The period of the embryoid body formation step is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 24 to 48 hours.
＜中胚葉誘導工程＞
　前記中胚葉誘導工程は、前記胚様体形成工程後の胚様体を浮遊培養し、中胚葉を誘導する工程である。 <Mesodermal induction process>
The mesoderm induction step is a step of inducing the mesoderm by suspension culture of the embryoid body after the embryoid body formation step.
＜＜培地＞＞
　前記中胚葉誘導工程における培地（以下、「中胚葉誘導工程用培地」、「中胚葉誘導工程用培養液」と称することがある）は、第２の分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地のいずれかであり、必要に応じて更にその他の成分を含む。 << Medium >>
The medium in the mesoderm induction process (hereinafter sometimes referred to as “medium mesoderm induction process medium” or “mesodermal induction process culture medium”) contains a Wnt signal activator in the second differentiation induction medium. Either of the added medium and the one-part differentiation induction medium, a medium added with a Wnt signal activator, and further containing other components as necessary.
－第２の分化誘導培地－
　前記第２の分化誘導培地（以下、「第二培地」と称することがある）は、基礎培地に、培地添加剤（以下、「第２の分化誘導培地用培地添加剤」と称することがある）を加えた培地であり、必要に応じて更にその他の成分を含む。 -Second differentiation induction medium-
The second differentiation induction medium (hereinafter sometimes referred to as “second medium”) may be referred to as a medium additive (hereinafter referred to as “second medium for differentiation induction medium”) as a basal medium. ), And further contains other components as necessary.
－－基礎培地－－
　前記第２の分化誘導培地における基礎培地は、ＩＭＤＭである。
　前記基礎培地は、市販品を用いてもよいし、適宜調製したものを用いてもよい。 --Basic medium--
The basal medium in the second differentiation induction medium is IMDM.
As the basal medium, commercially available products may be used, or those prepared as appropriate may be used.
－－第２の分化誘導培地用培地添加剤－－
　前記第２の分化誘導培地用培地添加剤は、アルブミンと、ポリビニルアルコールと、エタノラミン塩酸塩と、亜セレン酸ナトリウムと、ヒドロコルチゾンと、ＤＬ－α－トコフェロール酢酸エステルと、Ｎ－アセチル－Ｌ－システインと、１－チオグリセロールと、アスコルビン酸とを少なくとも含み、必要に応じて更にその他の成分を含む。 --- Medium additive for second differentiation induction medium-
The second medium additive for differentiation induction medium is albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine. And at least 1-thioglycerol and ascorbic acid, and further contains other components as necessary.
　前記第２の分化誘導培地におけるアルブミンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５００ｍｇ／Ｌ超８，０００ｍｇ／Ｌ以下が好ましく、１，５００ｍｇ／Ｌ超７，０００ｍｇ／Ｌ以下がより好ましく、３，０００ｍｇ／Ｌ超６，０００ｍｇ／Ｌ以下が特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。
　前記アルブミンとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト血清アルブミン、牛血清アルブミン、リコンビナントヒトアルブミン、リコンビナント牛アルブミンなどが挙げられる。 The content of albumin in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably more than 500 mg / L and 8,000 mg / L or less, preferably 1,500 mg / L. More than 7,000 mg / L is more preferable, and more than 3,000 mg / L and 6,000 mg / L or less is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
There is no restriction | limiting in particular as said albumin, According to the objective, it can select suitably, For example, human serum albumin, bovine serum albumin, recombinant human albumin, recombinant bovine albumin etc. are mentioned.
　前記第２の分化誘導培地におけるポリビニルアルコールの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５００ｍｇ／Ｌ超４，０００ｍｇ／Ｌ以下が好ましく、６００ｍｇ／Ｌ超２,０００ｍｇ／Ｌがより好ましく、８００ｍｇ／Ｌ超１,５００ｍｇ／Ｌ以下が特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。
　前記ポリビニルアルコールの重量平均分子量としては、特に制限はなく、目的に応じて適宜選択することができるが、３０，０００～７０，０００が好ましい。 The content of polyvinyl alcohol in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably more than 500 mg / L and less than 4,000 mg / L, more than 600 mg / L. 2,000 mg / L is more preferable, and more than 800 mg / L and 1,500 mg / L or less is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
The weight average molecular weight of the polyvinyl alcohol is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 30,000 to 70,000.
　前記第２の分化誘導培地におけるポリビニルアルコールの含有量と、アルブミンの含有量との組合せとしては、特に制限はなく、目的に応じて適宜選択することができるが、ポリビニルアルコールの含有量が５００ｍｇ／Ｌ～１，５００ｍｇ／Ｌの場合は、アルブミンの含有量は、３，０００ｍｇ／Ｌ超が好ましく、ポリビニルアルコールの含有量が４，０００ｍｇ／Ｌの場合は、アルブミンの含有量は、５００ｍｇ／Ｌ以上が好ましい。 The combination of the polyvinyl alcohol content and the albumin content in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. In the case of L to 1,500 mg / L, the albumin content is preferably more than 3,000 mg / L, and when the polyvinyl alcohol content is 4,000 mg / L, the albumin content is 500 mg / L. The above is preferable.
　前記第２の分化誘導培地におけるエタノラミン塩酸塩の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、２ｍｇ／Ｌ～１８ｍｇ／Ｌが好ましく、５ｍｇ／Ｌ～１５ｍｇ／Ｌがより好ましく、７ｍｇ／Ｌ～１２ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of etanolamine hydrochloride in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 mg / L to 18 mg / L, and 5 mg / L to 15 mg / L L is more preferable, and 7 mg / L to 12 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第２の分化誘導培地における亜セレン酸ナトリウムの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００２ｍｇ／Ｌ～０．００８ｍｇ／Ｌが好ましく、０．００３ｍｇ／Ｌ～０．００７ｍｇ／Ｌがより好ましく、０．００４ｍｇ／Ｌ～０．００６ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of sodium selenite in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.002 mg / L to 0.008 mg / L, 0.003 mg / L to 0.007 mg / L is more preferable, and 0.004 mg / L to 0.006 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第２の分化誘導培地におけるヒドロコルチゾンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．０２ｍｇ／Ｌ～０．０８ｍｇ／Ｌが好ましく、０．０３ｍｇ／Ｌ～０．０７ｍｇ／Ｌがより好ましく、０．０４ｍｇ／Ｌ～０．０６ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of hydrocortisone in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.02 mg / L to 0.08 mg / L, preferably 0.03 mg / L L to 0.07 mg / L is more preferable, and 0.04 mg / L to 0.06 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第２の分化誘導培地におけるＤＬ－α－トコフェロール酢酸エステルの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．００１ｍｇ／Ｌ～０．００８ｍｇ／Ｌが好ましく、０．００５ｍｇ／Ｌ～０．０４ｍｇ／Ｌがより好ましく、０．０１ｍｇ／Ｌ～０．０３ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of DL-α-tocopherol acetate in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.001 mg / L to 0.008 mg / L. Preferably, 0.005 mg / L to 0.04 mg / L is more preferable, and 0.01 mg / L to 0.03 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第２の分化誘導培地におけるＮ－アセチル－Ｌ－システインの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５００ｍｇ／Ｌ～３，０００ｍｇ／Ｌが好ましく、１，０００ｍｇ／Ｌ～２，５００ｍｇ／Ｌがより好ましく、１，５００ｍｇ／Ｌ～２，０００ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of N-acetyl-L-cysteine in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 500 mg / L to 3,000 mg / L, 1,000 mg / L to 2,500 mg / L is more preferable, and 1,500 mg / L to 2,000 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第２の分化誘導培地における１－チオグリセロールの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、２０ｍｇ／Ｌ～８０ｍｇ／Ｌが好ましく、３０ｍｇ／Ｌ～７０ｍｇ／Ｌがより好ましく、４０ｍｇ／Ｌ～６０ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of 1-thioglycerol in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 20 mg / L to 80 mg / L, and preferably 30 mg / L to 70 mg. / L is more preferable, and 40 mg / L to 60 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第２の分化誘導培地におけるアスコルビン酸の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、２０ｍｇ／Ｌ～８０ｍｇ／Ｌが好ましく、３０ｍｇ／Ｌ～７０ｍｇ／Ｌがより好ましく、４０ｍｇ／Ｌ～６０ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of ascorbic acid in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 20 mg / L to 80 mg / L, and preferably 30 mg / L to 70 mg / L. Is more preferable, and 40 mg / L to 60 mg / L is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第２の分化誘導培地用培地添加剤におけるその他の成分としては、特に制限はなく、目的に応じて適宜選択することができるが、Ｌ－グルタミン、及びトランスフェリンを含むことが好ましい。 Other components in the second culture medium supplement for differentiation induction medium are not particularly limited and may be appropriately selected depending on the intended purpose, but preferably include L-glutamine and transferrin.
　前記第２の分化誘導培地におけるＬ－グルタミンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、５０ｍｇ／Ｌ～１，０００ｍｇ／Ｌが好ましく、１００ｍｇ／Ｌ～５００ｍｇ／Ｌがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of L-glutamine in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / L to 1,000 mg / L, preferably 100 mg / L to 500 mg / L is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第２の分化誘導培地におけるトランスフェリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｍｇ／Ｌ～１５０ｍｇ／Ｌが好ましく、２ｍｇ／Ｌ～１００ｍｇ／Ｌがより好ましく、３ｍｇ／Ｌ～５０ｍｇ／Ｌが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The transferrin content in the second differentiation induction medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 mg / L to 150 mg / L, and preferably 2 mg / L to 100 mg / L. More preferred is 3 mg / L to 50 mg / L. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第２の分化誘導培地用培地添加剤は、前記その他の成分としてインスリンを含んでもよいが、含まないことが好ましい。
　前記第２の分化誘導培地にインスリンを添加する場合におけるインスリンの含有量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、５ｎｇ／ｍＬ～１５ｎｇ／ｍＬなどが挙げられる。 The second culture medium additive for differentiation induction medium may contain insulin as the other component, but preferably does not contain insulin.
The content of insulin when adding insulin to the second differentiation-inducing medium is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include 5 ng / mL to 15 ng / mL. .
　前記第２の分化誘導培地用培地添加剤は、市販品を用いてもよいし、化学合成したものを用いてもよい。 The second medium for differentiation induction medium may be a commercially available product or a chemically synthesized product.
　前記第２の分化誘導培地用培地添加剤は、各成分を含む１剤として培地に添加してもよいし、成分ごとに別々の剤として培地に添加してもよいし、任意の複数の成分を含む剤として培地に添加してもよい。 The second culture medium addition agent for differentiation induction medium may be added to the medium as one agent containing each component, may be added to the medium as a separate agent for each component, or any plurality of components You may add to a culture medium as an agent containing.
－一液式分化誘導培地－
　前記一液式分化誘導培地は、上記胚様体形成工程の一液式分化誘導培地の項目に記載したものと同様である。 -One-part differentiation induction medium-
The one-part differentiation induction medium is the same as that described in the item of the one-part differentiation induction medium in the embryoid body formation step.
－分化誘導調節剤－
　前記中胚葉誘導工程用培地に添加する分化誘導調節剤（以下、「中胚葉誘導工程用分化誘導調節剤」と称することがある）としては、Ｗｎｔシグナル活性化物質を含む限り、特に制限はなく、目的に応じて適宜選択することができる。
　前記Ｗｎｔシグナル活性化物質としては、例えば、ＣＨＩＲ９９０２１、ＢＩＯ、Ｗｎｔ　アゴニスト（ＣＡＳ　８５３２２０－５２－７）、Ｗｎｔ　アゴニストＩＩ（ＳＫＬ２００１）などが挙げられる。これらの中でも、細胞毒性が弱く、分化誘導効率に優れる点で、ＣＨＩＲ９９０２１が好ましい。
　前記Ｗｎｔシグナル活性化物質は、１種単独で使用してもよいし、２種以上を併用してもよい。 -Differentiation induction regulator-
The differentiation induction regulator added to the medium for mesoderm induction process (hereinafter sometimes referred to as “differentiation induction regulator for mesoderm induction process”) is not particularly limited as long as it contains a Wnt signal activator. Can be appropriately selected according to the purpose.
Examples of the Wnt signal activator include CHIR99021, BIO, Wnt agonist (CAS 853220-52-7), Wnt agonist II (SKL2001) and the like. Among these, CHIR99021 is preferable because it has low cytotoxicity and excellent differentiation induction efficiency.
The Wnt signal activator may be used alone or in combination of two or more.
　前記中胚葉誘導工程用培地における分化誘導調節剤の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。例えば、前記中胚葉誘導工程用培地における分化誘導調節剤としてＣＨＩＲ９９０２１を用いる場合には、２μＭ～６μＭが好ましく、３μＭ～５μＭがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of the differentiation induction regulator in the medium for mesoderm induction process is not particularly limited and can be appropriately selected depending on the purpose. For example, when CHIR99021 is used as the differentiation induction regulator in the medium for the mesoderm induction process, it is preferably 2 μM to 6 μM, and more preferably 3 μM to 5 μM. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記中胚葉誘導工程用分化誘導調節剤は、Ｗｎｔシグナル活性化物質以外の分化誘導調節剤を含んでもよいし、含まなくてもよいが、心筋細胞を安価、かつ簡便に得ることができる点で、ＣＨＩＲ９９０２１以外の分化誘導調節剤を含まないことが好ましい。特に、Ｗｎｔシグナル活性化物質の中では、ＢＩＯ、Ｗｎｔ　アゴニスト、Ｗｎｔ　アゴニストＩＩを含まないことが好ましい。 The differentiation induction regulator for the mesoderm induction process may or may not contain a differentiation induction regulator other than the Wnt signal activator, but cardiomyocytes can be obtained inexpensively and easily. It is preferable that no differentiation induction regulator other than CHIR99021 is contained. In particular, the Wnt signal activator preferably does not contain BIO, Wnt agonist, or Wnt agonist II.
　前記中胚葉誘導工程用分化誘導調節剤におけるＷｎｔシグナル活性化物質以外の分化誘導調節剤としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、ｂＦＧＦなどが挙げられる。
　前記中胚葉誘導工程用培地にｂＦＧＦを添加する場合におけるｂＦＧＦの含有量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、１０ｎｇ／ｍＬ～１００ｎｇ／ｍＬなどが挙げられる。 The differentiation induction regulator other than the Wnt signal activator in the differentiation induction regulator for the mesoderm induction step is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected according to the purpose. For example, bFGF etc. are mentioned.
The content of bFGF in the case where bFGF is added to the medium for mesoderm induction process is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 10 ng / mL to 100 ng / mL. .
　前記中胚葉誘導工程用分化誘導調節剤は、市販品を用いてもよいし、化学合成したものを用いてもよい。 As the differentiation induction regulator for the mesoderm induction process, a commercially available product or a chemically synthesized product may be used.
　前記中胚葉誘導工程用分化誘導調節剤は、各成分を含む１剤として培地に添加してもよいし、成分ごとに別々の剤として培地に添加してもよいし、任意の複数の成分を含む剤として培地に添加してもよい。 The differentiation induction regulator for mesoderm induction process may be added to the medium as one agent containing each component, or may be added to the medium as a separate agent for each component, or any plurality of components may be added. You may add to a culture medium as an agent to contain.
　前記中胚葉誘導工程用培地の好ましい態様としては、下記中胚葉誘導工程用培地（１）、（２）が挙げられる。これらの中でも、中胚葉誘導工程用培地（１）がより好ましい。 Preferred embodiments of the medium for mesoderm induction process include the following mediums for mesoderm induction process (1) and (2). Among these, the medium (1) for mesoderm induction process is more preferable.
－中胚葉誘導工程用培地（１）－
　下記表３に記載の組成となるように、第２の分化誘導培地用培地添加剤を添加した第２の分化誘導培地に、ＣＨＩＲ９９０２１を４μＭとなるように添加した培地。 -Medium for mesoderm induction process (1)-
A medium in which CHIR99021 is added to 4 μM in the second differentiation induction medium to which the second medium for differentiation induction medium additive is added so as to have the composition shown in Table 3 below.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
－中胚葉誘導工程用培地（２）－
　上記表２－１及び２－２に記載の組成となるように、一液式分化誘導培地用培地添加剤を添加した一液式分化誘導培地に、ＣＨＩＲ９９０２１を４μＭとなるように添加した培地。 -Medium for mesoderm induction process (2)-
A medium in which CHIR99021 is added to 4 μM in a one-part differentiation induction medium supplemented with a medium additive for a one-part differentiation induction medium so as to have the composition described in Tables 2-1 and 2-2.
＜＜浮遊培養＞＞
　前記浮遊培養の方法、培養条件としては、特に制限はなく、公知の方法を適宜選択することができ、例えば、上記胚様体形成工程の浮遊培養の項目に記載したものと同様とすることができる。 << Floating culture >>
The suspension culture method and culture conditions are not particularly limited, and a known method can be appropriately selected. For example, the suspension culture method and the culture conditions may be the same as those described in the item of suspension culture in the embryoid body formation step. it can.
　前記中胚葉誘導工程の期間としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、胚様体形成工程開始１日目～３日目、胚様体形成工程開始１日目～４日目、胚様体形成工程開始２日目～４日目などとすることができる。 The duration of the mesoderm induction process is not particularly limited and may be appropriately selected depending on the purpose. For example, the embryoid body formation process starts on the first day to the third day, and the embryoid body formation process starts on the first day. The fourth day, the second day to the fourth day, and the like.
＜心筋細胞誘導工程＞
　前記心筋細胞誘導工程は、前記中胚葉誘導後の胚様体を浮遊培養し、心筋細胞を誘導する工程である。
　前記心筋細胞誘導工程は、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理とを少なくとも含み、必要に応じて更にその他の処理を含む。 <Cardiomyocyte induction process>
The cardiomyocyte inducing step is a step of inducing cardiomyocytes by suspension culture of the embryoid body after the mesoderm induction.
The cardiomyocyte induction step includes at least a first cardiomyocyte induction process and a second cardiomyocyte induction process, and further includes other processes as necessary.
＜＜第１の心筋細胞誘導処理＞＞
　前記第１の心筋細胞誘導処理は、前記中胚葉誘導後の胚様体を第１の心筋細胞誘導処理用培地（以下、「第１の心筋細胞誘導処理用培養液」と称することがある）で培養する処理である。 << First cardiomyocyte induction process >>
In the first cardiomyocyte induction treatment, the embryoid body after the mesoderm induction is referred to as a first cardiomyocyte induction treatment medium (hereinafter sometimes referred to as “first cardiomyocyte induction treatment medium”). It is the process which culture | cultivates by.
－培地－
　前記第１の心筋細胞誘導処理における培地（以下、「第１の心筋細胞誘導処理用培地」、「第１の心筋細胞誘導処理用培養液」と称することがある）は、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地のいずれかであり、必要に応じて更にその他の成分を含む。 -Culture medium-
The medium in the first cardiomyocyte induction treatment (hereinafter sometimes referred to as “first cardiomyocyte induction treatment medium” or “first cardiomyocyte induction treatment culture medium”) is used for the second differentiation induction. Medium supplemented with Wnt signal suppressor, TGF-β signal inhibitor, and estrogen-like substance in medium, and one-part differentiation induction medium, Wnt signal suppressor, TGF-β signal inhibitor, and estrogen-like action It is any medium added with substances, and further contains other components as necessary.
－－第２の分化誘導培地－－
　前記第２の分化誘導培地は、上記中胚葉誘導工程の第２の分化誘導培地の項目に記載したものと同様である。 --Second differentiation induction medium--
The second differentiation induction medium is the same as that described in the item of the second differentiation induction medium in the mesoderm induction step.
－－一液式分化誘導培地－－
　前記一液式分化誘導培地は、上記胚様体形成工程の一液式分化誘導培地の項目に記載したものと同様である。 --One-part differentiation induction medium--
The one-part differentiation induction medium is the same as that described in the item of the one-part differentiation induction medium in the embryoid body formation step.
－－分化誘導調節剤－－
　前記第１の心筋細胞誘導処理用培地に添加する分化誘導調節剤（以下、「第１の心筋細胞誘導処理用分化誘導調節剤」と称することがある）は、Ｗｎｔシグナル抑制物質と、ＴＧＦ－βシグナル阻害剤と、エストロゲン様作用物質とを少なくとも含み、必要に応じて更にその他の成分を含む。 --- Differentiation induction regulator ---
The differentiation-inducing regulator added to the first cardiomyocyte-inducing treatment medium (hereinafter sometimes referred to as “first cardiomyocyte-inducing treatment differentiation-inducing regulator”) is a Wnt signal inhibitor, TGF- It contains at least a β signal inhibitor and an estrogen-like substance, and further contains other components as necessary.
　前記第１の心筋細胞誘導処理用培地における分化誘導調節剤の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。 The content of the differentiation-inducing regulator in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose.
　前記Ｗｎｔシグナル抑制物質としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＩＷＰ２、ＩＷＲ１、ＸＡＶ９３９、ＫＹ０２１１１などが挙げられる。これらの中でも、ＩＷＰ２が、低濃度から優れた分化誘導効率を発揮する点で、好ましい。
　前記Ｗｎｔシグナル抑制物質は、１種単独で使用してもよいし、２種以上を併用してもよい。 There is no restriction | limiting in particular as said Wnt signal suppression substance, According to the objective, it can select suitably, For example, IWP2, IWR1, XAV939, KY02111 etc. are mentioned. Among these, IWP2 is preferable in that it exhibits excellent differentiation induction efficiency from a low concentration.
The Wnt signal inhibitor may be used alone or in combination of two or more.
　前記第１の心筋細胞誘導処理用培地におけるＷｎｔシグナル抑制物質の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１μＭ～１０μＭが好ましく、２μＭ～９μＭがより好ましく、４μＭ～７μＭが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of the Wnt signal inhibitor in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 μM to 10 μM, more preferably 2 μM to 9 μM. 4 μM to 7 μM is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記ＴＧＦ－βシグナル阻害剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＳＢ４３１５４２、ＳＢ５０５１２４、Ａ－８３－０１などが挙げられる。これらの中でも、ＳＢ４３１５４２が、分化誘導効率に優れる点で、好ましい。
　前記ＴＧＦ－βシグナル阻害剤は、１種単独で使用してもよいし、２種以上を併用してもよい。 The TGF-β signal inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include SB431542, SB505124, A-83-01 and the like. Among these, SB431542 is preferable because it is excellent in differentiation induction efficiency.
The TGF-β signal inhibitor may be used alone or in combination of two or more.
　前記第１の心筋細胞誘導処理用培地におけるＴＧＦ－βシグナル阻害剤の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１μＭ～１０μＭが好ましく、２μＭ～９μＭがより好ましく、３μＭ～８μＭが特に好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of the TGF-β signal inhibitor in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 μM to 10 μM, and 2 μM to 9 μM. More preferably, 3 μM to 8 μM is particularly preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記エストロゲン様作用物質とは、エストロゲン作用を示すホルモン、低分子化合物をいう。
　前記エストロゲン様作用物質としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、エストラジオール、エストロン、エストリオール、ゲニステインなどが挙げられる。これらの中でも、エストラジオールが、分化誘導効率に優れる点で、好ましい。
　前記エストロゲン様作用物質は、１種単独で使用してもよいし、２種以上を併用してもよい。 The estrogen-like substance refers to hormones and low molecular compounds that exhibit estrogen action.
The estrogen-like substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include estradiol, estrone, estriol, and genistein. Among these, estradiol is preferable because it is excellent in differentiation induction efficiency.
The said estrogen-like active substance may be used individually by 1 type, and may use 2 or more types together.
　前記第１の心筋細胞誘導処理用培地におけるエストロゲン様作用物質の含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、１ｎＭ～１０，０００ｎＭが好ましく、１０ｎＭ～１，０００ｎＭがより好ましい。前記好ましい範囲内であると、より心筋細胞への分化誘導効率に優れる点で、有利である。 The content of the estrogen-like substance in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 nM to 10,000 nM, 10 nM to 1, 000 nM is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
　前記第１の心筋細胞誘導処理用分化誘導調節剤は、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質以外の分化誘導調節剤を含んでもよいし、含まなくてもよい。
　前記エストロゲン様作用物質の代わりに、ＶＥＧＦを１０ｎｇ／ｍＬの濃度で用いることも可能であるが、心筋細胞を安価、かつ簡便に得ることができる点で、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質以外の分化誘導調節剤を含まないことが好ましい。 The first differentiation-inducing regulator for cardiomyocyte induction treatment may or may not contain a differentiation-inducing regulator other than a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like agent.
VEGF can be used at a concentration of 10 ng / mL in place of the estrogen-like substance, but a Wnt signal inhibitor, TGF-β signal inhibition can be obtained in that cardiomyocytes can be obtained inexpensively and easily. It is preferable that a differentiation induction regulator other than an agent and an estrogen-like agent is not included.
　前記第１の心筋細胞誘導処理用分化誘導調節剤におけるＷｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質以外の分化誘導調節剤としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、ｂＦＧＦなどが挙げられる。
　前記第１の心筋細胞誘導処理用培地にｂＦＧＦを添加する場合におけるｂＦＧＦの含有量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、１０ｎｇ／ｍＬ～１００ｎｇ／ｍＬなどが挙げられる。 The differentiation-inducing regulators other than the Wnt signal suppressor, TGF-β signal inhibitor, and estrogen-like agent in the differentiation-inducing regulator for the first cardiomyocyte induction treatment are not particularly limited as long as the effects of the present invention are not impaired. There is no restriction | limiting, According to the objective, it can select suitably, For example, bFGF etc. are mentioned.
The content of bFGF in the case of adding bFGF to the first cardiomyocyte induction treatment medium is not particularly limited and can be appropriately selected according to the purpose. For example, 10 ng / mL to 100 ng / mL, etc. Is mentioned.
　前記第１の心筋細胞誘導処理用分化誘導調節剤は、市販品を用いてもよいし、化学合成したものを用いてもよい。 As the first differentiation induction regulator for cardiomyocyte induction treatment, a commercially available product or a chemically synthesized product may be used.
　前記第１の心筋細胞誘導処理用分化誘導調節剤は、各成分を含む１剤として培地に添加してもよいし、成分ごとに別々の剤として培地に添加してもよいし、任意の複数の成分を含む剤として培地に添加してもよい。 The first differentiation-inducing regulator for cardiomyocyte induction treatment may be added to the medium as one agent containing each component, or may be added to the medium as a separate agent for each component. You may add to a culture medium as an agent containing these components.
　前記第１の心筋細胞誘導処理用培地の好ましい態様としては、下記第１の心筋細胞誘導処理用培地（１）、（２）が挙げられる。これらの中でも、第１の心筋細胞誘導処理用培地（１）がより好ましい。 Preferred embodiments of the first cardiomyocyte induction treatment medium include the following first cardiomyocyte induction treatment media (1) and (2). Among these, the first cardiomyocyte induction treatment medium (1) is more preferable.
－第１の心筋細胞誘導処理用培地（１）－
　上記表３に記載の組成となるように、第２の分化誘導培地用培地添加剤を添加した第２の分化誘導培地に、ＩＷＰ２を５μＭ、ＳＢ４３１５４２を５μＭ、及びエストラジオールを１００ｎＭとなるように添加した培地。 -First cardiomyocyte induction treatment medium (1)-
Add the second differentiation induction medium supplemented with the second medium for differentiation induction medium to the composition described in Table 3 above, so that IWP2 is 5 μM, SB431542 is 5 μM, and estradiol is 100 nM. Medium.
－第１の心筋細胞誘導処理用培地（２）－
　上記表２－１及び２－２に記載の組成となるように、一液式分化誘導培地用培地添加剤を添加した一液式分化誘導培地に、ＩＷＰ２を５μＭ、ＳＢ４３１５４２を５μＭ、及びエストラジオールを１００ｎＭとなるように添加した培地。 -First cardiomyocyte induction treatment medium (2)-
In a one-part differentiation induction medium supplemented with a medium additive for one-part differentiation induction medium so as to have the composition described in Tables 2-1 and 2-2 above, 5 μM IWP2, 5 μM SB431542, and estradiol Medium added to 100 nM.
－浮遊培養－
　前記浮遊培養の方法、培養条件としては、特に制限はなく、公知の方法を適宜選択することができ、例えば、上記胚様体形成工程の浮遊培養の項目に記載したものと同様とすることができる。 -Suspension culture-
The suspension culture method and culture conditions are not particularly limited, and a known method can be appropriately selected. For example, the suspension culture method and the culture conditions may be the same as those described in the item of suspension culture in the embryoid body formation step. it can.
　前記第１の心筋細胞誘導処理の期間としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、胚様体形成工程開始３日目～６日目、胚様体形成工程開始３日目～５日目、胚様体形成工程開始４日目～６日目などとすることができる。 The period of the first cardiomyocyte induction treatment is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the embryoid body formation step is the 3rd to 6th day from the start of the embryoid body formation step. The third day to the fifth day from the start, the fourth day to the sixth day from the start of the embryoid body formation process, and the like can be used.
＜＜第２の心筋細胞誘導処理＞＞
　前記第２の心筋細胞誘導処理は、前記第１の心筋細胞誘導処理後の胚様体を第２の心筋細胞誘導処理用培地（以下、「第２の心筋細胞誘導処理用培養液」と称することがある）で培養する処理である。 << Second cardiomyocyte induction process >>
In the second cardiomyocyte induction treatment, the embryoid body after the first cardiomyocyte induction treatment is referred to as a second cardiomyocyte induction treatment medium (hereinafter, “second cardiomyocyte induction treatment medium”). In some cases).
－培地－
　前記第２の心筋細胞誘導処理における培地（以下、「第２の心筋細胞誘導処理用培地」、「第２の心筋細胞誘導処理用培養液」と称することがある）は、第２の分化誘導培地に、エストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、エストロゲン様作用物質を加えた培地のいずれかであり、必要に応じて更にその他の成分を含む。 -Culture medium-
The medium in the second cardiomyocyte induction treatment (hereinafter sometimes referred to as “second cardiomyocyte induction treatment medium” or “second cardiomyocyte induction treatment culture medium”) is used for the second differentiation induction. It is either a medium obtained by adding an estrogen-like agent to a medium, or a medium obtained by adding an estrogen-like substance to a one-part differentiation induction medium, and further contains other components as necessary.
－－第２の分化誘導培地－－
　前記第２の分化誘導培地は、上記中胚葉誘導工程の第２の分化誘導培地の項目に記載したものと同様である。 --Second differentiation induction medium--
The second differentiation induction medium is the same as that described in the item of the second differentiation induction medium in the mesoderm induction step.
－－一液式分化誘導培地－－
　前記一液式分化誘導培地は、上記胚様体形成工程の一液式分化誘導培地の項目に記載したものと同様である。 --One-part differentiation induction medium--
The one-part differentiation induction medium is the same as that described in the item of the one-part differentiation induction medium in the embryoid body formation step.
－－分化誘導調節剤－－
　前記第２の心筋細胞誘導処理用培地に添加する分化誘導調節剤（以下、「第２の心筋細胞誘導処理用分化誘導調節剤」と称することがある）は、エストロゲン様作用物質を少なくとも含み、必要に応じて更にその他の成分を含む。 --- Differentiation induction regulator ---
The differentiation-inducing regulator added to the second cardiomyocyte-inducing treatment medium (hereinafter sometimes referred to as “second differentiation-inducing regulator for cardiomyocyte-inducing treatment”) contains at least an estrogenic agent, If necessary, it further contains other components.
　前記第２の心筋細胞誘導処理用培地における分化誘導調節剤の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。 The content of the differentiation induction regulator in the second cardiomyocyte induction treatment medium is not particularly limited and can be appropriately selected depending on the purpose.
　前記エストロゲン様作用物質、及びその含有量は、上記第１の心筋細胞誘導処理用培地の項目に記載したものと同様である。 The estrogen-like substance and the content thereof are the same as those described in the item of the first cardiomyocyte induction treatment medium.
　前記第２の心筋細胞誘導処理用分化誘導調節剤は、エストロゲン様作用物質以外の分化誘導調節剤を含んでもよいし、含まなくてもよい。
　前記エストロゲン様作用物質の代わりに、ＶＥＧＦを１０ｎｇ／ｍＬの濃度で用いることも可能であるが、心筋細胞を安価、かつ簡便に得ることができる点で、エストロゲン様作用物質以外の分化誘導調節剤を含まないことが好ましい。 The second differentiation induction regulator for cardiomyocyte induction treatment may or may not contain a differentiation induction regulator other than an estrogen-like agent.
VEGF can be used at a concentration of 10 ng / mL instead of the estrogen-like substance, but differentiation-inducing regulators other than estrogen-like substances can be obtained at low cost and easily. It is preferable not to contain.
　前記第２の心筋細胞誘導処理用分化誘導調節剤におけるエストロゲン様作用物質以外の分化誘導調節剤としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、ｂＦＧＦなどが挙げられる。
　前記第２の心筋細胞誘導処理用培地にｂＦＧＦを添加する場合におけるｂＦＧＦの含有量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、１０ｎｇ／ｍＬ～１００ｎｇ／ｍＬなどが挙げられる。 The differentiation induction regulator other than the estrogen-like agent in the second differentiation induction regulator for cardiomyocyte induction treatment is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. For example, bFGF etc. are mentioned.
The content of bFGF in the case of adding bFGF to the second cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose. For example, 10 ng / mL to 100 ng / mL, etc. Is mentioned.
　前記第２の心筋細胞誘導処理用分化誘導調節剤は、市販品を用いてもよいし、化学合成したものを用いてもよい。 The second cardiomyocyte induction treatment differentiation induction regulator may be a commercially available product or a chemically synthesized product.
　前記第２の心筋細胞誘導処理用分化誘導調節剤は、各成分を含む１剤として培地に添加してもよいし、成分ごとに別々の剤として培地に添加してもよいし、任意の複数の成分を含む剤として培地に添加してもよい。 The second differentiation-inducing regulator for cardiomyocyte induction treatment may be added to the medium as one agent containing each component, or may be added to the medium as a separate agent for each component. You may add to a culture medium as an agent containing these components.
　前記第２の心筋細胞誘導処理用培地の好ましい態様としては、下記第２の心筋細胞誘導処理用培地（１）、（２）が挙げられる。これらの中でも、第２の心筋細胞誘導処理用培地（１）がより好ましい。 Preferred embodiments of the second cardiomyocyte induction treatment medium include the following second cardiomyocyte induction treatment media (1) and (2). Among these, the second cardiomyocyte induction treatment medium (1) is more preferable.
－第２の心筋細胞誘導処理用培地（１）－
　上記表３に記載の組成となるように、第２の分化誘導培地用培地添加剤を添加した第２の分化誘導培地に、エストラジオールを１００ｎＭとなるように添加した培地。 -Second medium for cardiomyocyte induction treatment (1)-
A medium in which estradiol is added to 100 nM in the second differentiation induction medium to which the second differentiation medium medium additive is added so as to have the composition described in Table 3 above.
－第１の心筋細胞誘導処理用培地（２）－
　上記表２－１及び２－２に記載の組成となるように、一液式分化誘導培地用培地添加剤を添加した一液式分化誘導培地に、エストラジオールを１００ｎＭとなるように添加した培地。 -First cardiomyocyte induction treatment medium (2)-
A medium obtained by adding estradiol to 100 nM to a one-part differentiation induction medium to which a medium additive for one-part differentiation induction medium is added so as to have the composition described in Tables 2-1 and 2-2.
－浮遊培養－
　前記浮遊培養の方法、培養条件としては、特に制限はなく、公知の方法を適宜選択することができ、例えば、上記胚様体形成工程の浮遊培養の項目に記載したものと同様とすることができる。 -Suspension culture-
The suspension culture method and culture conditions are not particularly limited, and a known method can be appropriately selected. For example, the suspension culture method and the culture conditions may be the same as those described in the item of suspension culture in the embryoid body formation step. it can.
　前記第２の心筋細胞誘導処理の期間としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、胚様体形成工程開始６日目以降、胚様体形成工程開始８日目以降などとすることができる。 The period of the second cardiomyocyte induction treatment is not particularly limited and can be appropriately selected according to the purpose. For example, the embryoid body formation process start day 6 and the embryoid body formation process start day 8 It can be after eyes.
＜＜その他の処理＞＞
　前記その他の処理としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、更なる心筋細胞誘導処理、洗浄処理などが挙げられる。 << Other processing >>
The other treatment is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Examples thereof include further cardiomyocyte induction treatment and washing treatment.
－更なる心筋細胞誘導処理－
　前記更なる心筋細胞誘導処理としては、前記第１の心筋細胞誘導処理と、前記第２の心筋細胞誘導処理との間の心筋細胞誘導処理であり、前記第１の心筋細胞誘導処理後の胚様体を第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地（以下、「第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液」と称することがある）で培養する処理が挙げられる。 -Further cardiomyocyte induction treatment-
The further cardiomyocyte induction process is a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process, and the embryo after the first cardiomyocyte induction process A medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment (hereinafter referred to as “first cardiomyocyte induction treatment and second cardiomyocyte induction treatment” And a culture medium for cardiomyocyte induction treatment during the treatment).
－－培地－－
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地は、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質を加えた培地のいずれかであり、必要に応じて更にその他の成分を含む。 --Culture medium--
In the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment, a Wnt signal inhibitory substance and an estrogen-like agent were added to the second differentiation induction medium. It is either a medium or a medium obtained by adding a Wnt signal inhibitory substance and an estrogen-like agent to a one-part differentiation induction medium, and further contains other components as necessary.
－－－第２の分化誘導培地－－－
　前記第２の分化誘導培地は、上記中胚葉誘導工程の第２の分化誘導培地の項目に記載したものと同様である。 --- Second differentiation induction medium ---
The second differentiation induction medium is the same as that described in the item of the second differentiation induction medium in the mesoderm induction step.
－－－一液式分化誘導培地－－－
　前記一液式分化誘導培地は、上記胚様体形成工程の一液式分化誘導培地の項目に記載したものと同様である。 --- One-part differentiation induction medium ---
The one-part differentiation induction medium is the same as that described in the item of the one-part differentiation induction medium in the embryoid body formation step.
－－－分化誘導調節剤－－－
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地に添加する分化誘導調節剤（以下、「第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用分化誘導調節剤」と称することがある）は、Ｗｎｔシグナル抑制物質と、エストロゲン様作用物質とを少なくとも含み、必要に応じて更にその他の成分を含む。 --- Differentiation induction regulator ---
A differentiation-inducing regulator added to the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment (hereinafter, “first cardiomyocyte induction treatment, The cardiomyocyte induction treatment differentiation differentiation regulator for cardiomyocyte induction treatment ”may include at least a Wnt signal suppressor and an estrogen-like agent, and may further contain other components as necessary. Including.
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地における分化誘導調節剤の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。 The content of the differentiation-inducing regulator in the cardiomyocyte induction treatment medium between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is not particularly limited and is appropriately selected depending on the purpose. be able to.
　前記Ｗｎｔシグナル抑制物質、及びその含有量は、上記第１の心筋細胞誘導処理用培地の項目に記載したものと同様である。 The Wnt signal suppressing substance and the content thereof are the same as those described in the item of the first medium for cardiomyocyte induction treatment.
　前記エストロゲン様作用物質、及びその含有量は、上記第１の心筋細胞誘導処理用培地の項目に記載したものと同様である。 The estrogen-like substance and the content thereof are the same as those described in the item of the first cardiomyocyte induction treatment medium.
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用分化誘導調節剤を含んでもよいし、含まなくてもよい。
　前記エストロゲン様作用物質の代わりに、ＶＥＧＦを１０ｎｇ／ｍＬの濃度で用いることも可能であるが、心筋細胞を安価、かつ簡便に得ることができる点で、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質以外の分化誘導調節剤を含まないことが好ましい。 A differentiation-inducing regulator for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment may or may not be included.
VEGF can be used at a concentration of 10 ng / mL instead of the estrogen-like substance, but a Wnt signal inhibitor and estrogen-like substance can be obtained at low cost and easily. It is preferable not to contain any other differentiation-inducing regulator.
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用分化誘導調節剤におけるＷｎｔシグナル抑制物質、及びエストロゲン様作用物質以外の分化誘導調節剤としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、ｂＦＧＦなどが挙げられる。
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地にｂＦＧＦを添加する場合におけるｂＦＧＦの含有量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、１０ｎｇ／ｍＬ～１００ｎｇ／ｍＬなどが挙げられる。 As a differentiation induction regulator other than a Wnt signal inhibitory substance and an estrogen-like agent in a differentiation induction regulator for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment, As long as the effects of the present invention are not impaired, there is no particular limitation, and it can be appropriately selected according to the purpose. Examples thereof include bFGF.
The content of bFGF in the case where bFGF is added to the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is not particularly limited and depends on the purpose. For example, it can be 10 ng / mL to 100 ng / mL.
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用分化誘導調節剤は、市販品を用いてもよいし、化学合成したものを用いてもよい。 As the differentiation induction regulator for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment, a commercially available product or a chemically synthesized product may be used. .
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用分化誘導調節剤は、各成分を含む１剤として培地に添加してもよいし、成分ごとに別々の剤として培地に添加してもよいし、任意の複数の成分を含む剤として培地に添加してもよい。 The differentiation-inducing regulator for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment may be added to the medium as one agent containing each component, or for each component May be added to the medium as separate agents, or may be added to the medium as agents containing any of a plurality of components.
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地の好ましい態様としては、下記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地（１）、（２）が挙げられる。これらの中でも、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地（１）がより好ましい。 As a preferred embodiment of the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment, the following first cardiomyocyte induction treatment and second cardiomyocyte induction treatment are described below. Medium for cardiomyocyte induction treatment between (1) and (2). Among these, the culture medium for cardiomyocyte induction treatment (1) between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is more preferable.
－第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地（１）－
　上記表３に記載の組成となるように、第２の分化誘導培地用培地添加剤を添加した第２の分化誘導培地に、ＩＷＰ２を５μＭ、及びエストラジオールを１００ｎＭとなるように添加した培地。 -Medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment (1)-
A medium obtained by adding 5 μM of IWP2 and 100 nM of estradiol to the second differentiation-inducing medium to which the second culture medium additive for differentiation-inducing medium is added so as to have the composition described in Table 3 above.
－第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地（２）－
　上記表２－１及び２－２に記載の組成となるように、一液式分化誘導培地用培地添加剤を添加した一液式分化誘導培地に、ＩＷＰ２を５μＭ、及びエストラジオールを１００ｎＭとなるように添加した培地。 -Medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment (2)-
In a one-part differentiation induction medium supplemented with a medium additive for one-part differentiation induction medium so that the composition described in Tables 2-1 and 2-2 is obtained, IWP2 is 5 μM and estradiol is 100 nM. Medium added to
－－浮遊培養－－
　前記浮遊培養の方法、培養条件としては、特に制限はなく、公知の方法を適宜選択することができ、例えば、上記胚様体形成工程の浮遊培養の項目に記載したものと同様とすることができる。 --- Suspension culture--
The suspension culture method and culture conditions are not particularly limited, and a known method can be appropriately selected. For example, the suspension culture method and the culture conditions may be the same as those described in the item of suspension culture in the embryoid body formation step. it can.
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理の期間としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、胚様体形成工程開始６日目～８日目、胚様体形成工程開始５日目～８日目などとすることができる。 The period of the cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process is not particularly limited and can be appropriately selected according to the purpose. It may be the 6th to 8th day from the start of the body formation process, the 5th to 8th day from the start of the embryoid body formation process.
－洗浄処理－
　前記洗浄処理は、前記第１の心筋細胞誘導処理、前記第２の心筋細胞誘導処理、及び前記更なる心筋細胞誘導処理の少なくともいずれかの後に、前記胚様体を洗浄する処理であり、後述する洗浄工程と同様にして行うことができる。 -Cleaning treatment-
The washing process is a process of washing the embryoid body after at least one of the first cardiomyocyte induction process, the second cardiomyocyte induction process, and the further cardiomyocyte induction process, which will be described later. It can carry out similarly to the washing | cleaning process to perform.
＜その他の工程＞
　前記その他の工程としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、フィーダー細胞培養工程、洗浄工程などが挙げられる。 <Other processes>
The other steps are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Examples thereof include a feeder cell culture step and a washing step.
＜＜フィーダー細胞培養工程＞＞
　前記フィーダー細胞培養工程は、多能性幹細胞と、フィーダー細胞とを共に培養し、多能性幹細胞を未分化な状態で維持する工程である。
　前記フィーダー細胞としては、特に制限はなく、公知のものを適宜選択することができる。
　前記フィーダー細胞培養工程の培養条件としては、特に制限はなく、目的に応じて適宜選択することができる。 << Feeder cell culture process >>
The feeder cell culturing step is a step of culturing pluripotent stem cells and feeder cells together to maintain the pluripotent stem cells in an undifferentiated state.
There is no restriction | limiting in particular as said feeder cell, A well-known thing can be selected suitably.
There is no restriction | limiting in particular as culture conditions of the said feeder cell culture process, According to the objective, it can select suitably.
＜＜洗浄工程＞＞
　前記洗浄工程は、上述した胚様体形成工程、中胚葉誘導工程、及び心筋細胞誘導工程の少なくともいずれかの後に、前記胚様体を洗浄する工程である。
　前記洗浄の方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、培養後の胚様体を培養液ごと容器に回収し、静置して前記胚様体を沈殿させた後、上清を除去し、次いで、培養液又は緩衝液を加え、再度静置して前記胚様体を沈殿させた後、上清を除去する方法などが挙げられる。
　前記洗浄の回数としては、特に制限はなく、目的に応じて適宜選択することができるが、複数回行うことが好ましい。 << Cleaning process >>
The washing step is a step of washing the embryoid body after at least one of the aforementioned embryoid body forming step, mesoderm induction step, and cardiomyocyte induction step.
The washing method is not particularly limited and can be appropriately selected depending on the purpose. For example, the cultured embryoid bodies are collected in a container together with the culture solution and allowed to stand to precipitate the embryoid bodies. After the treatment, the supernatant is removed, and then a culture solution or a buffer solution is added, and the mixture is allowed to stand again to precipitate the embryoid body, and then the supernatant is removed.
There is no restriction | limiting in particular as the frequency | count of the said washing | cleaning, Although it can select suitably according to the objective, It is preferable to perform in multiple times.
　前記各工程における培地の組合せの態様としては、特に制限はなく、目的に応じて適宜選択することができるが、胚様体形成工程における培地が、第１の分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地であり、中胚葉誘導工程における培地が、第２の分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地であり、第１の心筋細胞誘導処理における培地が、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地であり、第２の心筋細胞誘導処理における培地が、第２の分化誘導培地に、エストロゲン様作用物質を加えた培地である態様、胚様体形成工程における培地が、一液式分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地であり、中胚葉誘導工程における培地が、一液式分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地であり、第１の心筋細胞誘導処理における培地が、一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地であり、第２の心筋細胞誘導処理における培地が、一液式分化誘導培地に、エストロゲン様作用物質を加えた培地である態様が好ましく、胚様体形成工程における培地が、第１の分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地であり、中胚葉誘導工程における培地が、第２の分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地であり、第１の心筋細胞誘導処理における培地が、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地であり、第２の心筋細胞誘導処理における培地が、第２の分化誘導培地に、エストロゲン様作用物質を加えた培地である態様がより好ましい。 The medium combination in each step is not particularly limited and can be appropriately selected depending on the purpose. The medium in the embryoid body formation step is a first differentiation-inducing medium, and a ROCK inhibitor is added to the medium. The medium in the mesoderm induction step is a medium obtained by adding a Wnt signal activator to the second differentiation induction medium, and the medium in the first cardiomyocyte induction treatment is the second differentiation induction. A medium in which a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like agent are added to a medium, and the medium in the second cardiomyocyte induction treatment is added to the second differentiation-inducing medium. The medium in the embryoid body formation step is a medium in which a ROCK inhibitor is added to the one-part differentiation induction medium, and the medium in the mesoderm induction step is one. A medium obtained by adding a Wnt signal activator to a formula differentiation-inducing medium, and the medium in the first cardiomyocyte induction treatment includes a one-part differentiation induction medium, a Wnt signal inhibitor, a TGF-β signal inhibitor, and An embodiment in which an estrogen-like agent is added, and the medium in the second cardiomyocyte induction treatment is preferably a medium in which an estrogen-like agent is added to a one-part differentiation induction medium, in the embryoid body formation step The medium is a medium obtained by adding a ROCK inhibitor to the first differentiation induction medium, the medium in the mesoderm induction step is a medium obtained by adding a Wnt signal activator to the second differentiation induction medium, The medium for the cardiomyocyte induction treatment of 1 is a medium obtained by adding a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like agent to the second differentiation induction medium. Ri, media in the second cardiomyocyte induction process, the second differentiation-inducing medium, aspects and more preferably a medium with estrogenic agents.
　上記方法により、多能性幹細胞から心筋細胞が得られたか否かは、胚様体が拍動しているか否かにより確認することができる。
　また、得られた心筋細胞が高品質であるか否かを確認する方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、パッチクランプ法などが挙げられる。前記パッチクランプ法により、大きく明瞭なプラトー相を有する活動電位（活動電位振幅１４０ｍＶ以上）、大きなピークカリウム電流（３００ｐＡ以上）、大きなピークカルシウム電流（１ｎＡ以上）、大きなピークナトリウム電流（６．５ｎＡ以上）電流が確認された場合には、高品質な心筋細胞が得られたと判断することができる。 Whether or not cardiomyocytes are obtained from pluripotent stem cells by the above method can be confirmed by whether or not the embryoid body is beating.
Moreover, there is no restriction | limiting in particular as a method of confirming whether the obtained cardiomyocytes are high quality, According to the objective, it can select suitably, For example, the patch clamp method etc. are mentioned. By the patch clamp method, an action potential having a large and clear plateau phase (action potential amplitude of 140 mV or more), a large peak potassium current (300 pA or more), a large peak calcium current (1 nA or more), a large peak sodium current (6.5 nA or more) ) If the current is confirmed, it can be determined that high-quality cardiomyocytes have been obtained.
（培地添加剤）
　本発明の培地添加剤は、多能性幹細胞から心筋細胞を分化誘導するための培地に添加する培地添加剤であって、アルブミン及び１－チオグリセロールの少なくともいずれかを含み、必要に応じて更にその他の成分を含む。
　前記培地添加剤は、本発明の多能性幹細胞から心筋細胞を分化誘導する方法に好適に用いることができる。 (Medium additive)
The medium additive of the present invention is a medium additive added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells, and contains at least one of albumin and 1-thioglycerol, and further if necessary Contains other ingredients.
The medium additive can be suitably used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
　前記その他の成分としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、トランスフェリン、インスリン、ポリビニルアルコール、エタノラミン塩酸塩、亜セレン酸ナトリウム、ヒドロコルチゾン、ＤＬ－α－トコフェロール酢酸エステル、Ｎ－アセチル－Ｌ－システイン、アスコルビン酸、グリシン、Ｌ－アラニン、Ｌ－アスパラギン・Ｈ２Ｏ、Ｌ－アスパラギン酸、Ｌ－グルタミン、Ｌ－グルタミン酸、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、ＺｒＯＣｌ２・８Ｈ２Ｏなどが挙げられる。
　前記その他の成分は、１種単独で使用してもよいし、２種以上を併用してもよいが、トランスフェリン、インスリン、ポリビニルアルコール、エタノラミン塩酸塩、亜セレン酸ナトリウム、ヒドロコルチゾン、ＤＬ－α－トコフェロール酢酸エステル、Ｎ－アセチル－Ｌ－システイン、及びアスコルビン酸からなる群から選択される少なくとも１種を含む態様、グリシン、Ｌ－アラニン、Ｌ－アスパラギン・Ｈ２Ｏ、Ｌ－アスパラギン酸、Ｌ－グルタミン、Ｌ－グルタミン酸、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏからなる群から選択される少なくとも１種を含む態様が好ましい。 The other components are not particularly limited and may be appropriately selected depending on the purpose. For example, transferrin, insulin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate N-acetyl-L-cysteine, ascorbic acid, glycine, L-alanine, L-asparagine / H 2 O, L-aspartic acid, L-glutamine, L-glutamic acid, L-histidine, L-isoleucine, L-methionine , L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, magnesium salt of ascorbic acid 2-2PO 4 , AgNO 3 , AlC l 3 · 6H 2 O, Ba (C 2 H 3 O 2 ) 2 , 3CdSO 4 · 8H 2 O, CoCl 2 · 6H 2 O, Cr 2 (SO 4 ) 3 · H 2 O, GeO 2 , Na 2 SeO 3 , KBr, KI, MnCl 2 .4H 2 O, NaF, Na 2 SiO 3 .9H 2 O, NaVO 3 , (NH 4 ) 6 Mo 7 O 24 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl, SnCl 2 .2H 2 O, ZrOCl 2 .8H 2 O and the like can be mentioned.
The other components may be used alone or in combination of two or more. Transferrin, insulin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α- An embodiment comprising at least one selected from the group consisting of tocopherol acetate, N-acetyl-L-cysteine, and ascorbic acid, glycine, L-alanine, L-asparagine / H 2 O, L-aspartic acid, L- Glutamine, L-glutamic acid, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine , Thiamine, reduced glutathione, ascorbic acid-2-2P Magnesium salt of O 4 , AgNO 3 , AlCl 3 .6H 2 O, Ba (C 2 H 3 O 2 ) 2 , 3CdSO 4 · 8H 2 O, CoCl 2 · 6H 2 O, Cr 2 (SO 4 ) 3 · H 2 O, GeO 2 , Na 2 SeO 3 , KBr, KI, MnCl 2 .4H 2 O, NaF, Na 2 SiO 3 .9H 2 O, NaVO 3 , (NH 4 ) 6 Mo 7 O 24 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl , SnCl 2 · 2H 2 O, and embodiments comprising at least one member selected from the group consisting of ZrOCl 2 · 8H 2 O preferred.
　前記培地添加剤の態様としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができるが、下記培地添加剤（１）～（３）の少なくともいずれかの態様が好ましい。 The mode of the medium additive is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the purpose. At least one of the following medium additives (1) to (3) This embodiment is preferred.
＜培地添加剤（１）＞
　前記培地添加剤（１）は、インスリンと、トランスフェリンと、アルブミンと、１－チオグリセロールとを少なくとも含み、必要に応じて更にその他の成分を含む。
　前記培地添加剤（１）におけるその他の成分としては、特に制限はなく、目的に応じて適宜選択することができるが、グリシン、Ｌ－アラニン、Ｌ－アスパラギン・Ｈ２Ｏ、Ｌ－アスパラギン酸、Ｌ－グルタミン、Ｌ－グルタミン酸、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏを含むことが好ましい。
　前記培地添加剤（１）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の分化誘導培地における第１の分化誘導培地用培地添加剤として好適に用いることができ、培地における含有量等も上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の分化誘導培地用培地添加剤と同様とすることができる。 <Medium medium additive (1)>
The medium additive (1) contains at least insulin, transferrin, albumin, and 1-thioglycerol, and further contains other components as necessary.
The other components in the medium additive (1) are not particularly limited and may be appropriately selected depending on the intended purpose. However, glycine, L-alanine, L-asparagine / H 2 O, L-aspartic acid, L-glutamine, L-glutamic acid, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L - valine, thiamine, reduced glutathione, magnesium salt of ascorbic acid -2-2PO 4, AgNO 3, AlCl 3 · 6H 2 O, Ba (C 2 H 3 O 2) 2, 3CdSO 4 · 8H 2 O, CoCl 2 · 6H 2 O, Cr 2 ( SO 4) 3 · H 2 O, GeO 2, Na 2 SeO 3, KBr, KI, nCl 2 · 4H 2 O, NaF , Na 2 SiO 3 · 9H 2 O, NaVO 3, (NH 4) 6 Mo 7 O 24 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl, SnCl 2 · 2H 2 O And ZrOCl 2 .8H 2 O.
The culture medium additive (1) can be suitably used as a first culture medium addition medium for differentiation induction medium in the first differentiation induction medium of the above-described method for inducing differentiation of cardiomyocytes from pluripotent stem cells. The content and the like in can also be the same as those in the first medium-inducing medium for differentiation-inducing medium of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
＜培地添加剤（２）＞
　前記培地添加剤（２）は、アルブミンと、ポリビニルアルコールと、エタノラミン塩酸塩と、亜セレン酸ナトリウムと、ヒドロコルチゾンと、ＤＬ－α－トコフェロール酢酸エステルと、Ｎ－アセチル－Ｌ－システインと、１－チオグリセロールと、アスコルビン酸とを少なくとも含み、必要に応じて更にその他の成分を含む。
　前記培地添加剤（２）におけるその他の成分としては、特に制限はなく、目的に応じて適宜選択することができるが、Ｌ－グルタミン、及びトランスフェリンを含むことが好ましい。
　前記培地添加剤（２）は、前記その他の成分としてインスリンを含んでもよいが、含まないことが好ましい。
　前記培地添加剤（２）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第２の分化誘導培地における第２の分化誘導培地用培地添加剤として好適に用いることができ、培地における含有量等も上述した多能性幹細胞から心筋細胞を分化誘導する方法の第２の分化誘導培地用培地添加剤と同様とすることができる。 <Medium medium additive (2)>
The medium additive (2) comprises albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine, 1- It contains at least thioglycerol and ascorbic acid, and further contains other components as necessary.
Other components in the medium additive (2) are not particularly limited and may be appropriately selected depending on the intended purpose, but preferably include L-glutamine and transferrin.
The medium additive (2) may contain insulin as the other component, but preferably does not contain insulin.
The medium additive (2) can be suitably used as the second medium for medium for inducing differentiation in the second medium for inducing differentiation of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. The content and the like in can also be the same as those of the second medium-inducing medium for differentiation-inducing medium of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells.
＜培地添加剤（３）＞
　前記培地添加剤（３）は、トランスフェリンと、アルブミンと、ポリビニルアルコールと、エタノラミン塩酸塩と、亜セレン酸ナトリウムと、ヒドロコルチゾンと、ＤＬ－α－トコフェロール酢酸エステルと、Ｎ－アセチル－Ｌ－システインと、１－チオグリセロールと、アスコルビン酸とを少なくとも含み、必要に応じて更にその他の成分を含む。
　前記培地添加剤（３）におけるその他の成分としては、特に制限はなく、目的に応じて適宜選択することができるが、グリシン、Ｌ－グルタミン、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏを含むことが好ましい。
　前記培地添加剤（３）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の一液式分化誘導培地における一液式分化誘導培地用培地添加剤として好適に用いることができ、培地における含有量等も上述した多能性幹細胞から心筋細胞を分化誘導する方法の一液式分化誘導培地用培地添加剤と同様とすることができる。 <Medium medium additive (3)>
The medium additive (3) includes transferrin, albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine, , 1-thioglycerol and ascorbic acid, and further contains other components as necessary.
Other components in the medium additive (3) are not particularly limited and may be appropriately selected depending on the intended purpose. However, glycine, L-glutamine, L-histidine, L-isoleucine, L-methionine, L -Phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, magnesium salt of ascorbic acid 2-2PO 4 , AgNO 3 , AlCl 3 · 6H 2 O, Ba (C 2 H 3 O 2 ) 2 , 3CdSO 4 · 8H 2 O, CoCl 2 · 6H 2 O, Cr 2 (SO 4 ) 3 · H 2 O, GeO 2 , Na 2 SeO 3 , KBr, KI, MnCl 2 .4H 2 O, NaF, Na 2 SiO 3 .9H 2 O, NaVO 3 , (NH 4 ) 6 Mo 7 O 24 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl, SnCl 2 · 2H 2 O, and ZrOCl 2 · 8H 2 O are preferably included.
The medium additive (3) can be suitably used as a medium additive for a one-part differentiation induction medium in a one-part differentiation induction medium for the method of inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. The content and the like in can also be the same as the medium additive for a one-part differentiation induction medium for the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
　前記培地添加剤は、市販品を用いてもよいし、化学合成したものを用いてもよい。 The medium additive may be a commercially available product or a chemically synthesized product.
　前記培地添加剤は、各成分を含む１剤であってもよいし、成分ごとに別々の剤としてもよいし、任意の複数の成分を含む剤を組み合わせたものであってもよい。 The medium additive may be one agent containing each component, may be a separate agent for each component, or may be a combination of agents containing any plural components.
（分化誘導調節剤）
　本発明の分化誘導調節剤は、多能性幹細胞から心筋細胞を分化誘導するための培地に添加する分化誘導調節剤であって、ＲＯＣＫ阻害剤、Ｗｎｔシグナル活性化物質、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質からなる群から選択される少なくとも１種を含み、必要に応じて更にその他の成分を含む。
　前記分化誘導調節剤は、本発明の多能性幹細胞から心筋細胞を分化誘導する方法に好適に用いることができる。 (Differentiation induction regulator)
The differentiation-inducing regulator of the present invention is a differentiation-inducing regulator added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells, which is a ROCK inhibitor, a Wnt signal activator, a Wnt signal suppressor, TGF -It contains at least one member selected from the group consisting of β signal inhibitor and estrogen-like substance, and further contains other components as necessary.
The differentiation-inducing regulator can be suitably used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
＜ＲＯＣＫ阻害剤＞
　前記ＲＯＣＫ阻害剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、Ｙ２７６３２、Ｆａｓｕｄｉｌ　Ｈｙｄｒｏｃｈｌｏｒｉｄｅなどが挙げられる。これらの中でも、心筋細胞への分化誘導効率に優れる点で、Ｙ２７６３２が好ましい。
　前記ＲＯＣＫ阻害剤は、１種単独で使用してもよいし、２種以上を併用してもよい。 <ROCK inhibitor>
There is no restriction | limiting in particular as said ROCK inhibitor, According to the objective, it can select suitably, For example, Y27632, Fastil Hydrochloride, etc. are mentioned. Among these, Y27632 is preferable because it is excellent in efficiency of inducing differentiation into cardiomyocytes.
The ROCK inhibitor may be used alone or in combination of two or more.
＜Ｗｎｔシグナル活性化物質＞
　前記Ｗｎｔシグナル活性化物質としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＣＨＩＲ９９０２１、ＢＩＯ、Ｗｎｔ　アゴニスト（ＣＡＳ　８５３２２０－５２－７）、Ｗｎｔ　アゴニストＩＩ（ＳＫＬ２００１）などが挙げられる。これらの中でも、分化誘導効率に優れる点で、ＣＨＩＲ９９０２１が好ましい。
　前記Ｗｎｔシグナル活性化物質は、１種単独で使用してもよいし、２種以上を併用してもよい。 <Wnt signal activator>
The Wnt signal activator is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include CHIR99021, BIO, Wnt agonist (CAS 853220-52-7), Wnt agonist II (SKL2001) and the like. Can be mentioned. Among these, CHIR99021 is preferable because it is excellent in differentiation induction efficiency.
The Wnt signal activator may be used alone or in combination of two or more.
＜Ｗｎｔシグナル抑制物質＞
　前記Ｗｎｔシグナル抑制物質としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＩＷＰ２、ＩＷＲ１、ＸＡＶ９３９、ＫＹ０２１１１などが挙げられる。これらの中でも、低濃度から優れた分化誘導効率を発揮する点で、ＩＷＰ２が好ましい。
　前記Ｗｎｔシグナル抑制物質は、１種単独で使用してもよいし、２種以上を併用してもよい。 <Wnt signal suppressor>
There is no restriction | limiting in particular as said Wnt signal suppression substance, According to the objective, it can select suitably, For example, IWP2, IWR1, XAV939, KY02111 etc. are mentioned. Among these, IWP2 is preferable in that it exhibits excellent differentiation induction efficiency from a low concentration.
The Wnt signal inhibitor may be used alone or in combination of two or more.
＜ＴＧＦ－βシグナル阻害剤＞
　前記ＴＧＦ－βシグナル阻害剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＳＢ４３１５４２、ＳＢ５０５１２４、Ａ－８３－０１などが挙げられる。これらの中でも、分化誘導効率に優れる点で、ＳＢ４３１５４２が好ましい。
　前記ＴＧＦ－βシグナル阻害剤は、１種単独で使用してもよいし、２種以上を併用してもよい。 <TGF-β signal inhibitor>
The TGF-β signal inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include SB431542, SB505124, A-83-01 and the like. Among these, SB431542 is preferable because it is excellent in differentiation induction efficiency.
The TGF-β signal inhibitor may be used alone or in combination of two or more.
＜エストロゲン様作用物質＞
　前記エストロゲン様作用物質とは、エストロゲン作用を示すホルモン、低分子化合物をいう。
　前記エストロゲン様作用物質としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、エストラジオール、エストロン、エストリオール、ゲニステインなどが挙げられる。これらの中でも、分化誘導効率に優れる点で、エストラジオールが好ましい。
　前記エストロゲン様作用物質は、１種単独で使用してもよいし、２種以上を併用してもよい。 <Estrogen-like substance>
The estrogen-like substance refers to hormones and low molecular compounds that exhibit estrogen action.
The estrogen-like substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include estradiol, estrone, estriol, and genistein. Among these, estradiol is preferable because it is excellent in differentiation induction efficiency.
The said estrogen-like active substance may be used individually by 1 type, and may use 2 or more types together.
　前記その他の成分としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ｂＦＧＦ、ＶＥＧＦなどが挙げられる。 The other components are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include bFGF and VEGF.
　前記分化誘導調節剤の態様としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができるが、下記分化誘導調節剤（１）～（５）の少なくともいずれかの態様が好ましい。 The mode of the differentiation induction regulator is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. At least one of the following differentiation induction regulators (1) to (5) Either embodiment is preferred.
＜分化誘導調節剤（１）＞
　前記分化誘導調節剤（１）は、ＲＯＣＫ阻害剤を少なくとも含み、必要に応じて更にその他の成分を含む。
　前記分化誘導調節剤（１）は、ＲＯＣＫ阻害剤以外の分化誘導調節剤を含んでもよいが、含まないことが好ましい。
　前記分化誘導調節剤（１）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の胚様体形成工程用分化誘導調節剤として好適に用いることができ、培地における含有量等も上述した多能性幹細胞から心筋細胞を分化誘導する方法の胚様体形成工程用分化誘導調節剤と同様とすることができる。 <Differentiation induction regulator (1)>
The differentiation-inducing regulator (1) contains at least a ROCK inhibitor, and further contains other components as necessary.
Although the differentiation-inducing regulator (1) may contain a differentiation-inducing regulator other than the ROCK inhibitor, it is preferably not contained.
The differentiation-inducing regulator (1) can be suitably used as a differentiation-inducing regulator for the embryoid body formation step in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above, and the content in the medium is also described above. It can be the same as the differentiation-inducing regulator for the embryoid body formation step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells.
＜分化誘導調節剤（２）＞
　前記分化誘導調節剤（２）は、Ｗｎｔシグナル活性化物質を少なくとも含み、必要に応じて更にその他の成分を含む。
　前記分化誘導調節剤（２）は、Ｗｎｔシグナル活性化物質以外の分化誘導調節剤を含んでもよいが、含まないことが好ましい。特に、Ｗｎｔシグナル活性化物質の中では、ＢＩＯ、Ｗｎｔ　アゴニスト、Ｗｎｔ　アゴニストＩＩを含まないことが好ましい。
　前記分化誘導調節剤（２）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の中胚葉誘導工程用分化誘導調節剤として好適に用いることができ、培地における含有量等も上述した多能性幹細胞から心筋細胞を分化誘導する方法の中胚葉誘導工程用分化誘導調節剤と同様とすることができる。 <Differentiation induction regulator (2)>
The differentiation-inducing regulator (2) contains at least a Wnt signal activator, and further contains other components as necessary.
The differentiation-inducing regulator (2) may contain a differentiation-inducing regulator other than the Wnt signal activator, but preferably does not contain it. In particular, the Wnt signal activator preferably does not contain BIO, Wnt agonist, or Wnt agonist II.
The differentiation-inducing regulator (2) can be suitably used as a differentiation-inducing regulator for the mesoderm-inducing process for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above, and the content in the medium is also described above. The method of inducing differentiation of cardiomyocytes from pluripotent stem cells can be the same as the differentiation induction regulator for the mesoderm induction process.
＜分化誘導調節剤（３）＞
　前記分化誘導調節剤（３）は、Ｗｎｔシグナル抑制物質と、ＴＧＦ－βシグナル阻害剤と、エストロゲン様作用物質とを少なくとも含み、必要に応じて更にその他の成分を含む。
　前記分化誘導調節剤（３）は、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質以外の分化誘導調節剤を含んでもよいが、含まないことが好ましい。
　前記分化誘導調節剤（３）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理用分化誘導調節剤として好適に用いることができ、培地における含有量等も上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理用分化誘導調節剤と同様とすることができる。 <Differentiation induction regulator (3)>
The differentiation induction regulator (3) contains at least a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like substance, and further contains other components as necessary.
The differentiation induction regulator (3) may contain, but preferably does not contain, a differentiation induction regulator other than a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like agent.
The differentiation induction regulator (3) can be suitably used as the first differentiation induction regulator for cardiomyocyte induction treatment in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. Can also be the same as the first differentiation-inducing regulator for cardiomyocyte induction treatment in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
＜分化誘導調節剤（４）＞
　前記分化誘導調節剤（４）は、エストロゲン様作用物質を少なくとも含み、必要に応じて更にその他の成分を含む。
　前記分化誘導調節剤（４）は、エストロゲン様作用物質以外の分化誘導調節剤を含んでもよいが、含まないことが好ましい。
　前記分化誘導調節剤（４）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第２の心筋細胞誘導処理用分化誘導調節剤として好適に用いることができ、培地における含有量等も上述した多能性幹細胞から心筋細胞を分化誘導する方法の第２の心筋細胞誘導処理用分化誘導調節剤と同様とすることができる。 <Differentiation induction regulator (4)>
The differentiation-inducing regulator (4) contains at least an estrogen-like substance, and further contains other components as necessary.
The differentiation-inducing regulator (4) may contain a differentiation-inducing regulator other than an estrogen-like agent, but preferably does not contain it.
The differentiation-inducing regulator (4) can be suitably used as the second differentiation-inducing regulator for cardiomyocyte induction treatment in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. Can also be the same as the second differentiation-inducing regulator for cardiomyocyte induction in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above.
＜分化誘導調節剤（５）＞
　前記分化誘導調節剤（５）は、Ｗｎｔシグナル抑制物質と、エストロゲン様作用物質とを少なくとも含み、必要に応じて更にその他の成分を含む。
　前記分化誘導調節剤（５）は、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質以外の分化誘導調節剤を含んでもよいが、含まないことが好ましい。
　前記分化誘導調節剤（５）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用分化誘導調節剤として好適に用いることができ、培地における含有量等も上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用分化誘導調節剤と同様とすることができる。 <Differentiation induction regulator (5)>
The differentiation induction regulator (5) contains at least a Wnt signal inhibitor and an estrogen-like substance, and further contains other components as necessary.
The differentiation-inducing regulator (5) may contain a differentiation-inducing regulator other than a Wnt signal suppressor and an estrogen-like agent, but preferably does not contain it.
The differentiation induction regulator (5) is used for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. The first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment of the method of inducing differentiation of cardiomyocytes from the pluripotent stem cells described above, which can be suitably used as a differentiation induction regulator and whose content in the medium is also described above It can be the same as the differentiation-inducing regulator for cardiomyocyte induction treatment.
　前記分化誘導調節剤は、市販品を用いてもよいし、化学合成したものを用いてもよい。 The differentiation induction regulator may be a commercially available product or a chemically synthesized product.
　前記分化誘導調節剤は、各成分を含む１剤であってもよいし、成分ごとに別々の剤としてもよいし、任意の複数の成分を含む剤を組み合わせたものであってもよい。 The differentiation-inducing regulator may be one agent containing each component, may be a separate agent for each component, or may be a combination of agents containing any plural components.
（培地）
　本発明の培地は、多能性幹細胞から心筋細胞を分化誘導するための培地であって、本発明の培地添加剤と、本発明の分化誘導調節剤とを少なくとも含み、必要に応じて更にその他の成分を含む。
　前記培地は、本発明の多能性幹細胞から心筋細胞を分化誘導する方法に好適に用いることができる。 (Culture medium)
The medium of the present invention is a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells, and includes at least the medium additive of the present invention and the differentiation-inducing regulator of the present invention, and if necessary, other Contains the ingredients.
The medium can be suitably used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
　前記培地に用いる基礎培地は、ＤＭＥＭ／Ｆ１２、ＤＭＥＭ、ＩＭＤＭ、ＲＰＭＩ１６４０培地、及びαＭＥＭ培地からなる群から選択される少なくとも１種である。
　前記ＤＭＥＭは、高グルコースであってもよいし、低グルコースであってもよい。 The basal medium used for the medium is at least one selected from the group consisting of DMEM / F12, DMEM, IMDM, RPMI1640 medium, and αMEM medium.
The DMEM may be high glucose or low glucose.
　前記培地の態様としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができるが、下記培地（１）～（１０）の少なくともいずれかの態様が好ましい。 The form of the medium is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. However, at least one of the following mediums (1) to (10) is preferable. .
＜培地（１）＞
　前記培地（１）は、基礎培地として、ＤＭＥＭ／Ｆ１２、ＤＭＥＭ、ＩＭＤＭ、ＲＰＭＩ１６４０培地、及びαＭＥＭ培地からなる群から選択される少なくとも１種を用い、前記培地添加剤として、前記培地添加剤（１）を含み、前記分化誘導調節剤として、前記分化誘導調節剤（１）を含み、必要に応じて更にその他の成分を含む。
　前記基礎培地の中でも、ＤＭＥＭ／Ｆ１２が好ましい。
　前記培地（１）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の胚様体形成工程における第１の分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地として好適に用いることができる。
　前記培地（１）の好ましい態様としては、上記胚様体形成工程用培地（１）が挙げられる。 <Medium (1)>
The medium (1) is at least one selected from the group consisting of DMEM / F12, DMEM, IMDM, RPMI1640 medium, and αMEM medium as a basal medium, and the medium additive (1 ), And the differentiation-inducing regulator (1) as the differentiation-inducing regulator, and further containing other components as necessary.
Among the basal media, DMEM / F12 is preferable.
The medium (1) can be suitably used as a medium in which a ROCK inhibitor is added to the first differentiation induction medium in the embryoid body formation step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. .
As a preferable aspect of the said culture medium (1), the said culture medium (1) for embryoid body formation processes is mentioned.
＜培地（２）＞
　前記培地（２）は、基礎培地として、ＩＭＤＭを用い、前記培地添加剤として、前記培地添加剤（３）を含み、前記分化誘導調節剤として、前記分化誘導調節剤（１）を含み、必要に応じて更にその他の成分を含む。
　前記培地（２）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の胚様体形成工程における一液式分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地として好適に用いることができる。
　前記培地（２）の好ましい態様としては、上記胚様体形成工程用培地（２）が挙げられる。 <Medium (2)>
The medium (2) uses IMDM as a basal medium, includes the medium additive (3) as the medium additive, and includes the differentiation induction regulator (1) as the differentiation induction regulator. Depending on the above, other components are further included.
The medium (2) can be suitably used as a medium in which a ROCK inhibitor is added to the one-part differentiation induction medium in the embryoid body formation step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. .
As a preferable aspect of the said culture medium (2), the said culture medium (2) for embryoid body formation processes is mentioned.
＜培地（３）＞
　前記培地（３）は、基礎培地として、ＩＭＤＭを用い、前記培地添加剤として、前記培地添加剤（２）を含み、前記分化誘導調節剤として、前記分化誘導調節剤（２）を含み、必要に応じて更にその他の成分を含む。
　前記培地（３）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の中胚葉誘導工程における第２の分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地として好適に用いることができる。
　前記培地（３）の好ましい態様としては、上記中胚葉誘導工程用培地（１）が挙げられる。 <Medium (3)>
The medium (3) uses IMDM as a basal medium, includes the medium additive (2) as the medium additive, and includes the differentiation induction regulator (2) as the differentiation induction regulator. Depending on the above, other components are further included.
The medium (3) is preferably used as a medium in which a Wnt signal activator is added to the second differentiation induction medium in the mesoderm induction step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. it can.
As a preferable aspect of the said culture medium (3), the said culture medium (1) for mesoderm induction processes is mentioned.
＜培地（４）＞
　前記培地（４）は、基礎培地として、ＩＭＤＭを用い、前記培地添加剤として、前記培地添加剤（３）を含み、前記分化誘導調節剤として、前記分化誘導調節剤（２）を含み、必要に応じて更にその他の成分を含む。
　前記培地（４）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の中胚葉誘導工程における一液式分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地として好適に用いることができる。
　前記培地（４）の好ましい態様としては、上記中胚葉誘導工程用培地（２）が挙げられる。 <Medium (4)>
The medium (4) uses IMDM as a basal medium, includes the medium additive (3) as the medium additive, and includes the differentiation induction regulator (2) as the differentiation induction regulator. Depending on the above, other components are further included.
The medium (4) is preferably used as a medium in which a Wnt signal activator is added to the one-component differentiation induction medium in the mesoderm induction step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. it can.
As a preferable aspect of the said culture medium (4), the said culture medium (2) for mesoderm induction processes is mentioned.
＜培地（５）＞
　前記培地（５）は、基礎培地として、ＩＭＤＭを用い、前記培地添加剤として、前記培地添加剤（２）を含み、前記分化誘導調節剤として、前記分化誘導調節剤（３）を含み、必要に応じて更にその他の成分を含む。
　前記培地（５）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理における第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地として好適に用いることができる。
　前記培地（５）の好ましい態様としては、上記第１の心筋細胞誘導処理用培地（１）が挙げられる。 <Medium (5)>
The medium (5) uses IMDM as a basal medium, includes the medium additive (2) as the medium additive, and includes the differentiation induction regulator (3) as the differentiation induction regulator. Depending on the above, other components are further included.
The medium (5) includes a Wnt signal inhibitor, a TGF-β signal inhibitor, a second differentiation induction medium in the first cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above, And an estrogen-like substance-added medium.
As a preferable aspect of the culture medium (5), the first cardiomyocyte induction treatment medium (1) may be mentioned.
＜培地（６）＞
　前記培地（６）は、基礎培地として、ＩＭＤＭを用い、前記培地添加剤として、前記培地添加剤（３）を含み、前記分化誘導調節剤として、前記分化誘導調節剤（３）を含み、必要に応じて更にその他の成分を含む。
　前記培地（６）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理における一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地として好適に用いることができる。
　前記培地（６）の好ましい態様としては、上記第１の心筋細胞誘導処理用培地（２）が挙げられる。 <Medium (6)>
The medium (6) uses IMDM as a basal medium, includes the medium additive (3) as the medium additive, and includes the differentiation induction regulator (3) as the differentiation induction regulator. Depending on the above, other components are further included.
The medium (6) includes a Wnt signal suppressor, a TGF-β signal inhibitor, a one-part differentiation induction medium in the first cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above, And an estrogen-like substance-added medium.
As a preferable aspect of the said culture medium (6), the said culture medium (2) for said 1st cardiomyocyte induction is mentioned.
＜培地（７）＞
　前記培地（７）は、基礎培地として、ＩＭＤＭを用い、前記培地添加剤として、前記培地添加剤（２）を含み、前記分化誘導調節剤として、前記分化誘導調節剤（４）を含み、必要に応じて更にその他の成分を含む。
　前記培地（７）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第２の心筋細胞誘導処理における第２の分化誘導培地に、エストロゲン様作用物質を加えた培地として好適に用いることができる。
　前記培地（７）の好ましい態様としては、上記第２の心筋細胞誘導処理用培地（１）が挙げられる。 <Medium (7)>
The medium (7) uses IMDM as a basal medium, includes the medium additive (2) as the medium additive, and includes the differentiation induction regulator (4) as the differentiation induction regulator. Depending on the above, other components are further included.
The medium (7) is suitably used as a medium in which an estrogen-like agent is added to the second differentiation inducing medium in the second cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. be able to.
As a preferable aspect of the culture medium (7), the second culture medium for cardiomyocyte induction treatment (1) may be mentioned.
＜培地（８）＞
　前記培地（８）は、基礎培地として、ＩＭＤＭを用い、前記培地添加剤として、前記培地添加剤（３）を含み、前記分化誘導調節剤として、前記分化誘導調節剤（４）を含み、必要に応じて更にその他の成分を含む。
　前記培地（８）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第２の心筋細胞誘導処理における一液式分化誘導培地に、エストロゲン様作用物質を加えた培地として好適に用いることができる。
　前記培地（８）の好ましい態様としては、上記第１の心筋細胞誘導処理用培地（２）が挙げられる。 <Medium (8)>
The medium (8) uses IMDM as a basal medium, includes the medium additive (3) as the medium additive, and includes the differentiation induction regulator (4) as the differentiation induction regulator. Depending on the above, other components are further included.
The medium (8) is preferably used as a medium in which an estrogen-like agent is added to the one-part differentiation induction medium in the second cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. be able to.
As a preferable aspect of the culture medium (8), the first culture medium for cardiomyocyte induction treatment (2) may be mentioned.
＜培地（９）＞
　前記培地（９）は、基礎培地として、ＩＭＤＭを用い、前記培地添加剤として、前記培地添加剤（２）を含み、前記分化誘導調節剤として、前記分化誘導調節剤（５）を含み、必要に応じて更にその他の成分を含む。
　前記培地（９）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理と、前記第２の心筋細胞誘導処理との間の心筋細胞誘導処理における第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質を加えた培地として好適に用いることができる。
　前記培地（９）の好ましい態様としては、上記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地（１）が挙げられる。 <Medium (9)>
The medium (9) uses IMDM as a basal medium, includes the medium additive (2) as the medium additive, and includes the differentiation induction regulator (5) as the differentiation induction regulator. Depending on the above, other components are further included.
The medium (9) is a second cardiomyocyte induction process between the first cardiomyocyte induction process of the above-described method for inducing differentiation of cardiomyocytes from the pluripotent stem cells and the second cardiomyocyte induction process. Can be suitably used as a medium obtained by adding a Wnt signal inhibitory substance and an estrogen-like agent to the differentiation induction medium.
A preferred embodiment of the medium (9) includes a medium for cardiomyocyte induction treatment (1) between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment.
＜培地（１０）＞
　前記培地（１０）は、基礎培地として、ＩＭＤＭを用い、前記培地添加剤として、前記培地添加剤（３）を含み、前記分化誘導調節剤として、前記分化誘導調節剤（５）を含み、必要に応じて更にその他の成分を含む。
　前記培地（１０）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理と、前記第２の心筋細胞誘導処理との間の心筋細胞誘導処理における一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質を加えた培地として好適に用いることができる。
　前記培地（１０）の好ましい態様としては、上記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地（２）が挙げられる。 <Medium (10)>
The medium (10) uses IMDM as a basal medium, includes the medium additive (3) as the medium additive, and includes the differentiation induction regulator (5) as the differentiation induction regulator. Depending on the above, other components are further included.
The medium (10) is a solution in the cardiomyocyte induction process between the first cardiomyocyte induction process of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above and the second cardiomyocyte induction process. It can be suitably used as a medium obtained by adding a Wnt signal inhibitory substance and an estrogen-like substance to a formula differentiation-inducing medium.
A preferable embodiment of the culture medium (10) includes a culture medium for cardiomyocyte induction treatment (2) between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment.
（培地作製用キット）
　本発明の培地作製用キットは、多能性幹細胞から心筋細胞を分化誘導するための培地作製用キットであって、本発明の培地添加剤と、本発明の分化誘導調節剤と、基礎培地とを少なくとも含み、必要に応じて更にその他の構成を含む。
　前記培地作製用キットは、本発明の多能性幹細胞から心筋細胞を分化誘導する方法に好適に用いることができる。
　前記その他の構成としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、培地を作製するための方法を教示する説明書などが挙げられる。 (Medium preparation kit)
The medium preparation kit of the present invention is a medium preparation kit for inducing differentiation of cardiomyocytes from pluripotent stem cells, the medium additive of the present invention, the differentiation induction regulator of the present invention, a basal medium, And at least further other configurations as necessary.
The medium preparation kit can be suitably used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
There is no restriction | limiting in particular as said other structure, According to the objective, it can select suitably, For example, the manual etc. which teach the method for producing a culture medium are mentioned.
　前記培地作製用キットにおける基礎培地は、ＤＭＥＭ／Ｆ１２、ＤＭＥＭ、ＩＭＤＭ、ＲＰＭＩ１６４０培地、及びαＭＥＭ培地からなる群から選択される少なくとも１種である。
　前記ＤＭＥＭは、高グルコースであってもよいし、低グルコースであってもよい。 The basal medium in the medium preparation kit is at least one selected from the group consisting of DMEM / F12, DMEM, IMDM, RPMI1640 medium, and αMEM medium.
The DMEM may be high glucose or low glucose.
　前記培地作製用キットの態様としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができるが、下記培地作製用キット（１）～（１０）の少なくともいずれかの態様が好ましい。 The form of the medium preparation kit is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. At least one of the following medium preparation kits (1) to (10) can be used. Either embodiment is preferred.
＜培地作製用キット（１）＞
　前記培地作製用キット（１）は、前記培地添加剤（１）と、前記分化誘導調節剤（１）と、基礎培地として、ＤＭＥＭ／Ｆ１２、ＤＭＥＭ、ＩＭＤＭ、ＲＰＭＩ１６４０培地、及びαＭＥＭ培地からなる群から選択される少なくとも１種とを含み、必要に応じて更にその他の構成を含む。
　前記基礎培地の中でも、ＤＭＥＭ／Ｆ１２が好ましい。
　前記培地作製用キット（１）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の胚様体形成工程における第１の分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地の作製用キットとして好適に用いることができる。
　前記培地作製用キット（１）により作製する培地の好ましい態様としては、上記胚様体形成工程用培地（１）が挙げられる。 <Medium preparation kit (1)>
The medium preparation kit (1) comprises the medium additive (1), the differentiation-inducing regulator (1), and DMEM / F12, DMEM, IMDM, RPMI1640 medium, and αMEM medium as a basal medium. At least one selected from the above, and further includes other configurations as necessary.
Among the basal media, DMEM / F12 is preferable.
The medium preparation kit (1) is a medium preparation kit obtained by adding a ROCK inhibitor to the first differentiation induction medium in the embryoid body formation step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. Can be suitably used.
As a preferable aspect of the culture medium prepared by the culture medium preparation kit (1), the embryoid body formation process culture medium (1) may be mentioned.
＜培地作製用キット（２）＞
　前記培地作製用キット（２）は、前記培地添加剤（３）と、前記分化誘導調節剤（１）と、基礎培地として、ＩＭＤＭとを含み、必要に応じて更にその他の構成を含む。
　前記培地作製用キット（２）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の胚様体形成工程における一液式分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地の作製用キットとして好適に用いることができる。
　前記培地作製用キット（２）により作製する培地の好ましい態様としては、上記胚様体形成工程用培地（２）が挙げられる。 <Medium preparation kit (2)>
The medium preparation kit (2) includes the medium additive (3), the differentiation induction regulator (1), and IMDM as a basal medium, and further includes other components as necessary.
The medium preparation kit (2) is a medium preparation kit in which a ROCK inhibitor is added to the one-part differentiation induction medium in the embryoid body formation step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. Can be suitably used.
As a preferred embodiment of the medium prepared by the medium preparation kit (2), the above-mentioned medium for embryoid body formation step (2) can be mentioned.
＜培地作製用キット（３）＞
　前記培地作製用キット（３）は、前記培地添加剤（２）と、前記分化誘導調節剤（２）と、基礎培地として、ＩＭＤＭとを含み、必要に応じて更にその他の構成を含む。
　前記培地作製用キット（３）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の中胚葉誘導工程における第２の分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地の作製用キットとして好適に用いることができる。
　前記培地作製用キット（３）により作製する培地の好ましい態様としては、上記中胚葉誘導工程用培地（１）が挙げられる。 <Medium preparation kit (3)>
The medium preparation kit (3) includes the medium additive (2), the differentiation induction regulator (2), and IMDM as a basal medium, and further includes other components as necessary.
The medium preparation kit (3) is for preparing a medium in which a Wnt signal activator is added to the second differentiation induction medium in the mesoderm induction step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a kit.
As a preferable embodiment of the medium prepared by the medium preparation kit (3), the medium for mesoderm induction process (1) can be mentioned.
＜培地作製用キット（４）＞
　前記培地作製用キット（４）は、前記培地添加剤（３）と、前記分化誘導調節剤（２）と、基礎培地として、ＩＭＤＭとを含み、必要に応じて更にその他の構成を含む。
　前記培地作製用キット（４）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の中胚葉誘導工程における一液式分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地の作製用キットとして好適に用いることができる。
　前記培地作製用キット（４）により作製する培地の好ましい態様としては、上記中胚葉誘導工程用培地（２）が挙げられる。 <Medium preparation kit (4)>
The medium preparation kit (4) includes the medium additive (3), the differentiation induction regulator (2), and IMDM as a basal medium, and further includes other components as necessary.
The medium preparation kit (4) is for preparation of a medium in which a Wnt signal activator is added to a one-part differentiation induction medium in the mesoderm induction step of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a kit.
As a preferable aspect of the medium prepared by the medium preparation kit (4), the medium for mesoderm induction process (2) can be mentioned.
＜培地作製用キット（５）＞
　前記培地作製用キット（５）は、前記培地添加剤（２）と、前記分化誘導調節剤（３）と、基礎培地として、ＩＭＤＭとを含み、必要に応じて更にその他の構成を含む。
　前記培地作製用キット（５）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理における第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地の作製用キットとして好適に用いることができる。
　前記培地作製用キット（５）により作製する培地の好ましい態様としては、上記第１の心筋細胞誘導処理用培地（１）が挙げられる。 <Medium preparation kit (5)>
The medium preparation kit (5) includes the medium additive (2), the differentiation induction regulator (3), and IMDM as a basal medium, and further includes other components as necessary.
The medium preparation kit (5) includes a Wnt signal inhibitor, a TGF-β signal, in the second differentiation induction medium in the first cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a kit for preparing a medium containing an inhibitor and an estrogen-like agent.
A preferred embodiment of the medium prepared by the medium preparation kit (5) includes the first cardiomyocyte induction treatment medium (1).
＜培地作製用キット（６）＞
　前記培地作製用キット（６）は、前記培地添加剤（３）と、前記分化誘導調節剤（３）と、基礎培地として、ＩＭＤＭとを含み、必要に応じて更にその他の構成を含む。
　前記培地作製用キット（６）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理における一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地の作製用キットとして好適に用いることができる。
　前記培地作製用キット（６）により作製する培地の好ましい態様としては、上記第１の心筋細胞誘導処理用培地（２）が挙げられる。 <Medium preparation kit (6)>
The medium preparation kit (6) includes the medium additive (3), the differentiation induction regulator (3), and IMDM as a basal medium, and further includes other components as necessary.
The medium preparation kit (6) includes a Wnt signal suppressor, a TGF-β signal, in a one-part differentiation induction medium in the first cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a kit for preparing a medium containing an inhibitor and an estrogen-like agent.
A preferred embodiment of the medium prepared by the medium preparation kit (6) is the first cardiomyocyte induction treatment medium (2).
＜培地作製用キット（７）＞
　前記培地作製用キット（７）は、前記培地添加剤（２）と、前記分化誘導調節剤（４）と、基礎培地として、ＩＭＤＭとを含み、必要に応じて更にその他の構成を含む。
　前記培地作製用キット（７）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第２の心筋細胞誘導処理における第２の分化誘導培地に、エストロゲン様作用物質を加えた培地の作製用キットとして好適に用いることができる。
　前記培地作製用キット（７）により作製する培地の好ましい態様としては、上記第２の心筋細胞誘導処理用培地（１）が挙げられる。 <Medium preparation kit (7)>
The medium preparation kit (7) includes the medium additive (2), the differentiation induction regulator (4), and IMDM as a basal medium, and further includes other components as necessary.
The medium preparation kit (7) comprises a medium obtained by adding an estrogen-like agent to the second differentiation inducing medium in the second cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a preparation kit.
A preferred embodiment of the medium prepared by the medium preparation kit (7) is the second cardiomyocyte induction treatment medium (1).
＜培地作製用キット（８）＞
　前記培地作製用キット（８）は、前記培地添加剤（３）と、前記分化誘導調節剤（４）と、基礎培地として、ＩＭＤＭとを含み、必要に応じて更にその他の構成を含む。
　前記培地作製用キット（８）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第２の心筋細胞誘導処理における一液式分化誘導培地に、エストロゲン様作用物質を加えた培地の作製用キットとして好適に用いることができる。
　前記培地作製用キット（８）により作製する培地の好ましい態様としては、上記第１の心筋細胞誘導処理用培地（２）が挙げられる。 <Medium preparation kit (8)>
The medium preparation kit (8) includes the medium additive (3), the differentiation induction regulator (4), and IMDM as a basal medium, and further includes other components as necessary.
The medium preparation kit (8) comprises a medium obtained by adding an estrogen-like agent to a one-part differentiation induction medium in the second cardiomyocyte induction treatment of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. It can be suitably used as a preparation kit.
A preferred embodiment of the medium prepared by the medium preparation kit (8) includes the first cardiomyocyte induction treatment medium (2).
＜培地作製用キット（９）＞
　前記培地作製用キット（９）は、前記培地添加剤（２）と、前記分化誘導調節剤（５）と、基礎培地として、ＩＭＤＭとを含み、必要に応じて更にその他の構成を含む。
　前記培地作製用キット（９）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理と、前記第２の心筋細胞誘導処理との間の心筋細胞誘導処理における第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質を加えた培地の作製用キットとして好適に用いることができる。
　前記培地作製用キット（９）により作製する培地の好ましい態様としては、上記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地（１）が挙げられる。 <Medium preparation kit (9)>
The medium preparation kit (9) includes the medium additive (2), the differentiation induction regulator (5), and IMDM as a basal medium, and further includes other components as necessary.
The medium preparation kit (9) is a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells described above. Can be suitably used as a kit for preparing a medium in which a Wnt signal inhibitory substance and an estrogen-like agent are added to the second differentiation-inducing medium.
A preferred embodiment of the medium prepared by the medium preparation kit (9) is a medium for cardiomyocyte induction treatment (1) between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment. It is done.
＜培地作製用キット（１０）＞
　前記培地作製用キット（１０）は、前記培地添加剤（３）と、前記分化誘導調節剤（５）と、基礎培地として、ＩＭＤＭとを含み、必要に応じて更にその他の構成を含む。
　前記培地作製用キット（１０）は、上述した多能性幹細胞から心筋細胞を分化誘導する方法の第１の心筋細胞誘導処理と、前記第２の心筋細胞誘導処理との間の心筋細胞誘導処理における一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質を加えた培地の作製用キットとして好適に用いることができる。
　前記培地作製用キット（１０）により作製する培地の好ましい態様としては、上記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地（２）が挙げられる。 <Medium preparation kit (10)>
The medium preparation kit (10) includes the medium additive (3), the differentiation induction regulator (5), and IMDM as a basal medium, and further includes other components as necessary.
The medium preparation kit (10) is a cardiomyocyte induction process between the first cardiomyocyte induction process of the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells and the second cardiomyocyte induction process. Can be suitably used as a kit for preparing a medium in which a Wnt signal inhibitory substance and an estrogen-like agent are added to the one-part differentiation induction medium.
A preferred embodiment of the medium prepared by the medium preparation kit (10) is a cardiomyocyte induction treatment medium (2) between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment. It is done.
（多能性幹細胞から心筋細胞を分化誘導するためのキット）
　本発明の多能性幹細胞から心筋細胞を分化誘導するためのキットは、本発明の培地、及び本発明の培地作製用キットの少なくともいずれかを含み、必要に応じて更にその他の構成を含む。
　前記多能性幹細胞から心筋細胞を分化誘導するためのキットは、本発明の多能性幹細胞から心筋細胞を分化誘導する方法に好適に用いることができる。 (Kit for inducing differentiation of cardiomyocytes from pluripotent stem cells)
The kit for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention includes at least one of the medium of the present invention and the medium preparation kit of the present invention, and further includes other components as necessary.
The kit for inducing differentiation of cardiomyocytes from the pluripotent stem cells can be suitably used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
　前記培地、及び前記培地作製用キットは、どちらか一方を含んでいてもよいし、両者を含んでいてもよい。 The medium and the medium preparation kit may contain either one or both.
　前記培地としては、特に制限はなく、目的に応じて適宜選択することができるが、上述した本発明の培地の項目に記載した前記培地（１）～（１０）の少なくともいずれかを含むことが好ましく、前記培地（１）、（３）、（５）、及び（７）を含むことがより好ましく、前記培地（１）、（３）、（５）、（７）、及び（９）を含むことが特に好ましい。 The medium is not particularly limited and may be appropriately selected depending on the intended purpose. However, the medium includes at least one of the mediums (1) to (10) described in the item of the medium of the present invention described above. Preferably, the medium (1), (3), (5), and (7) are more preferably included, and the medium (1), (3), (5), (7), and (9) are included. It is particularly preferable to include it.
　前記培地作製用キットとしては、特に制限はなく、目的に応じて適宜選択することができるが、上述した本発明の培地作製用キットの項目に記載した前記培地作製用キット（１）～（１０）の少なくともいずれかを含むことが好ましく、前記培地作製用キット（１）、（３）、（５）、及び（７）を含むことがより好ましく、前記培地作製用キット（１）、（３）、（５）、（７）、及び（９）を含むことが特に好ましい。 The medium preparation kit is not particularly limited and may be appropriately selected depending on the intended purpose. However, the medium preparation kits (1) to (10) described in the item of the medium preparation kit of the present invention described above. ), More preferably the medium preparation kits (1), (3), (5), and (7), and the medium preparation kits (1), (3 ), (5), (7) and (9) are particularly preferred.
　前記その他の構成としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、培養皿、本発明の多能性幹細胞から心筋細胞を分化誘導する方法を教示する説明書などが挙げられる。これらの中でも、本発明の多能性幹細胞から心筋細胞を分化誘導する方法を教示する説明書を含むことが好ましい。 The other configuration is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include a culture dish and instructions for teaching a method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention. Can be mentioned. Among these, it is preferable to include instructions that teach a method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention.
　以下に試験例を挙げて本発明を具体的に説明するが、本発明はこれらの試験例に何ら限定されるものではない。 The present invention will be specifically described below with reference to test examples, but the present invention is not limited to these test examples.
（試験例１）
　中胚葉誘導工程における培地に添加するＷｎｔシグナル活性化物質の検討を以下のようにして行った。 (Test Example 1)
The Wnt signal activator added to the medium in the mesoderm induction process was examined as follows.
＜多能性幹細胞＞
　多能性幹細胞として、国立大学法人　東京大学医学部附属病院にて樹立した２つのヒトｉＰＳ細胞（クローン１、クローン２）を用いた。 <Pluripotent stem cells>
Two human iPS cells (clone 1 and clone 2) established at the University of Tokyo Hospital were used as pluripotent stem cells.
＜胚様体形成工程＞
　常法に則って未分化状態で維持されている前記ヒトｉＰＳ細胞をコロニー状に解離し、下記培地（以下、「胚様体形成工程用培養液」と称することがある）に懸濁した後、低接着加工を行った培養皿（Ｃｏｒｎｉｎｇ超低接着加工表面（コーニング社製））で浮遊培養（培養条件：３７℃、５％ＣＯ２、５％Ｏ２）を開始し（０日目）、１日間培養することで、前記ヒトｉＰＳ細胞の凝集体（胚様体（Ｅｍｂｒｙｏｉｄ　Ｂｏｄｙ：ＥＢ）を形成させた。
－胚様体形成工程用培養液－
　基礎培地として、ＤＭＥＭ／Ｆ１２（ナカライテスク株式会社製）を用い、前記基礎培地に、分化誘導調節剤として、ＲＯＣＫ阻害剤であるＹ２７６３２（シグマ社製、５μＭ）、培地添加剤として、下記表４－１～表４－２に記載の濃度となるように各成分を添加し、胚様体形成工程用培養液とした。 <Embryoid body formation process>
After the human iPS cells maintained in an undifferentiated state according to a conventional method are dissociated into colonies and suspended in the following medium (hereinafter, sometimes referred to as “culture medium for embryoid body formation process”) The suspension culture (culture conditions: 37 ° C., 5% CO 2 , 5% O 2 ) was started on the culture dish (Corning ultra-low adhesion processed surface (manufactured by Corning)) subjected to the low adhesion process (Day 0) By culturing for 1 day, the human iPS cell aggregate (embryoid body (EB)) was formed.
-Culture fluid for embryoid body formation process-
As a basal medium, DMEM / F12 (manufactured by Nacalai Tesque Co., Ltd.) was used. As a differentiation induction regulator, Y27632 (manufactured by Sigma, 5 μM) as a differentiation induction regulator, and as a medium additive, Table 4 below. Each component was added so that the concentrations described in -1 to Table 4-2 were obtained, and used as a culture solution for embryoid body formation process.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
＜洗浄工程＞
　前記胚様体を培養液ごと遠心管に回収し、静置して前記胚様体を沈殿させた後、上清を除去した。次いで、ＩＭＤＭを加え、再度静置して前記胚様体を沈殿させた後、上清を除去した。この作業を２回繰り返し行った。 <Washing process>
The embryoid bodies were collected in a centrifuge tube together with the culture solution, and allowed to stand to precipitate the embryoid bodies, and then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
＜中胚葉誘導工程＞
　前記洗浄工程後、下記培地（以下、「中胚葉誘導工程用培養液」と称することがある）を加え、前記培養皿で、浮遊培養を再開した（胚様体形成工程開始１日目～３日目）。
－中胚葉誘導工程用培養液－
　基礎培地として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）を用い、前記基礎培地に、分化誘導調節剤として、Ｗｎｔシグナル活性化物質であるＣＨＩＲ９９０２１（ミリポア社製、１μＭ、３μＭ、５μＭ、又は７μＭ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加し、中胚葉誘導工程用培養液とした。
　なお、ネガティブコントロールとして、Ｗｎｔシグナル活性化物質を含まない培養液も調製した。 <Mesodermal induction process>
After the washing step, the following medium (hereinafter sometimes referred to as “culture medium for mesoderm induction process”) was added, and suspension culture was resumed in the culture dish (embryoid body formation process starting day 1 to 3 Day).
-Medium for mesoderm induction process-
StemPro (registered trademark) 34 (manufactured by Invitrogen) is used as the basal medium, and CHIR99021 (manufactured by Millipore, 1 μM, 3 μM, 5 μM, or 7 μM) is used as the differentiation induction regulator in the basal medium. ), L-glutamine (manufactured by Invitrogen, 2 mM), transferrin (manufactured by Sigma, 150 μg / mL), ascorbic acid (manufactured by Sigma, 50 μg / mL), and 1-thioglycerol (manufactured by Sigma) , 50 μg / mL) was added to obtain a culture solution for the mesoderm induction process.
As a negative control, a culture solution containing no Wnt signal activator was also prepared.
＜洗浄工程＞
　前記中胚葉誘導後の胚様体を培養液ごと遠心管に回収し、静置して前記胚様体を沈殿させた後、上清を除去した。次いで、ＩＭＤＭを加え、再度静置して前記胚様体を沈殿させた後、上清を除去した。この作業を２回繰り返し行った。 <Washing process>
The embryoid body after the mesoderm induction was collected in a centrifuge tube together with the culture solution, allowed to stand to precipitate the embryoid body, and then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　前記洗浄工程後、下記培地（以下、「第１の心筋細胞誘導処理用培養液」と称することがある）を加え、前記培養皿で、浮遊培養を再開した（胚様体形成工程開始３日目～６日目）。
－第１の心筋細胞誘導処理用培養液－
　基礎培地として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）を用い、前記基礎培地に、分化誘導調節剤として、ＩＷＰ２（シグマ社製、５μＭ）、ＳＢ４３１５４２（シグマ社製、５μＭ）及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加し、第１の心筋細胞誘導処理用培養液とした。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
After the washing step, the following medium (hereinafter also referred to as “first culture medium for cardiomyocyte induction treatment”) was added, and suspension culture was resumed in the culture dish (embryoid body formation process start day 3 Eyes to 6th).
-First culture solution for cardiomyocyte induction treatment-
StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, and IWP2 (manufactured by Sigma, 5 μM), SB431542 (manufactured by Sigma, 5 μM) and VEGF (R & D) were used as the differentiation induction regulators in the basal medium. 10 ng / mL), L-glutamine (manufactured by Invitrogen, 2 mM), transferrin (manufactured by Sigma, 150 μg / mL), ascorbic acid (manufactured by Sigma, 50 μg / mL), and 1- Thioglycerol (manufactured by Sigma, 50 μg / mL) was added to obtain a first culture solution for cardiomyocyte induction treatment.
＜＜洗浄処理＞＞
　前記第１の心筋細胞誘導処理後の胚様体を培養液ごと遠心管に回収し、静置して前記胚様体を沈殿させた後、上清を除去した。次いで、ＩＭＤＭを加え、再度静置して前記胚様体を沈殿させた後、上清を除去した。この作業を２回繰り返し行った。 << Cleaning process >>
The embryoid body after the first cardiomyocyte induction treatment was collected in a centrifuge tube together with the culture solution, allowed to stand to precipitate the embryoid body, and then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　前記洗浄処理後、下記培地（以下、「第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液」と称することがある）を加え、前記培養皿で、浮遊培養を再開した（胚様体形成工程開始６日目～８日目）。
－第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液－
　基礎培地として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）を用い、前記基礎培地に、分化誘導調節剤として、ＩＷＰ２（シグマ社製、５μＭ）、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加し、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液とした。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
After the washing treatment, the following medium (hereinafter sometimes referred to as “a culture solution for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment”) is added, and the culture is performed. Suspension culture was resumed in the dish (embryoid body formation process from day 6 to day 8).
-Culture medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment-
StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, and IWP2 (manufactured by Sigma, 5 μM) and VEGF (manufactured by R & D, 10 ng / mL) as differentiation regulators. As media additives, L-glutamine (manufactured by Invitrogen, 2 mM), transferrin (manufactured by Sigma, 150 μg / mL), ascorbic acid (manufactured by Sigma, 50 μg / mL), and 1-thioglycerol (manufactured by Sigma, 50 μg) / ML) to obtain a culture solution for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment.
＜＜洗浄処理＞＞
　前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を培養液ごと遠心管に回収し、静置して前記胚様体を沈殿させた後、上清を除去した。次いで、ＩＭＤＭを加え、再度静置して前記胚様体を沈殿させた後、上清を除去した。この作業を２回繰り返し行った。 << Cleaning process >>
The embryoid body after the cardiomyocyte inducing process between the first cardiomyocyte inducing process and the second cardiomyocyte inducing process is collected in a centrifuge tube together with the culture solution and allowed to stand to precipitate the embryoid body And then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
＜＜第２の心筋細胞誘導処理＞＞
　前記洗浄処理後、下記培地（以下、「第２の心筋細胞誘導処理用培養液」と称することがある）を加え、前記培養皿で、浮遊培養を再開した（胚様体形成工程開始８日目～１６日目）。
－第２の心筋細胞誘導処理用培養液－
　基礎培地として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）を用い、前記基礎培地に、分化誘導調節剤として、ＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、及びｂＦＧＦ（和光純薬工業株式会社製、１０ｎｇ／ｍＬ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加し、第２の心筋細胞誘導処理用培養液とした。 << Second cardiomyocyte induction process >>
After the washing treatment, the following medium (hereinafter sometimes referred to as “second culture medium for cardiomyocyte induction treatment”) was added, and suspension culture was resumed in the culture dish (embryoid body formation process start day 8 Eyes to 16th day).
-Second culture solution for cardiomyocyte induction treatment-
StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, and VEGF (manufactured by R & D, 10 ng / mL) and bFGF (manufactured by Wako Pure Chemical Industries, Ltd.) as the differentiation induction regulator. 10 ng / mL), L-glutamine (manufactured by Invitrogen, 2 mM), transferrin (manufactured by Sigma, 150 μg / mL), ascorbic acid (manufactured by Sigma, 50 μg / mL), and 1-thioglycerol (Manufactured by Sigma, 50 μg / mL) was added to obtain a second culture solution for cardiomyocyte induction treatment.
＜評価＞
　前記第２の心筋細胞誘導処理後の胚様体を倒立顕微鏡（カール・ツァイス社製）により観察し、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図５に示す。 <Evaluation>
The embryoid body after the second cardiomyocyte induction treatment is observed with an inverted microscope (manufactured by Carl Zeiss), and the proportion of beating embryoid bodies in 50 arbitrarily selected embryoid bodies is obtained. It was. The results are shown in FIG.
　図５中、ＣＨＩＲは、ＣＨＩＲ９９０２１を表し、各項目における左側（黒色）は、クローン１、右側（灰色）は、クローン２の結果を表す。
　図５の結果から、中胚葉誘導工程における培地にＷｎｔシグナル活性化物質を添加することにより、拍動している胚様体が得られることが示された。 In FIG. 5, CHIR represents CHIR99021, and the left side (black) in each item represents the result of clone 1 and the right side (gray) represents the result of clone 2.
From the results of FIG. 5, it was shown that a beating embryoid body can be obtained by adding a Wnt signal activator to the medium in the mesoderm induction step.
（試験例２）
　中胚葉誘導工程における培地に添加するＣＨＩＲ９９０２１の至適濃度の検討を以下のようにして行った。 (Test Example 2)
The optimum concentration of CHIR99021 added to the medium in the mesoderm induction process was examined as follows.
＜多能性幹細胞＞
　前記試験例１と同様に、国立大学法人　東京大学医学部附属病院にて樹立した２つのヒトｉＰＳ細胞（クローン１、クローン２）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 1, two human iPS cells (clone 1 and clone 2) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　前記試験例１と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
In the same manner as in Test Example 1, an embryoid body formation step was performed.
＜洗浄工程＞
　前記試験例１と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 1.
＜中胚葉誘導工程＞
　前記試験例１において、Ｗｎｔシグナル活性化物質をＣＨＩＲ９９０２１（濃度：１μＭ、３μＭ、３．５μＭ、４μＭ、４．５μＭ、５μＭ、５．５μＭ、６μＭ、又は７μＭ）とした以外は、前記試験例１と同様にして、中胚葉誘導工程を行った。 <Mesodermal induction process>
Test Example 1 except that the Wnt signal activator in Test Example 1 was CHIR99021 (concentration: 1 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, or 7 μM). In the same manner, a mesoderm induction process was performed.
＜洗浄工程＞
　前記試験例１と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 1, the embryoid body after the mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　前記試験例１と同様にして、第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
In the same manner as in Test Example 1, the first cardiomyocyte induction treatment was performed.
＜＜洗浄処理＞＞
　前記試験例１と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 1, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　前記試験例１と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
In the same manner as in Test Example 1, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed.
＜＜洗浄処理＞＞
　前記試験例１と同様にして、前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 1, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　前記試験例１と同様にして、第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
In the same manner as in Test Example 1, a second cardiomyocyte induction treatment was performed.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図６及び図７に示す。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIGS.
　図６は、クローン１の結果を示し、図７は、クローン２の結果を示す。
　図６及び図７の結果から、ＣＨＩＲ９９０２１の濃度が３μＭ以上６μＭ以下の範囲において、拍動している胚様体が得られることがわかった。
　また、図６の結果から、クローン１におけるＣＨＩＲ９９０２１の至適濃度は、３．９１μＭであり、図７の結果から、クローン２におけるＣＨＩＲ９９０２１の至適濃度は、４．３１μＭであることがわかった。 FIG. 6 shows the results of clone 1 and FIG. 7 shows the results of clone 2.
From the results of FIG. 6 and FIG. 7, it was found that a beating embryoid body was obtained when the concentration of CHIR99021 was in the range of 3 μM to 6 μM.
Further, from the results of FIG. 6, it was found that the optimum concentration of CHIR99021 in clone 1 was 3.91 μM, and from the results of FIG. 7, the optimum concentration of CHIR99021 in clone 2 was 4.31 μM.
（試験例３）
　第１の心筋細胞誘導処理、及び第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理における培地において添加するＷｎｔシグナル抑制物質の検討を以下のようにして行った。 (Test Example 3)
The examination of the Wnt signal inhibitor added in the medium in the first cardiomyocyte induction treatment and the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is as follows. went.
＜多能性幹細胞＞
　前記試験例１と同様に、国立大学法人　東京大学医学部附属病院にて樹立した２つのヒトｉＰＳ細胞（クローン１、クローン２）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 1, two human iPS cells (clone 1 and clone 2) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　前記試験例１と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
In the same manner as in Test Example 1, an embryoid body formation step was performed.
＜洗浄工程＞
　前記試験例１と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 1.
＜中胚葉誘導工程＞
　前記試験例１において、Ｗｎｔシグナル活性化物質をＣＨＩＲ９９０２１（濃度：４μＭ）とした以外は、前記試験例１と同様にして、中胚葉誘導工程を行った。 <Mesodermal induction process>
A mesoderm induction step was performed in the same manner as in Test Example 1 except that the Wnt signal activator in Test Example 1 was changed to CHIR99021 (concentration: 4 μM).
＜洗浄工程＞
　前記試験例１と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 1, the embryoid body after the mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　前記試験例１において、第１の心筋細胞誘導処理用培養液における分化誘導調節剤を、下記Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤であるＳＢ４３１５４２（シグマ社製、５μＭ）、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）に代えた以外は、前記試験例１と同様にして、第１の心筋細胞誘導処理を行った。
　なお、ネガティブコントロールとして、Ｗｎｔシグナル抑制物質に代えてＤＭＳＯ（シグマ社製）を添加した培養液も調製した。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
In Test Example 1, the differentiation-inducing regulator in the first culture solution for cardiomyocyte induction treatment was the following Wnt signal inhibitory substance, SB431542 (SIGMA, 5 μM) as a TGF-β signal inhibitor, and VEGF (R & D). The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 1 except that the product was changed to 10 ng / mL.
As a negative control, a culture solution to which DMSO (manufactured by Sigma) was added instead of the Wnt signal suppressing substance was also prepared.
－－Ｗｎｔシグナル抑制物質－－
　・　ＩＷＰ２（シグマ社製）
　　　濃度：１μＭ、２μＭ、３μＭ、４μＭ、５μＭ、６μＭ、又は１０μＭ
　・　ＩＷＲ１（シグマ社製）
　　　濃度：１μＭ、２μＭ、５μＭ、１０μＭ、又は１５μＭ
　・　ＸＡＶ９３９（シグマ社製）
　　　濃度：１μＭ、２μＭ、５μＭ、１０μＭ、１５μＭ、又は２０μＭ
　・　ＫＹ０２１１１（シグマ社製）
　　　濃度：１μＭ、２μＭ、５μＭ、１０μＭ、２０μＭ、又は４０μＭ --Wnt signal suppressor--
・ IWP2 (manufactured by Sigma)
Concentration: 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, or 10 μM
・ IWR1 (manufactured by Sigma)
Concentration: 1 μM, 2 μM, 5 μM, 10 μM, or 15 μM
・ XAV939 (manufactured by Sigma)
Concentration: 1 μM, 2 μM, 5 μM, 10 μM, 15 μM, or 20 μM
・ KY02111 (manufactured by Sigma)
Concentration: 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, or 40 μM
＜＜洗浄処理＞＞
　前記試験例１と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 1, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　前記試験例１において、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液における分化誘導調節剤を、前記第１の心筋細胞誘導処理用培養液に用いたＷｎｔシグナル抑制物質、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）に代えた以外は、前記試験例１と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。
　なお、ネガティブコントロールとして、Ｗｎｔシグナル抑制物質に代えてＤＭＳＯ（シグマ社製）を添加した培養液も調製した。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
In Test Example 1, the differentiation-inducing regulator in the culture solution for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is used as the culture for the first cardiomyocyte induction treatment. The first cardiomyocyte induction treatment and the second cardiomyocyte induction were carried out in the same manner as in Test Example 1 except that the Wnt signal inhibitory substance and VEGF (manufactured by R & D, 10 ng / mL) were used. Cardiomyocyte induction treatment between treatments was performed.
As a negative control, a culture solution to which DMSO (manufactured by Sigma) was added instead of the Wnt signal suppressing substance was also prepared.
＜＜洗浄処理＞＞
　前記試験例１と同様にして、前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 1, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　前記試験例１と同様にして、第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
In the same manner as in Test Example 1, a second cardiomyocyte induction treatment was performed.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図８に示す。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG.
　図８中、ｎｅｇａ　ｃｏｎ（ＤＭＳＯ）は、ネガティブコントロールを表し、各項目における左側（黒色）は、クローン１、右側（灰色）は、クローン２の結果を表す。
　図８の結果から、いずれのＷｎｔシグナル抑制物質を用いた場合にも、拍動している胚様体が得られることが示された。これらの中でも、ＩＷＰ２が低濃度帯から最も安定して分化誘導能を発揮した。 In FIG. 8, negative con (DMSO) represents a negative control. In each item, the left side (black) represents the result of clone 1 and the right side (gray) represents the result of clone 2.
From the results in FIG. 8, it was shown that a pulsating embryoid body can be obtained when any Wnt signal inhibitor is used. Among these, IWP2 exhibited the differentiation induction ability most stably from the low concentration zone.
（試験例４）
　第１の心筋細胞誘導処理、及び第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理における培地に添加するＩＷＰ２の至適濃度の検討を以下のようにして行った。 (Test Example 4)
The examination of the optimum concentration of IWP2 added to the medium in the first cardiomyocyte induction treatment and the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is as follows. I went.
＜多能性幹細胞＞
　前記試験例１と同様に、国立大学法人　東京大学医学部附属病院にて樹立した２つのヒトｉＰＳ細胞（クローン１、クローン２）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 1, two human iPS cells (clone 1 and clone 2) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　前記試験例１と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
In the same manner as in Test Example 1, an embryoid body formation step was performed.
＜洗浄工程＞
　前記試験例１と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 1.
＜中胚葉誘導工程＞
　前記試験例３と同様にして、中胚葉誘導工程を行った。 <Mesodermal induction process>
In the same manner as in Test Example 3, a mesoderm induction process was performed.
＜洗浄工程＞
　前記試験例１と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 1, the embryoid body after the mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　前記試験例３において、Ｗｎｔシグナル抑制物質をＩＷＰ２（濃度：１μＭ、２μＭ、３μＭ、４μＭ、５μＭ、６μＭ、又は１０μＭ）とした以外は、前記試験例３と同様にして、第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
The first cardiomyocyte induction was performed in the same manner as in Test Example 3 except that the Wnt signal inhibitory substance was IWP2 (concentration: 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, or 10 μM) in Test Example 3. Processed.
＜＜洗浄処理＞＞
　前記試験例１と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 1, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　前記試験例３において、Ｗｎｔシグナル抑制物質をＩＷＰ２（濃度：１μＭ、２μＭ、３μＭ、４μＭ、５μＭ、６μＭ、又は１０μＭ）とした以外は、前記試験例３と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
The first cardiomyocyte induction was performed in the same manner as in Test Example 3 except that the Wnt signal inhibitory substance was IWP2 (concentration: 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, or 10 μM) in Test Example 3. A cardiomyocyte induction process was performed between the treatment and the second cardiomyocyte induction process.
＜＜洗浄処理＞＞
　前記試験例１と同様にして、前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 1, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　前記試験例１と同様にして、第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
In the same manner as in Test Example 1, a second cardiomyocyte induction treatment was performed.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図９及び図１０に示す。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIGS.
　図９は、クローン１の結果を示し、図１０は、クローン２の結果を示す。
　図９及び図１０の結果から、少なくともＩＷＰ２の濃度が１μＭ以上１０μＭ以下の範囲において、拍動している胚様体が得られることがわかった。
　また、図９の結果から、クローン１におけるＩＷＰ２の至適濃度は、６．２２μＭであり、図１０の結果から、クローン２におけるＩＷＰ２の至適濃度は、５．１１μＭであることがわかった。 FIG. 9 shows the results of clone 1 and FIG. 10 shows the results of clone 2.
From the results shown in FIGS. 9 and 10, it was found that a beating embryoid body can be obtained at least in the range where the concentration of IWP2 is 1 μM or more and 10 μM or less.
Further, from the results of FIG. 9, it was found that the optimal concentration of IWP2 in clone 1 was 6.22 μM, and from the results of FIG. 10, the optimal concentration of IWP2 in clone 2 was 5.11 μM.
（試験例５）
　第１の心筋細胞誘導処理、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理、及び第２の心筋細胞誘導処理における培地に添加するＶＥＧＦに代わる物質の検討を以下のようにして行った。 (Test Example 5)
Substance in place of VEGF added to the medium in the first cardiomyocyte induction process, the cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process, and the second cardiomyocyte induction process Was examined as follows.
＜多能性幹細胞＞
　多能性幹細胞として、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
As pluripotent stem cells, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　前記試験例１と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
In the same manner as in Test Example 1, an embryoid body formation step was performed.
＜洗浄工程＞
　前記試験例１と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 1.
＜中胚葉誘導工程＞
　中胚葉誘導工程用培地として下記中胚葉誘導工程用培養液を用いた以外は、前記試験例１と同様にして、中胚葉誘導工程を行った。
－中胚葉誘導工程用培養液－
　基礎培地として、ＩＭＤＭ（ナカライテスク株式会社製）を用い、前記基礎培地に、分化誘導調節剤として、ＣＨＩＲ９９０２１（ミリポア社製、４μＭ）、培地添加剤として、下記表５－１～表５－２に記載の濃度となるように各成分を添加し、中胚葉誘導工程用培養液とした。 <Mesodermal induction process>
The mesoderm induction step was performed in the same manner as in Test Example 1 except that the following medium for mesoderm induction step was used as the medium for mesoderm induction step.
-Medium for mesoderm induction process-
IMDM (manufactured by Nacalai Tesque Co., Ltd.) was used as the basal medium, CHIR99021 (manufactured by Millipore, 4 μM) as the differentiation induction regulator, and Table 5-1 to Table 5-2 below as the medium additive. Each component was added so that it might become the density | concentration described in 1. It was set as the culture solution for mesoderm induction processes.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
＜洗浄工程＞
　前記試験例１と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 1, the embryoid body after the mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　第１の心筋細胞誘導処理用培地として下記第１の心筋細胞誘導処理用培養液を用いた以外は、前記試験例１と同様にして、第１の心筋細胞誘導処理を行った。
－第１の心筋細胞誘導処理用培養液－
　本試験例５の中胚葉誘導工程用培養液における分化誘導調節剤をＩＷＰ２（シグマ社製、５μＭ）、ＳＢ４３１５４２（シグマ社製、５μＭ）、及び下記候補物質に代えた以外は、前記中胚葉誘導工程用培養液と同様として、第１の心筋細胞誘導処理用培養液とした。
　なお、ネガティブコントロール（（－））として、下記候補物質を含まない培養液も調製した。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 1 except that the following first cardiomyocyte induction treatment culture medium was used as the first cardiomyocyte induction treatment medium.
-First culture solution for cardiomyocyte induction treatment-
The mesoderm induction except that the differentiation induction regulator in the culture solution for the mesodermal induction process of Test Example 5 was replaced with IWP2 (Sigma, 5 μM), SB431542 (Sigma, 5 μM), and the following candidate substances: The first culture solution for cardiomyocyte induction treatment was used in the same manner as the process culture solution.
As a negative control ((−)), a culture solution not containing the following candidate substances was also prepared.
－－候補物質－－
　・　ＶＥＧＦ（Ｒ＆Ｄ社製）
　　　濃度：１０ｎｇ／ｍＬ
　・　レチノイン酸（シグマ社製）
　　　濃度：１ｎＭ、又は１０ｎＭ
　・　エストラジオール（シグマ社製）
　　　濃度：１ｎＭ、１０ｎＭ、１００ｎＭ、又は１０μＭ --Candidate substance--
・ VEGF (R & D)
Concentration: 10 ng / mL
・ Retinoic acid (manufactured by Sigma)
Concentration: 1 nM or 10 nM
・ Estradiol (Sigma)
Concentration: 1 nM, 10 nM, 100 nM, or 10 μM
＜＜洗浄処理＞＞
　前記試験例１と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 1, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地として下記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液を用い、浮遊培養の期間を胚様体形成工程開始６日目～８日目とした以外は、本試験例５の前記第１の心筋細胞誘導処理と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。
－第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液－
　本試験例５の中胚葉誘導工程用培養液における分化誘導調節剤をＩＷＰ２（シグマ社製、５μＭ）、及び下記候補物質に代えた以外は、前記中胚葉誘導工程用培養液と同様として、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液とした。
　なお、ネガティブコントロール（（－））として、下記候補物質を含まない培養液も調製した。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
A cardiomyocyte between the following first cardiomyocyte induction process and the second cardiomyocyte induction process as a medium for cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process In the same manner as in the first cardiomyocyte induction treatment of Test Example 5, except that the culture solution for induction treatment was used and the period of suspension culture was changed to the sixth to eighth days from the start of the embryoid body formation step. A cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed.
-Culture medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment-
As in the culture medium for the mesoderm induction process, except that the differentiation induction regulator in the culture medium for the mesoderm induction process of Test Example 5 was replaced with IWP2 (manufactured by Sigma, 5 μM) and the following candidate substances: The culture solution for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was used.
As a negative control ((−)), a culture solution not containing the following candidate substances was also prepared.
－－候補物質－－
　・　ＶＥＧＦ（Ｒ＆Ｄ社製）
　　　濃度：１０ｎｇ／ｍＬ
　・　レチノイン酸（シグマ社製）
　　　濃度：１ｎＭ、又は１０ｎＭ
　・　エストラジオール（シグマ社製）
　　　濃度：１ｎＭ、１０ｎＭ、１００ｎＭ、又は１０μＭ --Candidate substance--
・ VEGF (R & D)
Concentration: 10 ng / mL
・ Retinoic acid (manufactured by Sigma)
Concentration: 1 nM or 10 nM
・ Estradiol (Sigma)
Concentration: 1 nM, 10 nM, 100 nM, or 10 μM
＜＜洗浄処理＞＞
　前記試験例１と同様にして、前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 1, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　第２の心筋細胞誘導処理用培地として下記第２の心筋細胞誘導処理用培養液を用いた以外は、前記試験例１と同様にして、第２の心筋細胞誘導処理を行った。
－第２の心筋細胞誘導処理用培養液－
　本試験例５の中胚葉誘導工程用培養液における分化誘導調節剤を下記候補物質に代えた以外は、前記中胚葉誘導工程用培養液と同様として、第２の心筋細胞誘導処理用培養液とした。
　なお、ネガティブコントロール（（－））として、下記候補物質を含まない培養液も調製した。 << Second cardiomyocyte induction process >>
A second cardiomyocyte induction treatment was performed in the same manner as in Test Example 1 except that the following second cardiomyocyte induction treatment medium was used as the second cardiomyocyte induction treatment medium.
-Second culture solution for cardiomyocyte induction treatment-
Except that the differentiation-inducing regulator in the culture medium for mesoderm induction process of Test Example 5 was replaced with the following candidate substances, the second culture medium for cardiomyocyte induction treatment was the same as the culture medium for mesoderm induction process, did.
As a negative control ((−)), a culture solution not containing the following candidate substances was also prepared.
－－候補物質－－
　・　ＶＥＧＦ（Ｒ＆Ｄ社製）
　　　濃度：１０ｎｇ／ｍＬ
　・　レチノイン酸（シグマ社製）
　　　濃度：１ｎＭ、又は１０ｎＭ
　・　エストラジオール（シグマ社製）
　　　濃度：１ｎＭ、１０ｎＭ、１００ｎＭ、又は１０μＭ --Candidate substance--
・ VEGF (R & D)
Concentration: 10 ng / mL
・ Retinoic acid (manufactured by Sigma)
Concentration: 1 nM or 10 nM
・ Estradiol (Sigma)
Concentration: 1 nM, 10 nM, 100 nM, or 10 μM
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図１１及び図１２に示す。
　なお、本試験例５では、前記候補物質は、各処理で同一の候補物質とし、その濃度も同一とした。即ち、候補物質としてエストラジオール（１ｎＭ）を用いた例では、第１の心筋細胞誘導処理、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理、及び第２の心筋細胞誘導処理の全ての処理において、エストラジオール（１ｎＭ）を使用した。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG. 11 and FIG.
In this Test Example 5, the candidate substance was the same candidate substance in each treatment, and the concentration was also the same. That is, in the example using estradiol (1 nM) as the candidate substance, the cardiomyocyte induction process between the first cardiomyocyte induction process, the first cardiomyocyte induction process, and the second cardiomyocyte induction process; Estradiol (1 nM) was used in all 2 cardiomyocyte induction treatments.
　図１１中、ＲＡは、レチノイン酸を表す。
　図１１及び図１２中、Ｅ２は、エストラジオールを表す。
　図１１及び図１２の結果から、エストラジオールを１ｎＭ～１０μＭの濃度で添加することにより、ＶＥＧＦを上回る分化効率が得られることが認められた。 In FIG. 11, RA represents retinoic acid.
11 and 12, E2 represents estradiol.
From the results shown in FIGS. 11 and 12, it was confirmed that differentiation efficiency exceeding VEGF can be obtained by adding estradiol at a concentration of 1 nM to 10 μM.
（試験例６）
　胚様体形成工程における培地に用いる基礎培地の検討を以下のようにして行った。 (Test Example 6)
The examination of the basal medium used for the medium in the embryoid body formation process was performed as follows.
＜多能性幹細胞＞
　前記試験例５と同様に、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 5, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　胚様体形成工程用培養液として、下記（１）～（６）の胚様体形成工程用培養液を用いた以外は、前記試験例１と同様にして、胚様体形成工程を行った。
－胚様体形成工程用培養液－
（１）～（５）：
　基礎培地　・・・　５ｍＬ
　ＫｎｏｃｋＯｕｔ　Ｓｅｒｕｍ　Ｒｅｐｌａｃｅｍｅｎｔ（インビトロジェン社製、以下、「ＫＳＲ」と称することがある）　・・・１ｍＬ
　ＭＥＭ　Ｎｏｎ－Ｅｓｓｅｎｔｉａｌ　Ａｍｉｎｏ　Ａｃｉｄｓ（ナカライテスク株式会社製、以下、「ＭＥＭ　ＮＥＡＡ」と称することがある）　・・・　６０μＬ
　１－チオグリセロール（シグマ社製）　・・・　５０ｍｇ／Ｌ
　Ｙ２７６３２（シグマ社製）　・・・　５μＭ
　ｂＦＧＦ（和光純薬工業株式会社製）　・・・　１０ｎｇ／ｍＬ
　前記基礎培地は、（１）ＤＭＥＭ（高グルコース）（ナカライテスク株式会社製）、（２）ＤＭＥＭ（低グルコース）（ナカライテスク株式会社製）、（３）ＤＭＥＭ／Ｆ１２（ナカライテスク株式会社製）、（４）ＩＭＤＭ（ナカライテスク株式会社製）、又は（５）αＭＥＭ培地（ナカライテスク株式会社製）を用いた。
（６）（コントロール）：
　ＲｅｐｒｏＦＦ２（株式会社リプロセル製）　・・・　５ｍＬ
　Ｙ２７６３２　・・・　５μＭ
　ｂＦＧＦ　・・・　１０ｎｇ／ｍＬ <Embryoid body formation process>
The embryoid body formation step was performed in the same manner as in Test Example 1 except that the following culture fluids for the embryoid body formation step (1) to (6) were used as the culture fluid for the embryoid body formation step. .
-Culture fluid for embryoid body formation process-
(1) to (5):
Basal medium ・ ・ ・ 5mL
KnockOut Serum Replacement (manufactured by Invitrogen, hereinafter, sometimes referred to as “KSR”) 1 mL
MEM Non-Essential Amino Acids (manufactured by Nacalai Tesque, Inc., hereinafter sometimes referred to as “MEM NEAA”) 60 μL
1-thioglycerol (manufactured by Sigma) 50 mg / L
Y27632 (manufactured by Sigma) 5 μM
bFGF (manufactured by Wako Pure Chemical Industries, Ltd.) ... 10 ng / mL
The basal medium is (1) DMEM (high glucose) (manufactured by Nacalai Tesque), (2) DMEM (low glucose) (manufactured by Nacalai Tesque), (3) DMEM / F12 (manufactured by Nacalai Tesque) (4) IMDM (manufactured by Nacalai Tesque) or (5) αMEM medium (manufactured by Nacalai Tesque) was used.
(6) (Control):
ReproFF2 (manufactured by Reprocell) 5 mL
Y27632 ... 5 μM
bFGF ... 10 ng / mL
＜洗浄工程＞
　前記試験例１と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 1.
＜中胚葉誘導工程＞
　中胚葉誘導工程用培地として下記中胚葉誘導工程用培養液を用いた以外は、前記試験例１と同様にして、中胚葉誘導工程を行った。
－中胚葉誘導工程用培養液－
　基礎培地として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）を用い、前記基礎培地に、分化誘導調節剤として、ＣＨＩＲ９９０２１（ミリポア社製、４μＭ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加し、中胚葉誘導工程用培養液とした。 <Mesodermal induction process>
The mesoderm induction step was performed in the same manner as in Test Example 1 except that the following medium for mesoderm induction step was used as the medium for mesoderm induction step.
-Medium for mesoderm induction process-
StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, CHIR99021 (manufactured by Millipore, 4 μM) as the differentiation induction regulator, and L-glutamine (manufactured by Invitrogen) as the medium additive. 2 mM), transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1-thioglycerol (Sigma, 50 μg / mL) are added for the mesoderm induction process. A culture solution was obtained.
＜洗浄工程＞
　前記試験例１と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 1, the embryoid body after the mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　第１の心筋細胞誘導処理用培地として下記第１の心筋細胞誘導処理用培養液を用いた以外は、前記試験例１と同様にして、第１の心筋細胞誘導処理を行った。
－第１の心筋細胞誘導処理用培養液－
　基礎培地として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）を用い、前記基礎培地に、分化誘導調節剤として、ＩＷＰ２（シグマ社製、５μＭ）、ＳＢ４３１５４２（シグマ社製、５μＭ）、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加し、第１の心筋細胞誘導処理用培養液とした。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 1 except that the following first cardiomyocyte induction treatment culture medium was used as the first cardiomyocyte induction treatment medium.
-First culture solution for cardiomyocyte induction treatment-
StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, and IWP2 (manufactured by Sigma, 5 μM), SB431542 (manufactured by Sigma, 5 μM), and VEGF (as a differentiation induction regulator) were used as the basal medium. R & D, 10 ng / mL), L-glutamine (Invitrogen, 2 mM), transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1 -Thioglycerol (manufactured by Sigma, 50 μg / mL) was added to obtain a first culture solution for cardiomyocyte induction treatment.
＜＜洗浄処理＞＞
　前記試験例１と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 1, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地として下記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液を用い、浮遊培養の期間を胚様体形成工程開始６日目～８日目とした以外は、本試験例６の第１の心筋細胞誘導処理と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。
－第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液－
　基礎培地として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）を用い、前記基礎培地に、分化誘導調節剤として、ＩＷＰ２（シグマ社製、５μＭ）、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加し、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液とした。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
A cardiomyocyte between the following first cardiomyocyte induction process and the second cardiomyocyte induction process as a medium for cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process The first treatment was performed in the same manner as in the first cardiomyocyte induction treatment of Test Example 6 except that the culture medium for induction treatment was used and the period of suspension culture was changed to the sixth to eighth days from the start of the embryoid body formation process. The cardiomyocyte induction process between the cardiomyocyte induction process and the second cardiomyocyte induction process was performed.
-Culture medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment-
StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, and IWP2 (manufactured by Sigma, 5 μM) and VEGF (manufactured by R & D, 10 ng / mL) as differentiation regulators. As media additives, L-glutamine (manufactured by Invitrogen, 2 mM), transferrin (manufactured by Sigma, 150 μg / mL), ascorbic acid (manufactured by Sigma, 50 μg / mL), and 1-thioglycerol (manufactured by Sigma, 50 μg) / ML) to obtain a culture solution for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment.
＜＜洗浄処理＞＞
　前記試験例１と同様にして、前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 1, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　第２の心筋細胞誘導処理用培地として下記第２の心筋細胞誘導処理用培養液を用いた以外は、前記試験例１と同様にして、第２の心筋細胞誘導処理を行った。
－第２の心筋細胞誘導処理用培養液－
　基礎培地として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）を用い、前記基礎培地に、分化誘導調節剤として、ＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加し、第２の心筋細胞誘導処理用培養液とした。 << Second cardiomyocyte induction process >>
A second cardiomyocyte induction treatment was performed in the same manner as in Test Example 1 except that the following second cardiomyocyte induction treatment medium was used as the second cardiomyocyte induction treatment medium.
-Second culture solution for cardiomyocyte induction treatment-
StemPro (registered trademark) 34 (manufactured by Invitrogen) was used as the basal medium, VEGF (manufactured by R & D, 10 ng / mL) as the differentiation induction regulator, and L-glutamine (Invitrogen) as the medium additive. 2 mM), transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1-thioglycerol (Sigma, 50 μg / mL) are added. A culture solution for cardiomyocyte induction treatment was used.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図１３に示す。
　図１３の結果から、基礎培地としてＤＭＥＭ／Ｆ１２を用いた場合には、コントロールであるＲｅｐｒｏＦＦ２よりも分化効率が優れていることが示された。また、ＤＭＥＭ／Ｆ１２以外の基礎培地であっても、拍動している胚様体が得られることが示された。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG.
From the results of FIG. 13, it was shown that when DMEM / F12 was used as the basal medium, the differentiation efficiency was superior to the control ReproFF2. In addition, it was shown that a pulsatile embryoid body can be obtained even with a basal medium other than DMEM / F12.
（試験例７）
　胚様体形成工程における培地に添加する分化誘導調節剤の検討を以下のようにして行った。 (Test Example 7)
The differentiation induction regulator added to the medium in the embryoid body formation step was examined as follows.
＜多能性幹細胞＞
　前記試験例５と同様に、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 5, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　胚様体形成工程用培養液として、前記試験例６の胚様体形成工程用培養液（３）（基礎培地として、ＤＭＥＭ／Ｆ１２を使用したもの）に、ＣＨＩＲ９９０２１（ミリポア社製）及びｂＦＧＦ（和光純薬工業株式会社製）の添加濃度を下記の濃度とした胚様体形成工程用培養液を用いた以外は、前記試験例６と同様にして、胚様体形成工程を行った。なお、コントロールとして、前記試験例６の胚様体形成工程用培養液（６）（ＲｅｐｒｏＦＦ２を使用したもの、以下、「ＲｅｐｒｏＦＦ２」と称することがある）についても同様にして、胚様体形成工程を行った。
－ｂＦＧＦ及びＣＨＩＲ９９０２１の添加濃度－
　・　ｂＦＧＦ：０ｎｇ／ｍＬ（以下、「ＦＧＦ　０」と称することがある）、５０ｎｇ／ｍＬ（以下、「ＦＧＦ　５０」と称することがある）、又は１００ｎｇ／ｍＬ（以下、「ＦＧＦ　１００」と称することがある）
　・　ＣＨＩＲ９９０２１：０．００５μＭ（以下、「ＣＨＩＲ　０．００５」と称することがある）、０．２５μＭ（以下、「ＣＨＩＲ　０．２５」と称することがある）、又は５μＭ（以下、「ＣＨＩＲ　５」と称することがある） <Embryoid body formation process>
As the culture medium for embryoid body formation process, the culture medium for embryoid body formation process of Test Example 6 (3) (using DMEM / F12 as the basal medium), CHIR99021 (manufactured by Millipore) and bFGF ( The embryoid body forming step was performed in the same manner as in Test Example 6 except that the culture solution for embryoid body forming step having the following concentration was added to Wako Pure Chemical Industries, Ltd.). As a control, the embryoid body formation step was similarly performed for the culture fluid for embryoid body formation step (6) of Test Example 6 (using ReproFF2, hereinafter sometimes referred to as “ReproFF2”). Went.
-Concentration of bFGF and CHIR99021-
BFGF: 0 ng / mL (hereinafter sometimes referred to as “FGF 0”), 50 ng / mL (hereinafter sometimes referred to as “FGF 50”), or 100 ng / mL (hereinafter referred to as “FGF 100”) Sometimes)
CHIR99021: 0.005 μM (hereinafter sometimes referred to as “CHIR 0.005”), 0.25 μM (hereinafter sometimes referred to as “CHIR 0.25”), or 5 μM (hereinafter referred to as “CHIR 5”) Sometimes called)
＜洗浄工程＞
　前記試験例６と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 6.
＜中胚葉誘導工程＞
　前記試験例６と同様にして、中胚葉誘導工程を行った。 <Mesodermal induction process>
In the same manner as in Test Example 6, a mesoderm induction step was performed.
＜洗浄工程＞
　前記試験例６と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 6, the embryoid body after mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　前記試験例６と同様にして、第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
In the same manner as in Test Example 6, the first cardiomyocyte induction treatment was performed.
＜＜洗浄処理＞＞
　前記試験例６と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 6, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　前記試験例６と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
In the same manner as in Test Example 6, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed.
＜＜洗浄処理＞＞
　前記試験例６と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 6, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　前記試験例６と同様にして、第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
In the same manner as in Test Example 6, a second cardiomyocyte induction treatment was performed.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図１４及び図１５に示す。
　前記試験例６の結果、及び図１４の結果から、ｂＦＧＦを１０ｎｇ／ｍＬ～１００ｎｇ／ｍＬ加えてもＲｅｐｒｏＦＦ２よりも分化効率が優れていることが示されたが、ｂＦＧＦを含まない場合が最も分化効率が優れていた。
　また、図１５の結果から、ＣＨＩＲ９９０２１を５ｎＭ～５μＭ加えてもＲｅｐｒｏＦＦ２よりも分化効率が優れていることが示された。なお、ＣＨＩＲ９９０２１を５ｎＭ加えた場合が最も分化効率が優れていたが、ＣＨＩＲ９９０２１を添加しなくても十分な分化効率が得られることから、コスト及び手間を考慮すると、ＣＨＩＲ９９０２１を添加しない態様が好ましいと考えられた。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIGS.
The results of Test Example 6 and the results of FIG. 14 showed that differentiation efficiency was superior to ReproFF2 even when bFGF was added at 10 ng / mL to 100 ng / mL, but the case where bFGF was not included was most differentiated. The efficiency was excellent.
Further, from the results of FIG. 15, it was shown that differentiation efficiency was superior to ReproFF2 even when 5 nM to 5 μM of CHIR99021 was added. When CHIR99021 was added at 5 nM, the differentiation efficiency was most excellent, but sufficient differentiation efficiency was obtained without adding CHIR99021. Therefore, in view of cost and labor, an embodiment in which CHIR99021 is not added is preferable. it was thought.
（試験例８）
　胚様体形成工程における培地の検討を以下のようにして行った。 (Test Example 8)
The examination of the culture medium in the embryoid body formation process was performed as follows.
＜多能性幹細胞＞
　前記試験例５と同様に、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 5, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　胚様体形成工程用培養液として、以下の胚様体形成工程用培養液Ａ又はＢを用いた以外は、前記試験例６と同様にして、胚様体形成工程を行った。
－胚様体形成工程用培養液Ａ－
　前記試験例１における胚様体形成工程用培養液。
－胚様体形成工程用培養液Ｂ－
　前記試験例６の胚様体形成工程用培養液（３）（（基礎培地として、ＤＭＥＭ／Ｆ１２を使用したもの）において、ｂＦＧＦを除いた培養液。 <Embryoid body formation process>
The embryoid body formation step was performed in the same manner as in Test Example 6 except that the following embryoid body formation step culture medium A or B was used as the embryoid body formation step culture medium.
-Culture fluid A for embryoid body formation process-
The culture solution for embryoid body formation process in Test Example 1.
-Culture solution B for embryoid body formation process-
The culture solution obtained by removing bFGF in the culture solution (3) for embryoid body formation process of Test Example 6 ((using DMEM / F12 as a basal medium)).
＜洗浄工程＞
　前記試験例６と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 6.
＜中胚葉誘導工程＞
　前記試験例６と同様にして、中胚葉誘導工程を行った。 <Mesodermal induction process>
In the same manner as in Test Example 6, a mesoderm induction step was performed.
＜洗浄工程＞
　前記試験例６と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 6, the embryoid body after mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　前記試験例６と同様にして、第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
In the same manner as in Test Example 6, the first cardiomyocyte induction treatment was performed.
＜＜洗浄処理＞＞
　前記試験例６と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 6, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　前記試験例６と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
In the same manner as in Test Example 6, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed.
＜＜洗浄処理＞＞
　前記試験例６と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 6, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　前記試験例６と同様にして、第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
In the same manner as in Test Example 6, a second cardiomyocyte induction treatment was performed.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図１６に示す。
　図１６の結果から、前記胚様体形成工程用培養液Ａを用いた場合のほうが、分化効率が優れていることが示された。前記胚様体形成工程用培養液Ａは、組成不明の脂質と結合したＢＳＡを使用した前記ＫＳＲ（インビトロジェン社製）が含まれていないため、例えば、心筋細胞の分化に脂質成分が与える影響を調べる実験など、実験の応用範囲が広がると考えられる。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG.
From the result of FIG. 16, it was shown that the differentiation efficiency was superior when the culture medium A for embryoid body formation process was used. Since the culture medium A for embryoid body formation process does not contain the KSR (manufactured by Invitrogen) using BSA combined with lipid of unknown composition, for example, the influence of lipid components on cardiomyocyte differentiation It is thought that the range of application of experiments, such as experiments to investigate, will expand.
（試験例９）
　中胚葉誘導工程、及び心筋細胞誘導工程における培地の検討を以下のようにして行った。 (Test Example 9)
The medium in the mesoderm induction process and the cardiomyocyte induction process was examined as follows.
＜多能性幹細胞＞
　前記試験例５と同様に、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 5, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　胚様体形成工程用培養液として、前記試験例８における胚様体形成工程用培養液Ｂを用いた以外は、前記試験例５と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
The embryoid body formation step was performed in the same manner as in Test Example 5 except that the culture fluid B for embryoid body formation step in Test Example 8 was used as the culture solution for embryoid body formation step.
＜洗浄工程＞
　前記試験例５と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 5.
＜中胚葉誘導工程＞
　中胚葉誘導工程用培地として、前記試験例５の中胚葉誘導工程用培養液におけるポリビニルアルコール（以下、「ＰＶＡ」と称することがある）及び牛血清アルブミン（以下、「ＢＳＡ」と称することがある）の濃度を下記の濃度とした培養液を用いた以外は、前記試験例５と同様にして、中胚葉誘導工程を行った。
　また、コントロール（以下、「ＳＰ３４」と称することがある）として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、分化誘導調節剤として、ＣＨＩＲ９９０２１（ミリポア社製、４μＭ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加した中胚葉誘導工程用培養液についても同様にして、中胚葉誘導工程を行った。
－ポリビニルアルコール及び牛血清アルブミンの濃度－
（１）ＰＶＡ　４，０００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ（以下、「ＰＶＡ４０００／ＢＳＡ５０００」と称することがある）
（２）ＰＶＡ　４，０００ｍｇ／Ｌ、ＢＳＡ　５００ｍｇ／Ｌ（以下、「ＰＶＡ４０００／ＢＳＡ５００」と称することがある）
（３）ＰＶＡ　４，０００ｍｇ／Ｌ、ＢＳＡ　０ｍｇ／Ｌ（以下、「ＰＶＡ４０００／ＢＳＡ０」と称することがある）
（４）ＰＶＡ　１，０００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ（以下、「ＰＶＡ１０００／ＢＳＡ５０００」と称することがある）
（５）ＰＶＡ　１，０００ｍｇ／Ｌ、ＢＳＡ　５００ｍｇ／Ｌ（以下、「ＰＶＡ１０００／ＢＳＡ５００」と称することがある）
（６）ＰＶＡ　１，０００ｍｇ／Ｌ、ＢＳＡ　０ｍｇ／Ｌ（以下、「ＰＶＡ１０００／ＢＳＡ０」と称することがある）
（７）ＰＶＡ　０ｍｇ／Ｌ、ＢＳＡ　０ｍｇ／Ｌ（以下、「ＰＶＡ０／ＢＳＡ０」と称することがある） <Mesodermal induction process>
As a medium for mesoderm induction process, polyvinyl alcohol (hereinafter sometimes referred to as “PVA”) and bovine serum albumin (hereinafter referred to as “BSA”) in the culture medium for mesoderm induction process of Test Example 5 may be referred to. The mesoderm induction step was performed in the same manner as in Test Example 5 except that the culture solution having the following concentration was used.
In addition, as a control (hereinafter also referred to as “SP34”), StemPro (registered trademark) 34 (manufactured by Invitrogen), differentiation induction regulator, CHIR99021 (manufactured by Millipore, 4 μM), medium additive, Added L-glutamine (Invitrogen, 2 mM), transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1-thioglycerol (Sigma, 50 μg / mL) The mesoderm induction step was performed in the same manner for the culture solution for the mesoderm induction step.
-Concentrations of polyvinyl alcohol and bovine serum albumin-
(1) PVA 4,000 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA4000 / BSA5000”)
(2) PVA 4,000 mg / L, BSA 500 mg / L (hereinafter sometimes referred to as “PVA4000 / BSA500”)
(3) PVA 4,000 mg / L, BSA 0 mg / L (hereinafter sometimes referred to as “PVA4000 / BSA0”)
(4) PVA 1,000 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA5000”)
(5) PVA 1,000 mg / L, BSA 500 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA500”)
(6) PVA 1,000 mg / L, BSA 0 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA0”)
(7) PVA 0 mg / L, BSA 0 mg / L (hereinafter sometimes referred to as “PVA0 / BSA0”)
＜洗浄工程＞
　前記試験例５と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 5, the embryoid body after mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　第１の心筋細胞誘導処理用培地として、本試験例９の前記中胚葉誘導工程用培養液におけるＣＨＩＲ９９０２１を、ＩＷＰ２（シグマ社製、５μＭ）、ＳＢ４３１５４２（シグマ社製、５μＭ）、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）に代え、インスリン（シグマ社製、１０ｎｇ／ｍＬ）を追加した以外は同様とした第１の心筋細胞誘導処理用培地を用い、浮遊培養の期間を胚様体形成工程開始３日目～５日目に変えた以外は、前記試験例５と同様にして、第１の心筋細胞誘導処理を行った。
　また、コントロールとして、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、分化誘導調節剤として、ＩＷＰ２（シグマ社製、５μＭ）、ＳＢ４３１５４２（シグマ社製、５μＭ）、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加した培養液についても同様にして、第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
As the first cardiomyocyte induction treatment medium, CHIR99021 in the culture solution for the mesoderm induction process of Test Example 9 was used as IWP2 (Sigma, 5 μM), SB431542 (Sigma, 5 μM), and VEGF (R & D). The first cardiomyocyte induction treatment medium was used in the same manner except that insulin (Sigma, 10 ng / mL) was added instead of 10 ng / mL, and the period of suspension culture was the embryoid body formation step. The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 5 except that the changes were made on the third to fifth days from the start.
As controls, StemPro (registered trademark) 34 (manufactured by Invitrogen), differentiation induction regulators as IWP2 (manufactured by Sigma, 5 μM), SB431542 (manufactured by Sigma, 5 μM), and VEGF (manufactured by R & D, 10 ng) / ML), L-glutamine (manufactured by Invitrogen, 2 mM), transferrin (manufactured by Sigma, 150 μg / mL), ascorbic acid (manufactured by Sigma, 50 μg / mL), and 1-thioglycerol (Sigma) The first myocardial cell induction treatment was performed in the same manner on the culture medium supplemented with 50 μg / mL).
＜＜洗浄処理＞＞
　前記試験例５と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 5, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地として、本試験例９の前記中胚葉誘導工程用培養液におけるＣＨＩＲ９９０２１を、ＩＷＰ２（シグマ社製、５μＭ）、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）に代え、インスリン（シグマ社製、１０ｎｇ／ｍＬ）を追加した以外は同様とした第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地を用い、浮遊培養の期間を胚様体形成工程開始５日目～８日目に変えた以外は、前記試験例５と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。
　また、コントロールとして、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、分化誘導調節剤として、ＩＷＰ２（シグマ社製、５μＭ）、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加した培養液についても同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
As a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment, CHIR99021 in the culture solution for the mesoderm induction step of Test Example 9 was used as IWP2 (manufactured by Sigma). 5 μM) and VEGF (R & D, 10 ng / mL) instead of insulin (Sigma, 10 ng / mL), the same first cardiomyocyte induction treatment and second cardiomyocyte The same procedure as in Test Example 5 was conducted except that the medium for cardiomyocyte induction treatment during the induction treatment was used, and the suspension culture period was changed to the fifth to eighth days from the start of the embryoid body formation step. The cardiomyocyte induction process between the cardiomyocyte induction process and the second cardiomyocyte induction process was performed.
In addition, as a control, StemPro (registered trademark) 34 (manufactured by Invitrogen), as a differentiation induction regulator, IWP2 (manufactured by Sigma, 5 μM), VEGF (manufactured by R & D, 10 ng / mL), as a medium additive, Added L-glutamine (Invitrogen, 2 mM), transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1-thioglycerol (Sigma, 50 μg / mL) Similarly, the cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed on the culture medium.
＜＜洗浄処理＞＞
　前記試験例５と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 5, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　第２の心筋細胞誘導処理用培地として、本試験例９の前記中胚葉誘導工程用培養液におけるＣＨＩＲ９９０２１を、ＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）に代え、インスリン（シグマ社製、１０ｎｇ／ｍＬ）を追加した以外は同様とした第２の心筋細胞誘導処理用培地を用いた以外は、前記試験例５と同様にして、第２の心筋細胞誘導処理を行った。
　また、コントロールとして、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、分化誘導調節剤として、ＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加した培養液についても同様にして、第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
As the second culture medium for cardiomyocyte induction treatment, CHIR99021 in the culture solution for the mesoderm induction process of Test Example 9 was replaced with VEGF (R & D, 10 ng / mL), and insulin (Sigma, 10 ng / mL). The second cardiomyocyte induction treatment was performed in the same manner as in Test Example 5 except that the second medium for inducing cardiomyocyte induction treatment was used except that (2) was added.
As a control, StemPro (registered trademark) 34 (manufactured by Invitrogen), VEGF (manufactured by R & D, 10 ng / mL) as a differentiation induction regulator, and L-glutamine (manufactured by Invitrogen, 2 mM) as a medium additive In the same manner, the culture solution to which transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1-thioglycerol (Sigma, 50 μg / mL) was added was also the same. 2 cardiomyocyte induction treatment was performed.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図１７に示す。
　図１７の結果から、（１）ＰＶＡ　４，０００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ、及び（４）ＰＶＡ　１，０００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌの場合には、コントロールよりも優れた分化効率を示した。コントロールに用いたＳｔｅｍＰｒｏ（登録商標）３４には、毒物である２－メルカプトエタノール、成分が明らかでない脂質成分であるＨｕｍａｎ－ＥｘＣｙｔｅ、及び高価でロット間差が大きいヒト血清アルブミンが含まれているが、これらを含まない培地でも優れた分化効率を示す培地が得られることが示された。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG.
From the results of FIG. 17, (1) PVA 4,000 mg / L, BSA 5,000 mg / L, and (4) PVA 1,000 mg / L, BSA 5,000 mg / L were superior to the control. Differentiation efficiency was demonstrated. StemPro (registered trademark) 34 used as a control contains 2-mercaptoethanol which is a toxic substance, Human-ExCyte which is a lipid component whose component is not clear, and human serum albumin which is expensive and has a large lot-to-lot difference. It was shown that a medium showing excellent differentiation efficiency can be obtained even in a medium not containing these.
（試験例１０）
　中胚葉誘導工程、及び心筋細胞誘導工程における培地の検討を以下のようにして行った。 (Test Example 10)
The medium in the mesoderm induction process and the cardiomyocyte induction process was examined as follows.
＜多能性幹細胞＞
　前記試験例５と同様に、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 5, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　前記試験例９と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
In the same manner as in Test Example 9, an embryoid body formation step was performed.
＜洗浄工程＞
　前記試験例９と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 9.
＜中胚葉誘導工程＞
　中胚葉誘導工程用培地として、前記試験例５の中胚葉誘導工程用培養液におけるポリビニルアルコール（以下、「ＰＶＡ」と称することがある）及び牛血清アルブミン（以下、「ＢＳＡ」と称することがある）の濃度を下記の濃度とした培養液を用いた以外は、前記試験例９と同様にして、中胚葉誘導工程を行った。
　また、コントロール（以下、「ＳＰ３４」と称することがある）として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、分化誘導調節剤として、ＣＨＩＲ９９０２１（ミリポア社製、４μＭ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加した中胚葉誘導工程用培養液についても同様にして、中胚葉誘導工程を行った。
－ポリビニルアルコール及び牛血清アルブミンの濃度－
（８）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ（以下、「ＰＶＡ５００／ＢＳＡ５０００」と称することがある）
（９）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　１，５００ｍｇ／Ｌ（以下、「ＰＶＡ５００／ＢＳＡ１５００」と称することがある）
（１０）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　５００ｍｇ／Ｌ（以下、「ＰＶＡ５００／ＢＳＡ５００」と称することがある）
（１１）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　０ｍｇ／Ｌ（以下、「ＰＶＡ５００／ＢＳＡ０」と称することがある） <Mesodermal induction process>
As a medium for mesoderm induction process, polyvinyl alcohol (hereinafter sometimes referred to as “PVA”) and bovine serum albumin (hereinafter referred to as “BSA”) in the culture medium for mesoderm induction process of Test Example 5 may be referred to. The mesoderm induction step was performed in the same manner as in Test Example 9 except that the culture solution having the following concentration was used.
In addition, as a control (hereinafter also referred to as “SP34”), StemPro (registered trademark) 34 (manufactured by Invitrogen), differentiation induction regulator, CHIR99021 (manufactured by Millipore, 4 μM), medium additive, Added L-glutamine (Invitrogen, 2 mM), transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1-thioglycerol (Sigma, 50 μg / mL) The mesoderm induction step was performed in the same manner for the culture solution for the mesoderm induction step.
-Concentrations of polyvinyl alcohol and bovine serum albumin-
(8) PVA 500 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA500 / BSA5000”)
(9) PVA 500 mg / L, BSA 1,500 mg / L (hereinafter sometimes referred to as “PVA500 / BSA1500”)
(10) PVA 500 mg / L, BSA 500 mg / L (hereinafter sometimes referred to as “PVA500 / BSA500”)
(11) PVA 500 mg / L, BSA 0 mg / L (hereinafter sometimes referred to as “PVA500 / BSA0”)
＜洗浄工程＞
　前記試験例９と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 9, the embryoid body after mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理用培地を調製していた点を、本試験例１０の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理用培地を調製した以外は、試験例９と同様にして、第１の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
In Test Example 9, the first cardiomyocyte induction treatment medium was prepared on the basis of the culture medium for the mesodermal induction process of Test Example 9, and the culture medium for the mesoderm induction process of Test Example 10 was used. The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that the first cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, the first cardiomyocyte induction treatment was performed for the control.
＜＜洗浄処理＞＞
　前記試験例９と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 9, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地を調製していた点を、本試験例１０の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地を調製した以外は、試験例９と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
In Test Example 9, a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction process and the second cardiomyocyte induction process is prepared based on the culture medium for the mesodermal induction process of Test Example 9. Except that the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was prepared based on the medium for mesodermal induction process of Test Example 10 In the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed. Further, in the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed for the control.
＜＜洗浄処理＞＞
　前記試験例９と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 9, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第２の心筋細胞誘導処理用培地を調製していた点を、本試験例１０の中胚葉誘導工程用培養液に基づいて第２の心筋細胞誘導処理用培地を調製した以外は、試験例９と同様にして、第２の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
In the test example 9, the second cardiomyocyte induction treatment medium was prepared based on the culture medium for the mesodermal induction process of the test example 9, and the culture medium for the mesodermal induction process of the test example 10 was used. A second cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that a second cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, a second cardiomyocyte induction treatment was performed for the control.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図１８に示す。
　図１８の結果から、（８）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌの場合には、コントロールよりも優れた分化効率を示した。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG.
From the results of FIG. 18, in the case of (8) PVA 500 mg / L and BSA 5,000 mg / L, differentiation efficiency superior to the control was shown.
（試験例１１）
　中胚葉誘導工程、及び心筋細胞誘導工程における培地の検討を以下のようにして行った。 (Test Example 11)
The medium in the mesoderm induction process and the cardiomyocyte induction process was examined as follows.
＜多能性幹細胞＞
　前記試験例５と同様に、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 5, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　前記試験例９と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
In the same manner as in Test Example 9, an embryoid body formation step was performed.
＜洗浄工程＞
　前記試験例９と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 9.
＜中胚葉誘導工程＞
　中胚葉誘導工程用培地として、前記試験例５の中胚葉誘導工程用培養液におけるポリビニルアルコール（以下、「ＰＶＡ」と称することがある）及び牛血清アルブミン（以下、「ＢＳＡ」と称することがある）の濃度を下記の濃度とした培養液を用いた以外は、前記試験例９と同様にして、中胚葉誘導工程を行った。
　また、コントロール（以下、「ＳＰ３４」と称することがある）として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、分化誘導調節剤として、ＣＨＩＲ９９０２１（ミリポア社製、４μＭ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加した中胚葉誘導工程用培養液についても同様にして、中胚葉誘導工程を行った。
－ポリビニルアルコール及び牛血清アルブミンの濃度－
（１２）ＰＶＡ　１，５００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ（以下、「ＰＶＡ１５００／ＢＳＡ５０００」と称することがある）
（１３）ＰＶＡ　１，５００ｍｇ／Ｌ、ＢＳＡ　４，０００ｍｇ／Ｌ（以下、「ＰＶＡ１５００／ＢＳＡ４０００」と称することがある）
（１４）ＰＶＡ　１，５００ｍｇ／Ｌ、ＢＳＡ　３，０００ｍｇ／Ｌ（以下、「ＰＶＡ１５００／ＢＳＡ３０００」と称することがある）
（１５）ＰＶＡ　１，０００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ（以下、「ＰＶＡ１０００／ＢＳＡ５０００」と称することがある）
（１６）ＰＶＡ　１，０００ｍｇ／Ｌ、ＢＳＡ　４，０００ｍｇ／Ｌ（以下、「ＰＶＡ１０００／ＢＳＡ４０００」と称することがある）
（１７）ＰＶＡ　１，０００ｍｇ／Ｌ、ＢＳＡ　３，０００ｍｇ／Ｌ（以下、「ＰＶＡ１０００／ＢＳＡ３０００」と称することがある）
（１８）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ（以下、「ＰＶＡ５００／ＢＳＡ５０００」と称することがある）
（１９）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　４，０００ｍｇ／Ｌ（以下、「ＰＶＡ５００／ＢＳＡ４０００」と称することがある）
（２０）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　３，０００ｍｇ／Ｌ（以下、「ＰＶＡ５００／ＢＳＡ３０００」と称することがある） <Mesodermal induction process>
As a medium for mesoderm induction process, polyvinyl alcohol (hereinafter sometimes referred to as “PVA”) and bovine serum albumin (hereinafter referred to as “BSA”) in the culture medium for mesoderm induction process of Test Example 5 may be referred to. The mesoderm induction step was performed in the same manner as in Test Example 9 except that the culture solution having the following concentration was used.
In addition, as a control (hereinafter also referred to as “SP34”), StemPro (registered trademark) 34 (manufactured by Invitrogen), differentiation induction regulator, CHIR99021 (manufactured by Millipore, 4 μM), medium additive, Added L-glutamine (Invitrogen, 2 mM), transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1-thioglycerol (Sigma, 50 μg / mL) The mesoderm induction step was performed in the same manner for the culture solution for the mesoderm induction step.
-Concentrations of polyvinyl alcohol and bovine serum albumin-
(12) PVA 1,500 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA1500 / BSA5000”)
(13) PVA 1,500 mg / L, BSA 4,000 mg / L (hereinafter sometimes referred to as “PVA1500 / BSA4000”)
(14) PVA 1,500 mg / L, BSA 3,000 mg / L (hereinafter sometimes referred to as “PVA1500 / BSA3000”)
(15) PVA 1,000 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA5000”)
(16) PVA 1,000 mg / L, BSA 4,000 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA4000”)
(17) PVA 1,000 mg / L, BSA 3,000 mg / L (hereinafter sometimes referred to as “PVA1000 / BSA3000”)
(18) PVA 500 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA500 / BSA5000”)
(19) PVA 500 mg / L, BSA 4,000 mg / L (hereinafter sometimes referred to as “PVA500 / BSA4000”)
(20) PVA 500 mg / L, BSA 3,000 mg / L (hereinafter sometimes referred to as “PVA500 / BSA3000”)
＜洗浄工程＞
　前記試験例９と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 9, the embryoid body after mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理用培地を調製していた点を、本試験例１１の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理用培地を調製した以外は、試験例９と同様にして、第１の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
In the test example 9, the first cardiomyocyte induction treatment medium was prepared based on the culture medium for the mesodermal induction process of the test example 9, and the culture medium for the mesodermal induction process of the test example 11 was used. The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that the first cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, the first cardiomyocyte induction treatment was performed for the control.
＜＜洗浄処理＞＞
　前記試験例９と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 9, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地を調製していた点を、本試験例１１の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地を調製した以外は、試験例９と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
In Test Example 9, a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction process and the second cardiomyocyte induction process is prepared based on the culture medium for the mesodermal induction process of Test Example 9. Except that the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was prepared based on the culture solution for mesodermal induction process of Test Example 11 In the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed. Further, in the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed for the control.
＜＜洗浄処理＞＞
　前記試験例９と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 9, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第２の心筋細胞誘導処理用培地を調製していた点を、本試験例１１の中胚葉誘導工程用培養液に基づいて第２の心筋細胞誘導処理用培地を調製した以外は、試験例９と同様にして、第２の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
In Test Example 9, the second cardiomyocyte induction treatment medium was prepared based on the culture medium for mesoderm induction process of Test Example 9, and the culture medium for mesoderm induction process of Test Example 11 was used. A second cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that a second cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, a second cardiomyocyte induction treatment was performed for the control.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図１９に示す。
　図１９の結果から、（１２）ＰＶＡ　１，５００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ、（１３）ＰＶＡ　１，５００ｍｇ／Ｌ、ＢＳＡ　４，０００ｍｇ／Ｌ、（１５）ＰＶＡ　１，０００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ、（１６）ＰＶＡ　１，０００ｍｇ／Ｌ、ＢＳＡ　４，０００ｍｇ／Ｌ、（１８）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ、及び（１９）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　４，０００ｍｇ／Ｌの場合には、コントロールよりも優れた分化効率を示した。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG.
From the results of FIG. 19, (12) PVA 1,500 mg / L, BSA 5,000 mg / L, (13) PVA 1,500 mg / L, BSA 4,000 mg / L, (15) PVA 1,000 mg / L, BSA 5,000 mg / L, (16) PVA 1,000 mg / L, BSA 4,000 mg / L, (18) PVA 500 mg / L, BSA 5,000 mg / L, and (19) PVA 500 mg / L, BSA 4 In the case of 1,000 mg / L, differentiation efficiency superior to the control was shown.
（試験例１２）
　中胚葉誘導工程、及び心筋細胞誘導工程における培地の検討を以下のようにして行った。 (Test Example 12)
The medium in the mesoderm induction process and the cardiomyocyte induction process was examined as follows.
＜多能性幹細胞＞
　前記試験例５と同様に、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 5, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　前記試験例９と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
In the same manner as in Test Example 9, an embryoid body formation step was performed.
＜洗浄工程＞
　前記試験例９と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 9.
＜中胚葉誘導工程＞
　中胚葉誘導工程用培地として、前記試験例５の中胚葉誘導工程用培養液におけるポリビニルアルコール（以下、「ＰＶＡ」と称することがある）及び牛血清アルブミン（以下、「ＢＳＡ」と称することがある）の濃度を下記の濃度とした培養液を用いた以外は、前記試験例９と同様にして、中胚葉誘導工程を行った。
　また、コントロール（以下、「ＳＰ３４」と称することがある）として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、ＣＨＩＲ９９０２１（ミリポア社製、４μＭ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加した中胚葉誘導工程用培養液についても同様にして、中胚葉誘導工程を行った。
－ポリビニルアルコール及び牛血清アルブミンの濃度－
（２１）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ（以下、「ＰＶＡ５００／ＢＳＡ５０００」と称することがある）
（２２）ＰＶＡ　０ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌ（以下、「ＰＶＡ０／ＢＳＡ５０００」と称することがある）
（２３）ＰＶＡ　０ｍｇ／Ｌ、ＢＳＡ　４，０００ｍｇ／Ｌ（以下、「ＰＶＡ０／ＢＳＡ４０００」と称することがある）
（２４）ＰＶＡ　０ｍｇ／Ｌ、ＢＳＡ　３，０００ｍｇ／Ｌ（以下、「ＰＶＡ０／ＢＳＡ３０００」と称することがある） <Mesodermal induction process>
As a medium for mesoderm induction process, polyvinyl alcohol (hereinafter sometimes referred to as “PVA”) and bovine serum albumin (hereinafter referred to as “BSA”) in the culture medium for mesoderm induction process of Test Example 5 may be referred to. The mesoderm induction step was performed in the same manner as in Test Example 9 except that the culture solution having the following concentration was used.
Further, as a control (hereinafter sometimes referred to as “SP34”), StemPro (registered trademark) 34 (manufactured by Invitrogen), CHIR99021 (manufactured by Millipore, 4 μM), and L-glutamine (Invitrogen) as a medium additive. 2 mM), transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1-thioglycerol (Sigma, 50 μg / mL) for mesoderm induction process Similarly, the mesoderm induction process was performed for the culture solution.
-Concentrations of polyvinyl alcohol and bovine serum albumin-
(21) PVA 500 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA500 / BSA5000”)
(22) PVA 0 mg / L, BSA 5,000 mg / L (hereinafter sometimes referred to as “PVA0 / BSA5000”)
(23) PVA 0 mg / L, BSA 4,000 mg / L (hereinafter sometimes referred to as “PVA0 / BSA4000”)
(24) PVA 0 mg / L, BSA 3,000 mg / L (hereinafter sometimes referred to as “PVA0 / BSA3000”)
＜洗浄工程＞
　前記試験例９と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 9, the embryoid body after mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理用培地を調製していた点を、本試験例１２の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理用培地を調製した以外は、試験例９と同様にして、第１の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
In the test example 9, the first cardiomyocyte induction treatment medium was prepared based on the culture medium for the mesodermal induction process of the test example 9, and the culture medium for the mesodermal induction process of the test example 12 was used. The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that the first cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, the first cardiomyocyte induction treatment was performed for the control.
＜＜洗浄処理＞＞
　前記試験例９と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 9, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地を調製していた点を、本試験例１２の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地を調製した以外は、試験例９と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
In Test Example 9, a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction process and the second cardiomyocyte induction process is prepared based on the culture medium for the mesodermal induction process of Test Example 9. Except that the medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was prepared based on the culture solution for the mesoderm induction process of Test Example 12 In the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed. Further, in the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed for the control.
＜＜洗浄処理＞＞
　前記試験例９と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 9, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第２の心筋細胞誘導処理用培地を調製していた点を、本試験例１２の中胚葉誘導工程用培養液に基づいて第２の心筋細胞誘導処理用培地を調製した以外は、試験例９と同様にして、第２の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
In Test Example 9, the second cardiomyocyte induction treatment medium was prepared on the basis of the culture medium for the mesodermal induction process of Test Example 9, and the culture medium for the mesoderm induction process of Test Example 12 was used. A second cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that a second cardiomyocyte induction treatment medium was prepared. Further, in the same manner as in Test Example 9, a second cardiomyocyte induction treatment was performed for the control.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図２０に示す。
　図２０の結果から、（２１）ＰＶＡ　５００ｍｇ／Ｌ、ＢＳＡ　５，０００ｍｇ／Ｌの場合には、コントロールよりも優れた分化効率を示した。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG.
From the results of FIG. 20, in the case of (21) PVA 500 mg / L and BSA 5,000 mg / L, differentiation efficiency superior to the control was shown.
（試験例１３）
　中胚葉誘導工程、及び心筋細胞誘導工程における培地の検討を以下のようにして行った。 (Test Example 13)
The medium in the mesoderm induction process and the cardiomyocyte induction process was examined as follows.
＜多能性幹細胞＞
　前記試験例５と同様に、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 5, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　前記試験例９と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
In the same manner as in Test Example 9, an embryoid body formation step was performed.
＜洗浄工程＞
　前記試験例９と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 9.
＜中胚葉誘導工程＞
　中胚葉誘導工程用培地として、前記試験例５の中胚葉誘導工程用培養液におけるトランスフェリンの濃度を５ｍｇ／Ｌ、又は０ｍｇ／Ｌとした培養液を用いた以外は、前記試験例９と同様にして、中胚葉誘導工程を行った。
　また、コントロール（以下、「ＳＰ３４」と称することがある）として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、ＣＨＩＲ９９０２１（ミリポア社製、４μＭ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加した中胚葉誘導工程用培養液についても同様にして、中胚葉誘導工程を行った。 <Mesodermal induction process>
The same as in Test Example 9, except that the medium for transferrin in the culture medium for mesoderm induction process in Test Example 5 was 5 mg / L or 0 mg / L as the medium for mesoderm induction process. Then, a mesoderm induction process was performed.
Further, as a control (hereinafter sometimes referred to as “SP34”), StemPro (registered trademark) 34 (manufactured by Invitrogen), CHIR99021 (manufactured by Millipore, 4 μM), and L-glutamine (Invitrogen) as a medium additive. 2 mM), transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1-thioglycerol (Sigma, 50 μg / mL) for mesoderm induction process Similarly, the mesoderm induction process was performed for the culture solution.
＜洗浄工程＞
　前記試験例９と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 9, the embryoid body after mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理用培地を調製していた点を、本試験例１３の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理用培地を調製し、また、インスリン（１０ｎｇ／ｍＬ）の添加を行った場合と、行わなかった場合のそれぞれの培地を調製した以外は、試験例９と同様にして、第１の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
In Test Example 9, the first cardiomyocyte induction treatment medium was prepared on the basis of the culture medium for the mesodermal induction process of Test Example 9, and the culture medium for the mesodermal induction process of Test Example 13 was used. First, the same cardiomyocyte induction treatment medium was prepared, and the same as in Test Example 9 except that each medium was prepared with and without the addition of insulin (10 ng / mL). Thus, the first cardiomyocyte induction treatment was performed. Further, in the same manner as in Test Example 9, the first cardiomyocyte induction treatment was performed for the control.
＜＜洗浄処理＞＞
　前記試験例９と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 9, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地を調製していた点を、本試験例１３の中胚葉誘導工程用培養液に基づいて第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地を調製し、また、インスリン（１０ｎｇ／ｍＬ）の添加を行った場合と、行わなかった場合のそれぞれの培地を調製した以外は、試験例９と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
In Test Example 9, a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction process and the second cardiomyocyte induction process is prepared based on the culture medium for the mesodermal induction process of Test Example 9. A medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment is prepared based on the culture solution for the mesodermal induction process of Test Example 13, The first cardiomyocyte induction treatment and the second myocardial cell induction treatment were performed in the same manner as in Test Example 9 except that the respective media were prepared with and without insulin (10 ng / mL). Cardiomyocyte induction treatment between cell induction treatments was performed. Further, in the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed for the control.
＜＜洗浄処理＞＞
　前記試験例９と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 9, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　前記試験例９において、試験例９の中胚葉誘導工程用培養液に基づいて第２の心筋細胞誘導処理用培地を調製していた点を、本試験例１３の中胚葉誘導工程用培養液に基づいて第２の心筋細胞誘導処理用培地を調製し、また、インスリン（１０ｎｇ／ｍＬ）の添加を行った場合と、行わなかった場合のそれぞれの培地を調製した以外は、試験例９と同様にして、第２の心筋細胞誘導処理を行った。また、前記試験例９と同様にして、コントロールについても第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
In Test Example 9, the second cardiomyocyte induction treatment medium was prepared on the basis of the culture medium for mesoderm induction process of Test Example 9, and the culture medium for mesoderm induction process of Test Example 13 was used. A second cardiomyocyte induction treatment medium was prepared based on the same procedure as in Test Example 9 except that each medium was prepared with and without the addition of insulin (10 ng / mL). Then, the second cardiomyocyte induction treatment was performed. Further, in the same manner as in Test Example 9, a second cardiomyocyte induction treatment was performed for the control.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図２１に示す。
　なお、本試験例１３では、各処理におけるインスリンの添加の有無は同一とした。即ち、第１の心筋細胞誘導処理用培養液にインスリンを添加した場合には、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液、及び第２の心筋細胞誘導処理用培養液においてもインスリンを添加し、第１の心筋細胞誘導処理用培養液にインスリンを添加しなかった場合には、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液、及び第２の心筋細胞誘導処理用培養液においてもインスリンを添加しなかった。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG.
In Test Example 13, the presence or absence of addition of insulin in each treatment was the same. That is, when insulin is added to the first cardiomyocyte induction treatment medium, the cardiomyocyte induction treatment medium between the first cardiomyocyte induction process and the second cardiomyocyte induction process, and In the case where insulin is also added to the second cardiomyocyte induction treatment medium and no insulin is added to the first cardiomyocyte induction treatment medium, the first cardiomyocyte induction treatment, the second cardiomyocyte induction treatment, Neither insulin was added to the cardiomyocyte-inducing culture medium during the cardiomyocyte-inducing process or the second cardiomyocyte-inducing culture medium.
　図２１中、トランスフェリン、及びインスリンのそれぞれについて、培養液中に含まれる場合を＋、含まれない場合を－で示した。
　図２１の結果から、トランスフェリンを含まなくてもコントロール（従来使用されていたＳｔｅｍＰｒｏ３４）を上回る分化効率を示すが、トランスフェリンを含むほうが、分化効率が優れることがわかった。また、心筋細胞誘導工程で用いる培地にインスリンが含まれていてもコントロールを上回る分化効率を示すが、インスリンを含まないほうが、分化効率が優れることもわかった。 In FIG. 21, transferrin and insulin are indicated by + when they are contained in the culture solution and by-when they are not contained.
The results of FIG. 21 show that the differentiation efficiency is higher than that of the control (StemPro34 conventionally used) even without transferrin, but the differentiation efficiency is superior when transferrin is included. Moreover, although the differentiation efficiency exceeding control was shown even if insulin was contained in the culture medium used at the cardiomyocyte induction | guidance | derivation process, it turned out that differentiation efficiency is excellent when it does not contain insulin.
（試験例１４）
　浮遊培養時の酸素条件、及び中胚葉誘導工程の培地へのインスリンの添加の有無の検討を以下のようにして行った。 (Test Example 14)
The oxygen conditions during suspension culture and the presence / absence of insulin addition to the medium in the mesoderm induction process were examined as follows.
＜多能性幹細胞＞
　前記試験例５と同様に、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 5, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　浮遊培養時の酸素条件を、（１）低酸素条件（酸素濃度５％）、又は（２）低酸素でない条件（酸素濃度２１％）とした以外は、前記試験例９と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
The embryo was treated in the same manner as in Test Example 9 except that the oxygen condition during suspension culture was (1) hypoxic condition (oxygen concentration 5%) or (2) non-hypoxic condition (oxygen concentration 21%). A body forming step was performed.
＜洗浄工程＞
　前記試験例９と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 9.
＜中胚葉誘導工程＞
　中胚葉誘導工程用培地として、前記試験例５の中胚葉誘導工程用培養液に、インスリン（１０ｎｇ／ｍＬ）を添加した培養液、又は添加しなかった培養液を用い、また、浮遊培養時の酸素条件を、（１）低酸素条件（酸素濃度５%）、又は（２）低酸素でない条件（酸素濃度２１％）とした以外は、前記試験例９と同様にして、中胚葉誘導工程を行った。
　また、コントロール（以下、「ＳＰ３４」と称することがある）として、ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、ＣＨＩＲ９９０２１（ミリポア社製、４μＭ）、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加した中胚葉誘導工程用培養液についても同様にして、中胚葉誘導工程を行った。なお、コントロールにおける浮遊培養時の酸素条件は、低酸素条件である。 <Mesodermal induction process>
As a medium for mesoderm induction process, a culture medium to which insulin (10 ng / mL) was added or not added to the culture medium for mesoderm induction process of Test Example 5 was used. The mesoderm induction process was performed in the same manner as in Test Example 9 except that the oxygen condition was (1) low oxygen condition (oxygen concentration 5%) or (2) non-low oxygen condition (oxygen concentration 21%). went.
Further, as a control (hereinafter sometimes referred to as “SP34”), StemPro (registered trademark) 34 (manufactured by Invitrogen), CHIR99021 (manufactured by Millipore, 4 μM), and L-glutamine (Invitrogen) as a medium additive. 2 mM), transferrin (Sigma, 150 μg / mL), ascorbic acid (Sigma, 50 μg / mL), and 1-thioglycerol (Sigma, 50 μg / mL) for mesoderm induction process Similarly, the mesoderm induction process was performed for the culture solution. The oxygen condition during suspension culture in the control is a hypoxic condition.
＜洗浄工程＞
　前記試験例９と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 9, the embryoid body after mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　第１の心筋細胞誘導処理用培地として、インスリンを追加しなかった以外は、前記試験例９における第１の心筋細胞誘導処理用培養液と同様とした培地を用い、浮遊培養時の酸素条件を、（１）低酸素条件（酸素濃度５％）、又は（２）低酸素でない条件（酸素濃度２１％）とした以外は、前記試験例９と同様にして、第１の心筋細胞誘導処理を行った。
　また、前記試験例９と同様にして、コントロールについても第１の心筋細胞誘導処理を行った。なお、コントロールにおける浮遊培養時の酸素条件は、低酸素条件である。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
As the first medium for inducing cardiomyocyte treatment, a medium similar to that in the first cardiomyocyte inducing treatment medium in Test Example 9 was used except that insulin was not added. The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that (1) hypoxic conditions (oxygen concentration 5%) or (2) non-hypoxic conditions (oxygen concentration 21%) were used. went.
Further, in the same manner as in Test Example 9, the first cardiomyocyte induction treatment was performed for the control. The oxygen condition during suspension culture in the control is a hypoxic condition.
＜＜洗浄処理＞＞
　前記試験例９と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 9, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培地として、インスリンを追加しなかった以外は、前記試験例９における第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液と同様とした培地を用い、浮遊培養時の酸素条件を、（１）低酸素条件（酸素濃度５％）、又は（２）低酸素でない条件（酸素濃度２１％）とした以外は、前記試験例９と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。
　また、前記試験例９と同様にして、コントロールについても第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。なお、コントロールにおける浮遊培養時の酸素条件は、低酸素条件である。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
The first cardiomyocyte induction treatment in Test Example 9 except that insulin was not added as a medium for cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment. Using a medium similar to the culture solution for cardiomyocyte induction treatment during the second cardiomyocyte induction treatment, the oxygen condition at the time of suspension culture is (1) low oxygen condition (oxygen concentration 5%), or ( 2) A cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process in the same manner as in Test Example 9 except that the condition is not low oxygen (oxygen concentration 21%). Went.
Further, in the same manner as in Test Example 9, a cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed for the control. The oxygen condition during suspension culture in the control is a hypoxic condition.
＜＜洗浄処理＞＞
　前記試験例９と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 9, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　第２の心筋細胞誘導処理用培地として、インスリンを追加しなかった以外は、前記試験例９における第２の心筋細胞誘導処理用培養液と同様とした培地を用い、浮遊培養時の酸素条件を、（１）低酸素条件（酸素濃度５％）、又は（２）低酸素でない条件（酸素濃度２１％）とした以外は、前記試験例９と同様にして、第２の心筋細胞誘導処理を行った。
　また、前記試験例９と同様にして、コントロールについても第２の心筋細胞誘導処理を行った。なお、コントロールにおける浮遊培養時の酸素条件は、低酸素条件である。 << Second cardiomyocyte induction process >>
The medium for the second cardiomyocyte induction treatment was the same as that for the second cardiomyocyte induction treatment in Test Example 9 except that no insulin was added. The second cardiomyocyte induction treatment was performed in the same manner as in Test Example 9 except that (1) hypoxic condition (oxygen concentration 5%), or (2) non-hypoxic condition (oxygen concentration 21%). went.
Further, in the same manner as in Test Example 9, a second cardiomyocyte induction treatment was performed for the control. The oxygen condition during suspension culture in the control is a hypoxic condition.
＜評価＞
　前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図２２に示す。
　なお、本試験例１４では、各工程における浮遊培養時の酸素条件は同一とした。即ち、胚様体形成工程における酸素条件を低酸素条件とした場合には、中胚葉誘導工程、及び心筋細胞誘導工程における酸素条件は、低酸素条件とした。 <Evaluation>
In the same manner as in Test Example 1, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG.
In Test Example 14, the oxygen conditions during suspension culture in each step were the same. That is, when the oxygen condition in the embryoid body formation process is a low oxygen condition, the oxygen condition in the mesoderm induction process and the cardiomyocyte induction process is a low oxygen condition.
　図２２中、低酸素条件の場合を＋、低酸素でない条件の場合を－で示し、中胚葉誘導工程における培養液にインスリンが含まれる場合を＋、含まれない場合を－で示した。
　図２２の結果から、中胚葉誘導工程における培養液がインスリンを含まないことで、コントロール（従来使用されていたＳｔｅｍＰｒｏ３４）を上回る分化効率を示すことがわかった。また、従来の分化誘導方法は、低酸素条件下で行われていたが、本発明の培地を用いることにより、低酸素でない条件でもコントロールよりも優れた分化効率を示すことがわかった。 In FIG. 22, the case of hypoxic condition is indicated by +, the case of non-hypoxic condition is indicated by-, the case where insulin is contained in the culture medium in the mesoderm induction step is indicated by +, and the case where it is not included is indicated by-.
From the result of FIG. 22, it was found that the culture solution in the mesoderm induction step does not contain insulin, and thus shows a differentiation efficiency higher than that of the control (StemPro34 used conventionally). Moreover, although the conventional differentiation-inducing method was performed under hypoxic conditions, it was found that by using the medium of the present invention, differentiation efficiency superior to that of the control was exhibited even under non-hypoxic conditions.
（試験例１５）
　各種培地の検討を以下のようにして行った。 (Test Example 15)
Various media were examined as follows.
＜培地の調製＞
（１）　ＳｔｅｍＰｒｏ（登録商標）３４（インビトロジェン社製）に、培地添加剤として、Ｌ－グルタミン（インビトロジェン社製、２ｍＭ）、トランスフェリン（シグマ社製、１５０μｇ／ｍＬ）、アスコルビン酸（シグマ社製、５０μｇ／ｍＬ）、及び１－チオグリセロール（シグマ社製、５０μｇ／ｍＬ）を添加した培地（以下、「ＳｔｅｍＰｒｏ３４」と称することがある）を調製した。 <Preparation of medium>
(1) To StemPro (registered trademark) 34 (manufactured by Invitrogen), L-glutamine (manufactured by Invitrogen, 2 mM), transferrin (manufactured by Sigma, 150 μg / mL), ascorbic acid (manufactured by Sigma, 50 μg / mL) and 1-thioglycerol (manufactured by Sigma, 50 μg / mL) were added (hereinafter sometimes referred to as “StemPro34”).
（２）　ＲｅｐｒｏＦＦ２（株式会社リプロセル製、以下、「ＲｅｐｒｏＦＦ２」と称することがある）を用意した。 (2) ReproFF2 (manufactured by Reprocell, Inc., hereinafter sometimes referred to as “ReproFF2”) was prepared.
（３）　基礎培地として、ＩＭＤＭ（ナカライテスク株式会社製）を用い、前記基礎培地に、培地添加剤として、下記表６－１～表６－２に記載の濃度となるように各成分を添加した培地（以下、「一液式培地」と称することがある）を調製した。 (3) IMDM (manufactured by Nacalai Tesque Co., Ltd.) is used as the basal medium, and each component is added to the basal medium as a medium additive to the concentrations shown in Table 6-1 to Table 6-2 below. A prepared medium (hereinafter sometimes referred to as “one-component medium”) was prepared.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
（４）　基礎培地として、ＤＭＥＭ／Ｆ１２（ナカライテスク株式会社製）を用い、前記基礎培地に、培地添加剤として、下記表７－１～表７－２に記載の濃度となるように各成分を添加した培地（以下、「第一培地」と称することがある）を調製した。 (4) DMEM / F12 (manufactured by Nacalai Tesque Co., Ltd.) is used as the basal medium, and each component is added to the basal medium as the medium additive to the concentrations shown in Table 7-1 to Table 7-2 below. (Hereinafter sometimes referred to as “first medium”) was prepared.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
（５）　基礎培地として、ＩＭＤＭ（ナカライテスク株式会社製）を用い、前記基礎培地に、培地添加剤として、下記表８に記載の濃度となるように各成分を添加した培地（以下、「第二培地」と称することがある）を調製した。 (5) As a basal medium, IMDM (manufactured by Nacalai Tesque Co., Ltd.) was used, and a medium (hereinafter referred to as “No. 2 medium), sometimes referred to as “two media”.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
＜多能性幹細胞＞
　前記試験例５と同様に、国立大学法人　東京大学医学部附属病院にて樹立したヒトｉＰＳ細胞（クローン３）を用いた。 <Pluripotent stem cells>
In the same manner as in Test Example 5, human iPS cells (clone 3) established at the University of Tokyo Hospital were used.
＜胚様体形成工程＞
　上記で調製した培地（ＳｔｅｍＰｒｏ３４、ＲｅｐｒｏＦＦ２、一液式培地、又は第一培地）に、分化誘導調節剤として、Ｙ２７６３２（シグマ社製、５μＭ）を添加した培地を胚様体形成工程用培養液とした以外は、試験例５と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
A medium obtained by adding Y27632 (manufactured by Sigma, 5 μM) as a differentiation induction regulator to the medium prepared above (StemPro34, ReproFF2, one-part medium, or first medium) The embryoid body formation process was performed like Test Example 5 except having performed.
＜洗浄工程＞
　前記試験例５と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 5.
＜中胚葉誘導工程＞
　上記で調製した培地（ＳｔｅｍＰｒｏ３４、一液式培地、又は第二培地）に、分化誘導調節剤として、ＣＨＩＲ９９０２１（ミリポア社製、４μＭ）を添加した培地を中胚葉誘導工程用培養液とした以外は、試験例５と同様にして、中胚葉誘導工程を行った。 <Mesodermal induction process>
A medium prepared by adding CHIR99021 (Millipore, 4 μM) as a differentiation-inducing regulator to the medium prepared above (StemPro34, one-part medium, or second medium) was used as a medium for mesoderm induction process. In the same manner as in Test Example 5, a mesoderm induction step was performed.
＜洗浄工程＞
　前記試験例５と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 5, the embryoid body after mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　上記で調製した培地（ＳｔｅｍＰｒｏ３４、一液式培地、又は第二培地）に、分化誘導調節剤として、ＩＷＰ２（シグマ社製、５μＭ）、ＳＢ４３１５４２（シグマ社製、５μＭ）、及びエストラジオール（シグマ社製、１００ｎＭ）を添加した培地を第１の心筋細胞誘導処理用培養液とした以外は、試験例５と同様にして、第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
As a differentiation induction regulator, IWP2 (Sigma, 5 μM), SB431542 (Sigma, 5 μM), and estradiol (Sigma) are used as the differentiation induction regulators in the above-prepared medium (StemPro34, one-pack medium, or second medium). , 100 nM), the first cardiomyocyte induction treatment was performed in the same manner as in Test Example 5 except that the culture medium for the first cardiomyocyte induction treatment was used.
＜＜洗浄処理＞＞
　前記試験例５と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 5, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　上記で調製した培地（ＳｔｅｍＰｒｏ３４、一液式培地、又は第二培地）に、分化誘導調節剤として、ＩＷＰ２（シグマ社製、５μＭ）、及びエストラジオール（シグマ社製、１００ｎＭ）を添加した培地を第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液とした以外は、試験例５と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
A medium prepared by adding IWP2 (manufactured by Sigma, 5 μM) and estradiol (manufactured by Sigma, 100 nM) as a differentiation induction regulator to the medium prepared above (StemPro34, one-pack medium, or second medium) The first cardiomyocyte induction treatment, the second cardiomyocyte induction treatment, and the second cardiomyocyte induction treatment, in the same manner as in Test Example 5, except that the culture solution for the cardiomyocyte induction treatment is used. Cardiomyocyte induction treatment was performed between the cardiomyocyte induction treatment.
＜＜洗浄処理＞＞
　前記試験例５と同様にして、前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 5, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　上記で調製した培地（ＳｔｅｍＰｒｏ３４、一液式培地、又は第二培地）に、分化誘導調節剤として、エストラジオール（シグマ社製、１００ｎＭ）を添加した培地を第２の心筋細胞誘導処理用培養液とした以外は、試験例５と同様にして、第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
A medium obtained by adding estradiol (manufactured by Sigma, 100 nM) as a differentiation-inducing regulator to the medium prepared above (StemPro34, one-component medium, or second medium) is a second cardiomyocyte-inducing culture medium. Except that, the second cardiomyocyte induction treatment was performed in the same manner as in Test Example 5.
　本試験例１５の各工程における培地と、分化誘導調節剤とを下記表９に示す。 Table 9 below shows media and differentiation-inducing regulators in each step of Test Example 15.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
＜評価＞
　胚様体形成工程開始８日目、１２日目、及び１６日目に測定した以外は、前記試験例１と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図２３に示す。
　図２３の結果から、本発明の培地と、分化誘導調節剤との組合せを用いたＢ－１、及びＦ－１では、従来の培地を用いたＡ－１、Ｃ－１、Ｄ－１、及びＥ－１よりも優れた分化効率が得られることが示された。また、一液式の培地であるＢ－１よりも、二液式の培地としたＦ－１のほうが心筋細胞の分化誘導効率に優れていることが示された。 <Evaluation>
A beating embryo in 50 embryoid bodies arbitrarily selected in the same manner as in Test Example 1 except that measurement was performed on the 8th, 12th, and 16th days from the start of the embryoid body formation process. The ratio of the body was obtained. The results are shown in FIG.
From the results shown in FIG. 23, in B-1 and F-1 using the combination of the culture medium of the present invention and the differentiation-inducing regulator, A-1, C-1, D-1, It was shown that differentiation efficiency superior to that of E-1 was obtained. In addition, it was shown that the cardiomyocyte differentiation induction efficiency was superior to F-1, which was a two-part medium, than B-1, which was a one-part medium.
（試験例１６）
　分化誘導調節剤の検討を以下のようにして行った。 (Test Example 16)
The differentiation induction regulator was examined as follows.
＜培地の調製＞
　前記試験例１５と同様にして、第一培地、及び第二培地を調製した。 <Preparation of medium>
In the same manner as in Test Example 15, a first medium and a second medium were prepared.
＜多能性幹細胞＞
　多能性幹細胞として、前記試験例１で用いたヒトｉＰＳ細胞のうち、クローン１を用いた。 <Pluripotent stem cells>
Of the human iPS cells used in Test Example 1, clone 1 was used as the pluripotent stem cell.
＜胚様体形成工程＞
　上記で調製した第一培地に、分化誘導調節剤として、ＢＭＰ４（Ｒ＆Ｄ社製、０．５ｎｇ／ｍＬ）、又はＹ２７６３２（シグマ社製、５μＭ）を添加した培地を胚様体形成工程用培養液とした以外は、試験例５と同様にして、胚様体形成工程を行った。 <Embryoid body formation process>
A medium obtained by adding BMP4 (manufactured by R & D, 0.5 ng / mL) or Y27632 (manufactured by Sigma, 5 μM) as a differentiation-inducing regulator to the first medium prepared above is a culture solution for embryoid body formation process. An embryoid body formation step was performed in the same manner as in Test Example 5 except that.
＜洗浄工程＞
　前記試験例５と同様にして、前記胚様体を洗浄した。 <Washing process>
The embryoid body was washed in the same manner as in Test Example 5.
＜中胚葉誘導工程＞
　上記で調製した第二培地に、分化誘導調節剤として、（１）Ａｃｔｉｖｉｎ　Ａ（Ｒ＆Ｄ社製、６ｎｇ／ｍＬ）、ＢＭＰ４（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、及びｂＦＧＦ（ＦＧＦ２、Ｒ＆Ｄ社製、５ｎｇ／ｍＬ）、又は（２）ＣＨＩＲ９９０２１（ミリポア社製、４μＭ）を添加した培地を中胚葉誘導工程用培養液とし、浮遊培養の期間を胚様体形成工程開始１日目～３日目、又は１日目～４日目とした以外は、試験例５と同様にして、中胚葉誘導工程を行った。 <Mesodermal induction process>
In the second medium prepared above, as a differentiation induction regulator, (1) Activin A (R & D, 6 ng / mL), BMP4 (R & D, 10 ng / mL), and bFGF (FGF2, R & D, 5 ng / mL), or (2) a medium supplemented with CHIR99021 (Millipore, 4 μM) is used as a culture solution for the mesoderm induction process, and the period of suspension culture is defined as the embryoid body formation process from the first day to the third day, Alternatively, the mesoderm induction process was performed in the same manner as in Test Example 5 except that the first to fourth days were used.
＜洗浄工程＞
　前記試験例５と同様にして、前記中胚葉誘導後の胚様体を洗浄した。 <Washing process>
In the same manner as in Test Example 5, the embryoid body after mesoderm induction was washed.
＜心筋細胞誘導工程＞
＜＜第１の心筋細胞誘導処理＞＞
　上記で調製した第二培地に、分化誘導調節剤として、（１）Ｄｋｋ１（Ｒ＆Ｄ社製、１５０ｎｇ／ｍＬ）、ＳＢ４３１５４２（シグマ社製、５μＭ）、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、又は（２）ＩＷＰ２（シグマ社製、５μＭ）、ＳＢ４３１５４２（シグマ社製、５μＭ）、及びエストラジオール（シグマ社製、１００ｎＭ）を添加した培地を第１の心筋細胞誘導処理用培養液とし、浮遊培養の期間を胚様体形成工程開始３日目～６日目、又は４日目～６日目とした以外は、試験例５と同様にして、第１の心筋細胞誘導処理を行った。 <Cardiomyocyte induction process>
<< First cardiomyocyte induction process >>
In the second medium prepared above, as a differentiation induction regulator, (1) Dkk1 (R & D, 150 ng / mL), SB431542 (Sigma, 5 μM), and VEGF (R & D, 10 ng / mL), Or (2) Suspension culture using a medium supplemented with IWP2 (Sigma, 5 μM), SB431542 (Sigma, 5 μM), and estradiol (Sigma, 100 nM) as the first cardiomyocyte induction culture medium The first cardiomyocyte induction treatment was performed in the same manner as in Test Example 5 except that the period of 3 was 6 days or 4 days to 6 days from the start of the embryoid body formation process.
＜＜洗浄処理＞＞
　前記試験例５と同様にして、前記第１の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 5, the embryoid body after the first cardiomyocyte induction treatment was washed.
＜＜第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理＞＞
　上記で調製した第二培地に、分化誘導調節剤として、（１）Ｄｋｋ１（Ｒ＆Ｄ社製、１５０ｎｇ／ｍＬ）、ＳＢ４３１５４２（シグマ社製、５μＭ）、及びＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、又は（２）ＩＷＰ２（シグマ社製、５μＭ）、及びエストラジオール（シグマ社製、１００ｎＭ）を添加した培地を第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理用培養液とした以外は、試験例５と同様にして、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理を行った。 << Cardiomyocyte Inducing Process Between First Cardiomyocyte Inducing Process and Second Cardiomyocyte Inducing Process >>
In the second medium prepared above, as a differentiation induction regulator, (1) Dkk1 (R & D, 150 ng / mL), SB431542 (Sigma, 5 μM), and VEGF (R & D, 10 ng / mL), Or (2) cardiomyocyte induction between the first cardiomyocyte induction treatment and the medium containing IWP2 (Sigma, 5 μM) and estradiol (Sigma, 100 nM) added A cardiomyocyte induction process between the first cardiomyocyte induction process and the second cardiomyocyte induction process was performed in the same manner as in Test Example 5 except that the culture solution for treatment was used.
＜＜洗浄処理＞＞
　前記試験例５と同様にして、前記第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間の心筋細胞誘導処理後の胚様体を洗浄した。 << Cleaning process >>
In the same manner as in Test Example 5, the embryoid body after the cardiomyocyte induction treatment between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment was washed.
＜＜第２の心筋細胞誘導処理＞＞
　上記で調製した培地（ＳｔｅｍＰｒｏ３４、一液式培地、又は第二培地）に、分化誘導調節剤として、エストラジオール（シグマ社製、１００ｎＭ）を添加した培地を第２の心筋細胞誘導処理用培養液とした以外は、試験例５と同様にして、第２の心筋細胞誘導処理を行った。
　上記で調製した第二培地に、分化誘導調節剤として、（１）ＶＥＧＦ（Ｒ＆Ｄ社製、１０ｎｇ／ｍＬ）、及びｂＦＧＦ（ＦＧＦ２、Ｒ＆Ｄ社製、５ｎｇ／ｍＬ）、又は（２）エストラジオール（シグマ社製、１００ｎＭ）を添加した培地を第２の心筋細胞誘導処理用培養液とした以外は、試験例５と同様にして、第２の心筋細胞誘導処理を行った。 << Second cardiomyocyte induction process >>
A medium obtained by adding estradiol (manufactured by Sigma, 100 nM) as a differentiation-inducing regulator to the medium prepared above (StemPro34, one-component medium, or second medium) is a second cardiomyocyte-inducing culture medium. Except that, the second cardiomyocyte induction treatment was performed in the same manner as in Test Example 5.
In the second medium prepared above, (1) VEGF (manufactured by R & D, 10 ng / mL) and bFGF (FGF2, R & D, 5 ng / mL), or (2) estradiol (Sigma) A second cardiomyocyte induction treatment was performed in the same manner as in Test Example 5 except that the medium supplemented with 100 nM) was used as the second culture medium for cardiomyocyte induction treatment.
　本試験例１６の各工程における分化誘導調節剤と、培地とを下記表１０に示す。 Table 10 below shows the differentiation induction regulator and the medium in each step of Test Example 16.
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
＜評価－１＞
　前記試験例１５と同様にして、任意に選択した５０個の胚様体における拍動している胚様体の割合を求めた。結果を図２４に示す。
　図２４の結果から、本発明の培地と、分化誘導調節剤との組合せを用いたＣ－２では、本発明の培地と、分化誘導調節剤との組合せではないＡ－２、及びＢ－２よりも優れた分化効率が得られることが示された。 <Evaluation-1>
In the same manner as in Test Example 15, the ratio of beating embryoid bodies in 50 arbitrarily selected embryoid bodies was determined. The results are shown in FIG.
From the results of FIG. 24, in C-2 using the combination of the culture medium of the present invention and the differentiation induction regulator, A-2 and B-2 which are not combinations of the culture medium of the present invention and the differentiation induction regulator It was shown that better differentiation efficiency can be obtained.
＜評価－２＞
　本試験例１６の前記Ｃ－２により分化誘導を行い、得られた心筋細胞をパッチクランプ法により評価した。結果の一例を図２５Ａ～図２５Ｄに示す。また、これまでに報告されているデータを図２６（リプロセル株式会社のホームページより）及び図２７（Ａｍ　Ｊ　Ｐｈｙｓｉｏｌ　Ｈｅａｒｔ　Ｃｉｒｃ　Ｐｈｙｓｉｏｌ　３０１：　Ｈ２００６－Ｈ２０１７，　２０１１）に示す。
－パッチクランプ法－
　得られた心筋細胞をＥＢから単離し、ゼラチンをコートしたカバーグラス上に播種した。５～１０日間培養した後、自発的に拍動している単細胞をホールセル状態にし、活動電位は電流固定下（０ｐＡ）で自発性の活動電位を測定し、各種イオンチャネル電流は電位固定下で観察した。細胞外液の組成は、ＮａＣｌ　１５０ｍｍｏｌ／Ｌ、ＫＣｌ　４ｍｍｏｌ／Ｌ、ＣａＣｌ２　１．２ｍｍｏｌ／Ｌ、ＭｇＣｌ２　１ｍｍｏｌ／Ｌ、Ｄ（＋）－Ｇｌｕｃｏｓｅ　１０ｍｍｏｌ／Ｌ、ＨＥＰＥＳ　１０ｍｍｏｌ／Ｌ（ＮａＯＨを用いてｐＨ７．４に調整）のものを用いた。電極内液の組成は、ＫＣｌ　１４０ｍｍｏｌ／Ｌ、ＭｇＣｌ２　１ｍｍｏｌ／Ｌ、ＥＧＴＡ　５ｍｍｏｌ／Ｌ、ＭｇＡＴＰ　５ｍｍｏｌ／Ｌ、ＨＥＰＥＳ　１０ｍｍｏｌ／Ｌ（ＫＯＨを用いてｐＨ７．２に調整）のものを用いた。
　イオンチャネル電流を測定する際の電位プロトコルは以下を用いた。
　・　ナトリウム電流　：　保持電位－９０ｍＶから－２０ｍＶまで２０ミリ秒の脱分極刺激を１秒間隔で与えた。
　・　カルシウム電流　：　保持電位－４０ｍＶから－０ｍＶまで１００ミリ秒の脱分極刺激を１０秒間隔で与えた。
　・　カリウム電流　：　保持電位－９０ｍＶから－４０ｍＶまで５０ミリ秒の脱分極の後、２０ｍＶ、２秒の脱分極、更に－４０ｍＶ、０．５秒に再分極させる刺激を１５秒間隔で与えた。 <Evaluation-2>
Differentiation was induced by C-2 of Test Example 16, and the obtained cardiomyocytes were evaluated by the patch clamp method. An example of the results is shown in FIGS. 25A to 25D. The data reported so far is shown in FIG. 26 (from the website of Reprocell Corporation) and FIG. 27 (Am J Physiol Heart Circ Phys 301: H2006-H2017, 2011).
-Patch clamp method-
The obtained cardiomyocytes were isolated from EB and seeded on a gelatin-coated cover glass. After culturing for 5 to 10 days, spontaneously beating single cells are put into a whole-cell state, the action potential is measured at a fixed current (0 pA), and the spontaneous action potential is measured. Various ion channel currents are fixed at a potential. Observed at. The composition of the extracellular fluid was NaCl 150 mmol / L, KCl 4 mmol / L, CaCl 2 1.2 mmol / L, MgCl 2 1 mmol / L, D (+)-Glucose 10 mmol / L, HEPES 10 mmol / L (using NaOH adjusted to pH 7.4). The composition of the electrode internal solution used was KCl 140 mmol / L, MgCl 2 1 mmol / L, EGTA 5 mmol / L, MgATP 5 mmol / L, HEPES 10 mmol / L (adjusted to pH 7.2 using KOH).
The following was used as the potential protocol for measuring the ion channel current.
Sodium current: A depolarization stimulus of 20 milliseconds from a holding potential of −90 mV to −20 mV was applied at 1 second intervals.
Calcium current: A depolarization stimulus of 100 milliseconds was applied at intervals of 10 seconds from a holding potential of −40 mV to −0 mV.
Potassium current: After 50 milliseconds of depolarization from a holding potential of -90 mV to -40 mV, a stimulus of 20 mV, 2 seconds of depolarization, and further -40 mV, repolarization to 0.5 seconds was given at 15 second intervals.
　図２５Ａは、活動電位を測定した結果を示し、図２５Ｂは、ナトリウム電流を測定した結果を示し、図２５Ｃは、カリウム電流を測定した結果を示し、図２５Ｄは、カルシウム電流を測定した結果を示す。
　図２５Ａ～図２７の結果から、本発明の方法により分化誘導された心筋細胞は、９５％が活動電位振幅１４０ｍＶ以上の大きな活動電位を有しており、過去に報告されているｉＰＳ細胞由来心筋細胞と比べて、大きく明瞭なプラトー相を有する活動電位が確認された。また、本発明の方法により分化誘導された心筋細胞は、３００ｐＡ以上のピークカリウム電流、１ｎＡ以上のピークカルシウム電流、及び６．５ｎＡ以上のピークナトリウム電流を示していた。したがって、本発明の方法により分化誘導された心筋細胞は、高品質な心筋細胞であることが示された。 FIG. 25A shows the result of measuring action potential, FIG. 25B shows the result of measuring sodium current, FIG. 25C shows the result of measuring potassium current, and FIG. 25D shows the result of measuring calcium current. Show.
From the results of FIGS. 25A to 27, 95% of cardiomyocytes induced to differentiate by the method of the present invention have a large action potential with an action potential amplitude of 140 mV or more, and iPS cell-derived myocardium reported in the past. An action potential having a large and clear plateau phase as compared with the cells was confirmed. The cardiomyocytes induced to differentiate by the method of the present invention exhibited a peak potassium current of 300 pA or more, a peak calcium current of 1 nA or more, and a peak sodium current of 6.5 nA or more. Therefore, it was shown that the cardiomyocytes induced to differentiate by the method of the present invention are high-quality cardiomyocytes.
　また、Ｗｎｔシグナル活性化物質及びＷｎｔシグナル抑制物質だけでは、心筋細胞への分化誘導効率は非常に低効率であることから、ＴＧＦ－βシグナル阻害剤及びエストラジオールを追加することが、接着培養系を介することなく浮遊培養系のみで心筋細胞へ分化させるために必要であったと推察される。 In addition, the Wnt signal activator and Wnt signal suppressor alone have very low efficiency in inducing differentiation into cardiomyocytes. Therefore, the addition of a TGF-β signal inhibitor and estradiol can improve the adhesion culture system. It is presumed that it was necessary to differentiate into cardiomyocytes only in the suspension culture system without intervention.
　本発明の多能性幹細胞から心筋細胞を分化誘導する方法によれば、浮遊培養開始後に接着培養を介することなく心筋細胞を分化誘導することができるので、大量に、かつ簡便に高品質な心筋細胞を製造することができる。
　また、本発明の多能性幹細胞から心筋細胞を分化誘導する方法では、安定して入手可能であり、ロット間差が少ない成分を分化誘導調節剤として用いるため、安定して高品質な心筋細胞を製造することができる。
　また、本発明の多能性幹細胞から心筋細胞を分化誘導する方法に用いる培地は、安価な成分で構成されているため、安価に高品質な心筋細胞を製造することができる。 According to the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention, cardiomyocytes can be induced to differentiate without going through adhesion culture after the start of suspension culture. Cells can be produced.
Further, in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention, a stable and high-quality cardiomyocyte is obtained because a component that is stably available and has little difference between lots is used as a differentiation induction regulator. Can be manufactured.
In addition, since the medium used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention is composed of inexpensive components, high-quality cardiomyocytes can be produced at low cost.
　更に、本発明の多能性幹細胞から心筋細胞を分化誘導する方法に用いる培地は、毒物（例えば、２－メルカプトエタノール）や、成分が明らかでない脂質成分（例えば、Ｈｕｍａｎ－ＥｘＣｙｔｅ）を含まないので、安全性にも優れる。 Furthermore, since the medium used in the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention does not contain a toxic substance (for example, 2-mercaptoethanol) or a lipid component (for example, Human-ExCyte) whose component is not clear. Excellent safety.
　また、本発明の多能性幹細胞から心筋細胞を分化誘導する方法によれば、クローン間差を問わずに、高品質な心筋細胞を製造することができる。 Moreover, according to the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention, high-quality cardiomyocytes can be produced regardless of differences between clones.
　また、本発明の多能性幹細胞から心筋細胞を分化誘導する方法では、ｉＰＳ細胞の単細胞培養、細胞外基質の培養皿へのコーティング、剥離しやすい細胞の培地交換、厳密なタイミングの培地交換、分化心筋の再浮遊などの特別な技術を用いなくても高品質な心筋細胞を製造することができる。 In the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells of the present invention, iPS cell single-cell culture, coating of extracellular matrix on a culture dish, medium exchange of cells that are easily detached, medium exchange at strict timing, High-quality cardiomyocytes can be produced without using special techniques such as resuspension of differentiated myocardium.
　本発明の態様としては、例えば、以下のものなどが挙げられる。
　＜１＞　多能性幹細胞を浮遊培養し、胚様体を形成する胚様体形成工程と、
　前記胚様体を浮遊培養し、中胚葉を誘導する中胚葉誘導工程と、
　前記中胚葉誘導後の胚様体を浮遊培養し、心筋細胞を誘導する心筋細胞誘導工程とを含み、
　前記心筋細胞誘導工程が、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理とを含み、
　前記胚様体形成工程における培地が、第１の分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地、及び一液式分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地のいずれかであり、
　前記中胚葉誘導工程における培地が、第２の分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地のいずれかであり、
　前記第１の心筋細胞誘導処理における培地が、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地のいずれかであり、
　前記第２の心筋細胞誘導処理における培地が、第２の分化誘導培地に、エストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、エストロゲン様作用物質を加えた培地のいずれかであり、
　前記第１の分化誘導培地が、基礎培地に、インスリン、トランスフェリン、アルブミン、及び１－チオグリセロールを加えた培地であり、
　前記第２の分化誘導培地が、イスコフ改変ダルベッコ培地に、アルブミン、ポリビニルアルコール、エタノラミン塩酸塩、亜セレン酸ナトリウム、ヒドロコルチゾン、ＤＬ－α－トコフェロール酢酸エステル、Ｎ－アセチル－Ｌ－システイン、１－チオグリセロール、及びアスコルビン酸を加えた培地であり、
　前記一液式分化誘導培地が、イスコフ改変ダルベッコ培地に、トランスフェリン、アルブミン、ポリビニルアルコール、エタノラミン塩酸塩、亜セレン酸ナトリウム、ヒドロコルチゾン、ＤＬ－α－トコフェロール酢酸エステル、Ｎ－アセチル－Ｌ－システイン、１－チオグリセロール、及びアスコルビン酸を加えた培地であることを特徴とする多能性幹細胞から心筋細胞を分化誘導する方法である。
　＜２＞　胚様体形成工程における培地が、第１の分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地であり、
　中胚葉誘導工程における培地が、第２の分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地であり、
　第１の心筋細胞誘導処理における培地が、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地であり、
　第２の心筋細胞誘導処理における培地が、第２の分化誘導培地に、エストロゲン様作用物質を加えた培地である前記＜１＞に記載の多能性幹細胞から心筋細胞を分化誘導する方法である。
　＜３＞　胚様体形成工程における培地が、一液式分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地であり、
　中胚葉誘導工程における培地が、一液式分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地であり、
　第１の心筋細胞誘導処理における培地が、一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地であり、
　第２の心筋細胞誘導処理における培地が、一液式分化誘導培地に、エストロゲン様作用物質を加えた培地である前記＜１＞に記載の多能性幹細胞から心筋細胞を分化誘導する方法である。
　＜４＞　第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間に、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質を加えた培地のいずれかで培養する処理を含む前記＜１＞から＜３＞のいずれかに記載の多能性幹細胞から心筋細胞を分化誘導する方法である。
　＜５＞　第１の分化誘導培地が、更に、グリシン、Ｌ－アラニン、Ｌ－アスパラギン・Ｈ２Ｏ、Ｌ－アスパラギン酸、Ｌ－グルタミン、Ｌ－グルタミン酸、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏを含み、
　第２の分化誘導培地が、更に、Ｌ－グルタミン、及びトランスフェリンを含み、
　一液式分化誘導培地が、更に、グリシン、Ｌ－グルタミン、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏを含む前記＜１＞から＜４＞のいずれかに記載の多能性幹細胞から心筋細胞を分化誘導する方法である。
　＜６＞　多能性幹細胞から心筋細胞を分化誘導するための培地に添加する培地添加剤であって、
　アルブミン及び１－チオグリセロールの少なくともいずれかを含むことを特徴とする培地添加剤である。
　＜７＞　更に、トランスフェリン、インスリン、ポリビニルアルコール、エタノラミン塩酸塩、亜セレン酸ナトリウム、ヒドロコルチゾン、ＤＬ－α－トコフェロール酢酸エステル、Ｎ－アセチル－Ｌ－システイン、及びアスコルビン酸からなる群から選択される少なくとも１種を含む前記＜６＞に記載の培地添加剤である。
　＜８＞　更に、グリシン、Ｌ－アラニン、Ｌ－アスパラギン・Ｈ２Ｏ、Ｌ－アスパラギン酸、Ｌ－グルタミン、Ｌ－グルタミン酸、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏからなる群から選択される少なくとも１種を含む前記＜６＞から＜７＞のいずれかに記載の培地添加剤である。
　＜９＞　多能性幹細胞から心筋細胞を分化誘導するための培地に添加する分化誘導調節剤であって、
　ＲＯＣＫ阻害剤、Ｗｎｔシグナル活性化物質、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質からなる群から選択される少なくとも１種を含むことを特徴とする分化誘導調節剤である。
　＜１０＞　多能性幹細胞から心筋細胞を分化誘導するための培地であって、
　前記＜６＞から＜８＞のいずれかに記載の培地添加剤と、
　前記＜９＞に記載の分化誘導調節剤とを含み、
　基礎培地が、ダルベッコ改変イーグル培地／栄養混合物Ｆ－１２ハム、ダルベッコ改変イーグル培地、イスコフ改変ダルベッコ培地、ＲＰＭＩ１６４０培地、及びαＭＥＭ培地からなる群から選択される少なくとも１種であることを特徴とする培地である。
　＜１１＞　多能性幹細胞から心筋細胞を分化誘導するための培地作製用キットであって、
　前記＜６＞から＜８＞のいずれかに記載の培地添加剤と、
　前記＜９＞に記載の分化誘導調節剤と、
　ダルベッコ改変イーグル培地／栄養混合物Ｆ－１２ハム、ダルベッコ改変イーグル培地、イスコフ改変ダルベッコ培地、ＲＰＭＩ１６４０培地、及びαＭＥＭ培地からなる群から選択される少なくとも１種の基礎培地とを含むことを特徴とする培地作製用キットである。
　＜１２＞　前記＜１０＞に記載の培地、及び前記＜１１＞に記載の培地作製用キットの少なくともいずれかを含むことを特徴とする多能性幹細胞から心筋細胞を分化誘導するためのキットである。
　＜１３＞　前記＜１＞から＜５＞のいずれかに記載の多能性幹細胞から心筋細胞を分化誘導する方法を教示する説明書を含む前記＜１２＞に記載の幹細胞から心筋細胞を分化誘導するためのキットである。 Examples of the aspect of the present invention include the following.
<1> Embryoid body formation step of suspension culture of pluripotent stem cells to form embryoid bodies,
Mesoderm induction step of culturing the embryoid body in suspension and inducing mesoderm,
A suspension culture of the embryoid body after the mesoderm induction, and a cardiomyocyte induction step of inducing cardiomyocytes,
The cardiomyocyte induction step includes a first cardiomyocyte induction process and a second cardiomyocyte induction process;
The medium in the embryoid body formation step is any one of a medium in which a ROCK inhibitor is added to a first differentiation induction medium, and a medium in which a ROCK inhibitor is added to a one-part differentiation induction medium.
The medium in the mesoderm induction step is any one of a medium obtained by adding a Wnt signal activator to a second differentiation induction medium and a medium obtained by adding a Wnt signal activator to a one-part differentiation induction medium. ,
The medium in the first cardiomyocyte induction treatment is a medium obtained by adding a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like agent to a second differentiation induction medium, and a one-part differentiation induction medium. , A Wnt signal suppressor, a TGF-β signal inhibitor, and a medium containing an estrogen-like agent,
The medium in the second cardiomyocyte induction treatment is either a medium in which an estrogen-like agent is added to the second differentiation-inducing medium, or a medium in which an estrogen-like agent is added to a one-part differentiation induction medium. Yes,
The first differentiation induction medium is a medium obtained by adding insulin, transferrin, albumin, and 1-thioglycerol to a basal medium;
The second differentiation-inducing medium is an Iskov-modified Dulbecco medium that contains albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine, 1-thiol. A medium to which glycerol and ascorbic acid are added,
The one-part differentiation induction medium is transferred to Iscov modified Dulbecco medium with transferrin, albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine, 1 A method for inducing differentiation of cardiomyocytes from pluripotent stem cells, characterized in that the medium is a medium supplemented with thioglycerol and ascorbic acid.
<2> The medium in the embryoid body formation step is a medium obtained by adding a ROCK inhibitor to the first differentiation induction medium,
The medium in the mesoderm induction step is a medium obtained by adding a Wnt signal activator to the second differentiation induction medium,
The medium in the first cardiomyocyte induction treatment is a medium obtained by adding a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like agent to the second differentiation induction medium,
The method for inducing differentiation of cardiomyocytes from pluripotent stem cells according to <1>, wherein the medium in the second cardiomyocyte induction treatment is a medium obtained by adding an estrogen-like agent to the second differentiation induction medium. .
<3> The medium in the embryoid body formation step is a medium in which a ROCK inhibitor is added to a one-part differentiation induction medium,
The medium in the mesoderm induction step is a medium obtained by adding a Wnt signal activator to a one-part differentiation induction medium,
The medium in the first cardiomyocyte induction treatment is a medium in which a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like agent are added to a one-part differentiation induction medium,
The method for inducing differentiation of cardiomyocytes from pluripotent stem cells according to <1>, wherein the medium in the second cardiomyocyte induction treatment is a medium in which an estrogen-like agent is added to a one-part differentiation induction medium. .
<4> Medium obtained by adding a Wnt signal inhibitory substance and an estrogen-like substance to the second differentiation induction medium between the first cardiomyocyte induction process and the second cardiomyocyte induction process, and one solution The pluripotent stem cell to cardiomyocyte according to any one of <1> to <3>, which comprises a treatment of culturing in a medium in which a Wnt signal inhibitory substance and an estrogen-like agent are added to a formula differentiation induction medium Is a method for inducing differentiation.
<5> The first differentiation-inducing medium further contains glycine, L-alanine, L-asparagine / H 2 O, L-aspartic acid, L-glutamine, L-glutamic acid, L-histidine, L-isoleucine, L- Methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, ascorbic acid 2-2PO 4 magnesium Salt, AgNO 3 , AlCl 3 · 6H 2 O, Ba (C 2 H 3 O 2 ) 2 , 3CdSO 4 · 8H 2 O, CoCl 2 · 6H 2 O, Cr 2 (SO 4 ) 3 · H 2 O, GeO 2 , Na 2 SeO 3 , KBr, KI, MnCl 2 .4H 2 O, NaF, Na 2 SiO 3 .9H 2 O, NaVO 3 , (NH 4 ) 6 Mo 7 O 24 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl, SnCl 2 · 2H 2 O, and ZrOCl 2 · 8H 2 O,
The second differentiation induction medium further contains L-glutamine and transferrin;
The one-part differentiation induction medium further contains glycine, L-glutamine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L - tryptophan, L- tyrosine, L- valine, thiamine, reduced glutathione, magnesium salt of ascorbic acid -2-2PO 4, AgNO 3, AlCl 3 · 6H 2 O, Ba (C 2 H 3 O 2) 2, 3CdSO 4 · 8H 2 O, CoCl 2 · 6H 2 O, Cr 2 (SO 4) 3 · H 2 O, GeO 2, Na 2 SeO 3, KBr, KI, MnCl 2 · 4H 2 O, NaF, Na 2 SiO 3 9H 2 O, NaVO 3 , (NH 4 ) 6 Mo 7 O 24 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl, SnCl 2 · 2H 2 O, and a ZrOCl 2 · 8H method of inducing differentiation of cardiomyocytes from pluripotent stem cells according to any one of <4>, wherein the <1> containing 2 O.
<6> A medium additive added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
A medium additive comprising at least one of albumin and 1-thioglycerol.
<7> Further, at least selected from the group consisting of transferrin, insulin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine, and ascorbic acid It is a culture medium additive as described in said <6> containing 1 type.
<8> Further, glycine, L-alanine, L-asparagine / H 2 O, L-aspartic acid, L-glutamine, L-glutamic acid, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L- Proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, magnesium salt of ascorbic acid-2-2PO 4 , AgNO 3 , AlCl 3. 6H 2 O, Ba (C 2 H 3 O 2 ) 2 , 3CdSO 4 .8H 2 O, CoCl 2 .6H 2 O, Cr 2 (SO 4 ) 3 .H 2 O, GeO 2 , Na 2 SeO 3 , KBr , KI, MnCl 2 · 4H 2 O, NaF, Na 2 SiO 3 · 9H 2 O, NaVO 3, (NH 4) 6 Mo O 24 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl, SnCl 2 · 2H 2 O, and ZrOCl 2 · 8H 2 wherein comprising at least one O is selected from the group consisting of <6><7> The medium additive according to any one of the above.
<9> A differentiation-inducing regulator added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
A differentiation-inducing regulator comprising at least one selected from the group consisting of a ROCK inhibitor, a Wnt signal activator, a Wnt signal suppressor, a TGF-β signal inhibitor, and an estrogen-like agent .
<10> A medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
The medium additive according to any one of <6> to <8>,
Including the differentiation-inducing regulator according to <9> above,
The basal medium is at least one selected from the group consisting of Dulbecco's modified Eagle medium / nutrient mixture F-12 ham, Dulbecco's modified Eagle medium, Iskov's modified Dulbecco medium, RPMI 1640 medium, and αMEM medium. It is.
<11> A medium preparation kit for inducing differentiation of cardiomyocytes from pluripotent stem cells,
The medium additive according to any one of <6> to <8>,
The differentiation-inducing regulator according to <9>,
A medium comprising at least one basal medium selected from the group consisting of Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 Ham, Dulbecco's Modified Eagle Medium, Iskov's Modified Dulbecco Medium, RPMI 1640 Medium, and αMEM Medium This is a preparation kit.
<12> A kit for inducing differentiation of cardiomyocytes from pluripotent stem cells, comprising at least one of the medium according to <10> and the medium preparation kit according to <11>. is there.
<13> Inducing differentiation of cardiomyocytes from the stem cells according to <12>, including instructions for teaching differentiation of cardiomyocytes from the pluripotent stem cells according to any one of <1> to <5> It is a kit to do.
本発明によれば、従来における前記諸問題を解決し、前記目的を達成することができ、高品質な心筋細胞を、大量に、安定して、安価に、かつ簡便に製造することが可能な多能性幹細胞から心筋細胞を分化誘導する方法、並びに該方法に好適な培地添加剤、分化誘導調節剤、培地、培地作製用キット、及び多能性幹細胞から心筋細胞を分化誘導するためのキットを提供することができる。 According to the present invention, it is possible to solve the conventional problems and achieve the object, and to manufacture high-quality cardiomyocytes in a large amount stably, inexpensively and simply. Method for inducing differentiation of cardiomyocytes from pluripotent stem cells, medium additive suitable for the method, differentiation induction regulator, medium, kit for preparing medium, and kit for inducing differentiation of cardiomyocytes from pluripotent stem cells Can be provided.

Claims (13)

  1. 　多能性幹細胞を浮遊培養し、胚様体を形成する胚様体形成工程と、
    　前記胚様体を浮遊培養し、中胚葉を誘導する中胚葉誘導工程と、
    　前記中胚葉誘導後の胚様体を浮遊培養し、心筋細胞を誘導する心筋細胞誘導工程とを含み、
    　前記心筋細胞誘導工程が、第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理とを含み、
    　前記胚様体形成工程における培地が、第１の分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地、及び一液式分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地のいずれかであり、
    　前記中胚葉誘導工程における培地が、第２の分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地のいずれかであり、
    　前記第１の心筋細胞誘導処理における培地が、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地のいずれかであり、
    　前記第２の心筋細胞誘導処理における培地が、第２の分化誘導培地に、エストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、エストロゲン様作用物質を加えた培地のいずれかであり、
    　前記第１の分化誘導培地が、基礎培地に、インスリン、トランスフェリン、アルブミン、及び１－チオグリセロールを加えた培地であり、
    　前記第２の分化誘導培地が、イスコフ改変ダルベッコ培地に、アルブミン、ポリビニルアルコール、エタノラミン塩酸塩、亜セレン酸ナトリウム、ヒドロコルチゾン、ＤＬ－α－トコフェロール酢酸エステル、Ｎ－アセチル－Ｌ－システイン、１－チオグリセロール、及びアスコルビン酸を加えた培地であり、
    　前記一液式分化誘導培地が、イスコフ改変ダルベッコ培地に、トランスフェリン、アルブミン、ポリビニルアルコール、エタノラミン塩酸塩、亜セレン酸ナトリウム、ヒドロコルチゾン、ＤＬ－α－トコフェロール酢酸エステル、Ｎ－アセチル－Ｌ－システイン、１－チオグリセロール、及びアスコルビン酸を加えた培地であることを特徴とする多能性幹細胞から心筋細胞を分化誘導する方法。     Embryoid body formation process of suspension culture of pluripotent stem cells to form embryoid bodies,
    Mesoderm induction step of culturing the embryoid body in suspension and inducing mesoderm,
    A suspension culture of the embryoid body after the mesoderm induction, and a cardiomyocyte induction step of inducing cardiomyocytes,
    The cardiomyocyte induction step includes a first cardiomyocyte induction process and a second cardiomyocyte induction process;
    The medium in the embryoid body formation step is any one of a medium in which a ROCK inhibitor is added to a first differentiation induction medium, and a medium in which a ROCK inhibitor is added to a one-part differentiation induction medium.
    The medium in the mesoderm induction step is any one of a medium obtained by adding a Wnt signal activator to a second differentiation induction medium and a medium obtained by adding a Wnt signal activator to a one-part differentiation induction medium. ,
    The medium in the first cardiomyocyte induction treatment is a medium obtained by adding a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like agent to a second differentiation induction medium, and a one-part differentiation induction medium. , A Wnt signal suppressor, a TGF-β signal inhibitor, and a medium containing an estrogen-like agent,
    The medium in the second cardiomyocyte induction treatment is either a medium in which an estrogen-like agent is added to the second differentiation-inducing medium, or a medium in which an estrogen-like agent is added to a one-part differentiation induction medium. Yes,
    The first differentiation induction medium is a medium obtained by adding insulin, transferrin, albumin, and 1-thioglycerol to a basal medium;
    The second differentiation-inducing medium is an Iskov-modified Dulbecco medium that contains albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine, 1-thiol. A medium to which glycerol and ascorbic acid are added,
    The one-part differentiation induction medium is transferred to Iscov modified Dulbecco medium with transferrin, albumin, polyvinyl alcohol, ethanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine, 1 A method for inducing differentiation of cardiomyocytes from pluripotent stem cells, characterized in that the medium is a medium supplemented with thioglycerol and ascorbic acid.
  2. 　胚様体形成工程における培地が、第１の分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地であり、
    　中胚葉誘導工程における培地が、第２の分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地であり、
    　第１の心筋細胞誘導処理における培地が、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地であり、
    　第２の心筋細胞誘導処理における培地が、第２の分化誘導培地に、エストロゲン様作用物質を加えた培地である請求項１に記載の多能性幹細胞から心筋細胞を分化誘導する方法。     The medium in the embryoid body formation step is a medium obtained by adding a ROCK inhibitor to the first differentiation induction medium,
    The medium in the mesoderm induction step is a medium obtained by adding a Wnt signal activator to the second differentiation induction medium,
    The medium in the first cardiomyocyte induction treatment is a medium obtained by adding a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like agent to the second differentiation induction medium,
    The method for inducing differentiation of cardiomyocytes from pluripotent stem cells according to claim 1, wherein the medium in the second cardiomyocyte induction treatment is a medium obtained by adding an estrogen-like agent to the second differentiation induction medium.
  3. 　胚様体形成工程における培地が、一液式分化誘導培地に、ＲＯＣＫ阻害剤を加えた培地であり、
    　中胚葉誘導工程における培地が、一液式分化誘導培地に、Ｗｎｔシグナル活性化物質を加えた培地であり、
    　第１の心筋細胞誘導処理における培地が、一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質を加えた培地であり、
    　第２の心筋細胞誘導処理における培地が、一液式分化誘導培地に、エストロゲン様作用物質を加えた培地である請求項１に記載の多能性幹細胞から心筋細胞を分化誘導する方法。     The medium in the embryoid body formation step is a medium obtained by adding a ROCK inhibitor to a one-part differentiation induction medium,
    The medium in the mesoderm induction step is a medium obtained by adding a Wnt signal activator to a one-part differentiation induction medium,
    The medium in the first cardiomyocyte induction treatment is a medium in which a Wnt signal inhibitor, a TGF-β signal inhibitor, and an estrogen-like agent are added to a one-part differentiation induction medium,
    The method for inducing differentiation of cardiomyocytes from pluripotent stem cells according to claim 1, wherein the medium in the second cardiomyocyte induction treatment is a medium in which an estrogen-like agent is added to a one-part differentiation induction medium.
  4. 　第１の心筋細胞誘導処理と、第２の心筋細胞誘導処理との間に、第２の分化誘導培地に、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質を加えた培地、及び一液式分化誘導培地に、Ｗｎｔシグナル抑制物質、及びエストロゲン様作用物質を加えた培地のいずれかで培養する処理を含む請求項１から３のいずれかに記載の多能性幹細胞から心筋細胞を分化誘導する方法。 Between the first cardiomyocyte induction treatment and the second cardiomyocyte induction treatment, a medium obtained by adding a Wnt signal inhibitory substance and an estrogen-like agent to the second differentiation induction medium, and one-part differentiation induction The method for inducing differentiation of cardiomyocytes from pluripotent stem cells according to any one of claims 1 to 3, comprising a treatment of culturing the medium in any one of a medium in which a Wnt signal inhibitory substance and an estrogen-like agent are added.
  5. 　第１の分化誘導培地が、更に、グリシン、Ｌ－アラニン、Ｌ－アスパラギン・Ｈ２Ｏ、Ｌ－アスパラギン酸、Ｌ－グルタミン、Ｌ－グルタミン酸、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏを含み、
    　第２の分化誘導培地が、更に、Ｌ－グルタミン、及びトランスフェリンを含み、
    　一液式分化誘導培地が、更に、グリシン、Ｌ－グルタミン、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏを含む請求項１から４のいずれかに記載の多能性幹細胞から心筋細胞を分化誘導する方法。     The first differentiation induction medium further comprises glycine, L-alanine, L-asparagine / H 2 O, L-aspartic acid, L-glutamine, L-glutamic acid, L-histidine, L-isoleucine, L-methionine, L -Phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, magnesium salt of ascorbic acid 2-2PO 4 , AgNO 3 , AlCl 3 · 6H 2 O, Ba (C 2 H 3 O 2 ) 2 , 3CdSO 4 · 8H 2 O, CoCl 2 · 6H 2 O, Cr 2 (SO 4 ) 3 · H 2 O, GeO 2 , Na 2 SeO 3, KBr, KI, MnCl 2 · 4H 2 O, NaF, Na 2 SiO 3 · 9H 2 O, NaVO 3, (N 4) 6 Mo 7 O 24 · 4H 2 O, NiSO 4 · 6H 2 O, wherein RbCl, SnCl 2 · 2H 2 O , and ZrOCl 2 · 8H 2 O,
    The second differentiation induction medium further contains L-glutamine and transferrin;
    The one-part differentiation induction medium further contains glycine, L-glutamine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L - tryptophan, L- tyrosine, L- valine, thiamine, reduced glutathione, magnesium salt of ascorbic acid -2-2PO 4, AgNO 3, AlCl 3 · 6H 2 O, Ba (C 2 H 3 O 2) 2, 3CdSO 4 · 8H 2 O, CoCl 2 · 6H 2 O, Cr 2 (SO 4) 3 · H 2 O, GeO 2, Na 2 SeO 3, KBr, KI, MnCl 2 · 4H 2 O, NaF, Na 2 SiO 3 9H 2 O, NaVO 3 , (NH 4 ) 6 Mo 7 O 24 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl, SnCl 2 · 2H 2 O, and ZrOCl method of inducing differentiation of cardiomyocytes from pluripotent stem cells according to any one of claims 1 to 4, comprising 2 · 8H 2 O.
  6. 　多能性幹細胞から心筋細胞を分化誘導するための培地に添加する培地添加剤であって、
    　アルブミン及び１－チオグリセロールの少なくともいずれかを含むことを特徴とする培地添加剤。     A medium additive added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
    A medium additive comprising at least one of albumin and 1-thioglycerol.
  7. 　更に、トランスフェリン、インスリン、ポリビニルアルコール、エタノラミン塩酸塩、亜セレン酸ナトリウム、ヒドロコルチゾン、ＤＬ－α－トコフェロール酢酸エステル、Ｎ－アセチル－Ｌ－システイン、及びアスコルビン酸からなる群から選択される少なくとも１種を含む請求項６に記載の培地添加剤。 And at least one selected from the group consisting of transferrin, insulin, polyvinyl alcohol, etanolamine hydrochloride, sodium selenite, hydrocortisone, DL-α-tocopherol acetate, N-acetyl-L-cysteine, and ascorbic acid. The culture medium additive according to claim 6 comprising.
  8. 　更に、グリシン、Ｌ－アラニン、Ｌ－アスパラギン・Ｈ２Ｏ、Ｌ－アスパラギン酸、Ｌ－グルタミン、Ｌ－グルタミン酸、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－ヒドロキシプロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、チアミン、還元型グルタチオン、アスコルビン酸－２－２ＰＯ４のマグネシウム塩、ＡｇＮＯ３、ＡｌＣｌ３・６Ｈ２Ｏ、Ｂａ（Ｃ２Ｈ３Ｏ２）２、３ＣｄＳＯ４・８Ｈ２Ｏ、ＣｏＣｌ２・６Ｈ２Ｏ、Ｃｒ２（ＳＯ４）３・Ｈ２Ｏ、ＧｅＯ２、Ｎａ２ＳｅＯ３、ＫＢｒ、ＫＩ、ＭｎＣｌ２・４Ｈ２Ｏ、ＮａＦ、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ、ＮａＶＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏ、ＮｉＳＯ４・６Ｈ２Ｏ、ＲｂＣｌ、ＳｎＣｌ２・２Ｈ２Ｏ、及びＺｒＯＣｌ２・８Ｈ２Ｏからなる群から選択される少なくとも１種を含む請求項６又は７に記載の培地添加剤。 Furthermore, glycine, L-alanine, L-asparagine / H 2 O, L-aspartic acid, L-glutamine, L-glutamic acid, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L -Hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, magnesium salt of ascorbic acid-2-2PO 4 , AgNO 3 , AlCl 3 · 6H 2 O , Ba (C 2 H 3 O 2 ) 2 , 3CdSO 4 .8H 2 O, CoCl 2 .6H 2 O, Cr 2 (SO 4 ) 3 .H 2 O, GeO 2 , Na 2 SeO 3 , KBr, KI, MnCl 2 .4H 2 O, NaF, Na 2 SiO 3 .9H 2 O, NaVO 3 , (NH 4 ) 6 Mo 7 O 2 4 · 4H 2 O, NiSO 4 · 6H 2 O, RbCl, culture medium according to SnCl 2 · 2H 2 O, and claim 6 or 7 comprising at least one selected from the group consisting of ZrOCl 2 · 8H 2 O Additive.
  9. 　多能性幹細胞から心筋細胞を分化誘導するための培地に添加する分化誘導調節剤であって、
    　ＲＯＣＫ阻害剤、Ｗｎｔシグナル活性化物質、Ｗｎｔシグナル抑制物質、ＴＧＦ－βシグナル阻害剤、及びエストロゲン様作用物質からなる群から選択される少なくとも１種を含むことを特徴とする分化誘導調節剤。     A differentiation-inducing regulator added to a medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
    A differentiation induction regulator comprising at least one selected from the group consisting of a ROCK inhibitor, a Wnt signal activator, a Wnt signal suppressor, a TGF-β signal inhibitor, and an estrogen-like agent.
  10. 　多能性幹細胞から心筋細胞を分化誘導するための培地であって、
    　請求項６から８のいずれかに記載の培地添加剤と、
    　請求項９に記載の分化誘導調節剤とを含み、
    　基礎培地が、ダルベッコ改変イーグル培地／栄養混合物Ｆ－１２ハム、ダルベッコ改変イーグル培地、イスコフ改変ダルベッコ培地、ＲＰＭＩ１６４０培地、及びαＭＥＭ培地からなる群から選択される少なくとも１種であることを特徴とする培地。     A medium for inducing differentiation of cardiomyocytes from pluripotent stem cells,
    The culture medium additive according to any one of claims 6 to 8,
    A differentiation-inducing regulator according to claim 9,
    The basal medium is at least one selected from the group consisting of Dulbecco's modified Eagle medium / nutrient mixture F-12 ham, Dulbecco's modified Eagle medium, Iskov's modified Dulbecco medium, RPMI 1640 medium, and αMEM medium. .
  11. 　多能性幹細胞から心筋細胞を分化誘導するための培地作製用キットであって、
    　請求項６から８のいずれかに記載の培地添加剤と、
    　請求項９に記載の分化誘導調節剤と、
    　ダルベッコ改変イーグル培地／栄養混合物Ｆ－１２ハム、ダルベッコ改変イーグル培地、イスコフ改変ダルベッコ培地、ＲＰＭＩ１６４０培地、及びαＭＥＭ培地からなる群から選択される少なくとも１種の基礎培地とを含むことを特徴とする培地作製用キット。     A medium preparation kit for inducing differentiation of cardiomyocytes from pluripotent stem cells,
    The culture medium additive according to any one of claims 6 to 8,
    A differentiation-inducing regulator according to claim 9,
    A medium comprising at least one basal medium selected from the group consisting of Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 Ham, Dulbecco's Modified Eagle Medium, Iskov's Modified Dulbecco Medium, RPMI 1640 Medium, and αMEM Medium Preparation kit.
  12. 　請求項１０に記載の培地、及び請求項１１に記載の培地作製用キットの少なくともいずれかを含むことを特徴とする多能性幹細胞から心筋細胞を分化誘導するためのキット。 A kit for inducing differentiation of cardiomyocytes from pluripotent stem cells, comprising at least one of the medium according to claim 10 and the medium preparation kit according to claim 11.
  13. 　請求項１から５のいずれかに記載の多能性幹細胞から心筋細胞を分化誘導する方法を教示する説明書を含む請求項１２に記載の幹細胞から心筋細胞を分化誘導するためのキット。 13. A kit for inducing differentiation of cardiomyocytes from stem cells according to claim 12, comprising instructions for teaching the method for inducing differentiation of cardiomyocytes from the pluripotent stem cells according to any one of claims 1 to 5.
PCT/JP2015/074947 2014-09-02 2015-09-02 Method for inducing differentiation of pluripotent stem cells into cardiomyocytes, and culture medium additive, differentiation-induction regulator, culture medium, culture medium preparation kit, and kit for inducing differentiation of pluripotent stem cells into cardiomyocytes suitable for said method WO2016035816A1 (en)

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