WO2014181682A1 - Composition for manufacturing induced pluripotent stem cell and method for manufacturing induced pluripotent stem cell - Google Patents

Composition for manufacturing induced pluripotent stem cell and method for manufacturing induced pluripotent stem cell Download PDF

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WO2014181682A1
WO2014181682A1 PCT/JP2014/061378 JP2014061378W WO2014181682A1 WO 2014181682 A1 WO2014181682 A1 WO 2014181682A1 JP 2014061378 W JP2014061378 W JP 2014061378W WO 2014181682 A1 WO2014181682 A1 WO 2014181682A1
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gene
gene product
product
pluripotent stem
induced pluripotent
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PCT/JP2014/061378
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French (fr)
Japanese (ja)
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大敬 伊関
康司 岡崎
晶彦 奥田
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学校法人埼玉医科大学
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Priority claimed from JP2013100311A external-priority patent/JP2014217344A/en
Priority claimed from JP2013100312A external-priority patent/JP2014217345A/en
Application filed by 学校法人埼玉医科大学 filed Critical 学校法人埼玉医科大学
Publication of WO2014181682A1 publication Critical patent/WO2014181682A1/en

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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a composition for producing induced pluripotent stem cells and a method for producing induced pluripotent stem cells.
  • iPS induced pluripotent stem cells
  • induced pluripotent stem cells have properties such as pluripotency, self-replication, and high proliferation ability. It is known that differentiation can be differentiated into cardiomyocytes, blood cells, retinal pigment epithelial cells, nerve cells and the like by inducing differentiation. Therefore, for example, it is expected to be used as a regenerative medicine such as cell transplantation treatment, drug screening, and a tool for elucidating the cause of a disease.
  • a method for producing the induced pluripotent stem cell for example, a method for producing by introducing Oct3 / 4 gene, Sox2 gene, Klf4 gene, and c-Myc gene into somatic cells has been proposed (for example, patents).
  • Reference 1 a method for producing by introducing Oct3 / 4 gene, Sox2 gene, Klf4 gene, and c-Myc gene into somatic cells.
  • induced pluripotent stem cells can be produced, (1) production efficiency is low, (2) production period is long, and (3) human induced pluripotent stem cells are Leukemia inhibitory.
  • LIF naive-type induced pluripotent stem cells having a factor
  • the present invention provides a method for producing an induced pluripotent stem cell that is excellent in production efficiency, can be produced in a short period of time, and is excellent in quality, and a composition for producing induced pluripotent stem cells. Objective.
  • Means for solving the problems are as follows. That is, ⁇ 1> (A) Oct3 / 4 gene or its gene product; (B) the Sox2 gene or its gene product; (C) the Klf4 gene or its gene product; (D) the c-Myc gene or its gene product; (E) Jarid2 (Jumonji, AT rich interactive domain 2) mutant gene or its gene product, Prdm14 (PR domain containing 14) gene or its gene product, Esrrb (Estrogen-related receptor gene, or its gene) (Sal-like 4, transscript variant a) introducing into a somatic cell at least one selected from the group consisting of a gene or a gene product thereof, A method for producing an induced pluripotent stem cell, wherein the Jarid2 mutant gene or a gene product thereof is a gene encoding the N-terminal amino acids 1 to 551 of the Jarid2 protein or a gene product thereof.
  • ⁇ 2> comprising at least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product, A composition for producing induced pluripotent stem cells, wherein the Jarid2 mutant gene or its gene product is a gene encoding the N-terminal 1st to 551st amino acids of the Jarid2 protein or its gene product.
  • an artificial pluripotent stem cell that can solve the above-mentioned problems and can achieve the above-mentioned object, is excellent in production efficiency, can be produced in a short period of time, and is excellent in quality.
  • a production method and a composition for producing induced pluripotent stem cells can be provided.
  • FIG. 1-1 is a graph showing the results of Test Example 1-1.
  • FIG. 1-2 is a graph showing the results of Test Example 1-2.
  • FIG. 1-3 is a graph showing the results of Test Example 1-3.
  • FIG. 1-4 is a graph showing the results of Test Example 1-4.
  • FIG. 1-5 is a graph showing the results of Test Example 1-5.
  • FIG. 1-6 is a graph showing the results of Test Example 1-6.
  • FIG. 2-1 is a graph showing the results of Test Example 2-1.
  • FIG. 2-2 is a graph showing the results of Test Example 2-2.
  • FIG. 2-3 is a graph showing the results of Test Example 2-3.
  • FIG. 3A is a photograph showing an example of the results obtained when the virus solution of (1) in Test Example 3-1 was used.
  • FIG. 3A is a photograph showing an example of the results obtained when the virus solution of (1) in Test Example 3-1 was used.
  • FIG. 3A is a photograph showing an example of the results obtained when the virus solution of (1) in Test
  • FIG. 3B is a photograph showing another example of the result obtained when the virus solution of (1) in Test Example 3-1 was used.
  • FIG. 3C is a photograph showing an example of the results obtained when the virus solution of (2) in Test Example 3-1 was used.
  • FIG. 3D is a photograph showing another example of the results obtained when the virus solution of (2) in Test Example 3-1 was used.
  • FIG. 3E is a photograph showing an example of the results obtained when the virus solution of (1) in Test Example 3-2 was used.
  • FIG. 3F is a photograph showing another example of the results obtained when the virus solution of (1) in Test Example 3-2 was used.
  • FIG. 3G is a photograph showing an example of the results obtained when the virus solution of (2) in Test Example 3-2 was used.
  • FIG. 3H is a photograph showing another example of the results obtained when the virus solution of (2) in Test Example 3-2 was used.
  • the method for producing induced pluripotent stem cells of the present invention comprises (A) Oct3 / 4 gene or its gene product, (B) Sox2 gene or its gene product, (C) Klf4 gene or its gene product, (D Selected from the group consisting of :) c-Myc gene or its gene product, (E) Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, or Sall4a gene or its gene product.
  • a somatic cell hereinafter sometimes referred to as a “gene or gene product introduction process”
  • the gene or its gene product introduction step includes at least (A) Oct3 / 4 gene or its gene product, (B) Sox2 gene or its gene product, (C) Klf4 gene or its gene product, and (D) c At least selected from the group consisting of the Myc gene or its gene product, and (E) the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product.
  • the gene product refers to mRNA (messenger RNA) transcribed from a gene and protein translated from the mRNA.
  • (E) Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, and Sall4a gene or its gene product there is a particular limitation.
  • (1) an embodiment including a Jarid2 mutant gene or its gene product, (2) a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or An embodiment including the gene product, (3) an embodiment including the Jarid2 mutant gene through the gene product, the Prdm14 gene through the gene product, the Esrrb gene through the gene product, and the Sall4a gene through the gene product are preferred, and (3) Jarid Variant gene or its gene product, PRDM14 gene or its gene product, Esrrb gene or its gene product, and is more preferred embodiment including Sall4a gene or its gene product.
  • the more preferable embodiment is advantageous in that it is superior in the production efficiency of induced pluripotent stem cells, can be produced in a shorter period of time,
  • the origin of the Oct3 / 4 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the Oct3 / 4 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_002701 (human) and NM_013633 (mouse).
  • Sox2 gene- The origin of the Sox2 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the Sox2 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_003106 (human) and NM_011443 (mouse).
  • Klf4 gene- The origin of the Klf4 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • sequence information of the Klf4 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_004235 (human) and NM_0103737 (mouse).
  • c-Myc gene- The origin of the c-Myc gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the c-Myc gene can be obtained from a known database. For example, NCBI can obtain the accession numbers NM_002467 (human) and NM_010849 (mouse).
  • the Jarid2 mutant gene is a gene (see SEQ ID NO: 12) encoding the 1st to 551st amino acids at the N-terminus of the Jarid2 protein.
  • the origin of the Jarid2 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the Jarid2 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_004973 (human) and NM_021878 (mouse).
  • Prdm14 gene- The origin of the Prdm14 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the Prdm14 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_024504 (human) and NM_001081209 (mouse, see SEQ ID NO: 19).
  • -Esrrb gene- There is no restriction
  • the sequence information of the Esrrb gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_004452 (human) and NM_011934 (mouse, see SEQ ID NO: 20).
  • the origin of the Sall4a gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the Sall4a gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_020436 (human) and NM_175303 (mouse, see SEQ ID NO: 21).
  • Oct3 / 4 gene to its gene product Sox2 gene to its gene product, Klf4 gene to its gene product, c-Myc gene to its gene product, Jarid2 mutant gene to its gene product, Prdm14 gene to its gene product, Esrrb
  • the sequence of the gene or its gene product, and the sequence of the Sall4a gene or its gene product are not particularly limited as long as the effects of the present invention are not impaired, and may be only the portion of the sequence of each gene that is translated into a protein. However, other parts may be included. Further, the sequence of each gene or gene product thereof may contain a mutation.
  • the mutation examples include a mutation that does not affect the amino acid sequence of the protein of each gene, and one or several (2 to 5) amino acids are deleted from the amino acid sequence of the protein of each gene. Substitution, insertion, or mutation to be added.
  • the homology with the wild-type when each gene or its gene product has a mutation is not particularly limited and can be appropriately selected according to the purpose. 70% or more is preferable, 80% or more is more preferable, and 90% or more is particularly preferable.
  • tissue stem cells such as adipose tissue-derived stromal (stem) cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, sperm stem cells; tissue precursor cells; lymphocytes And already differentiated cells such as epithelial cells, muscle cells, fibroblasts, and the like.
  • the somatic cells may be recombinant so as to facilitate selection of induced pluripotent stem cells.
  • the recombinant cell include a recombinant cell in which at least one of a reporter gene and a drug resistance gene is incorporated into a gene locus that is specifically highly expressed in a pluripotent cell.
  • the gene that is specifically highly expressed in the pluripotent cells include Fbx15 gene, Nanog gene, Oct3 / 4 gene, and the like.
  • the reporter gene include a green fluorescent protein (GFP) gene and a ⁇ -galactosidase gene.
  • the drug resistance gene include a puromycin resistance gene and a neomycin resistance gene.
  • the individual that collects the somatic cells is not particularly limited and can be appropriately selected according to the purpose.However, when the obtained induced pluripotent stem cells are used for regenerative medicine, from the viewpoint of rejection, the individual itself or another individual with the same or substantially the same type of MHC is preferred.
  • the type of the MHC is substantially the same when the cells obtained by inducing differentiation from the somatic cell-derived induced pluripotent stem cells are transplanted into an individual by using an immunosuppressant or the like. It means that the MHC types match to such an extent that cells can be engrafted.
  • the somatic cell culture conditions are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a culture temperature of about 37 ° C. and a CO 2 concentration of about 2% to 5%.
  • the medium used for culturing the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a medium (DMEM), RPMI 1640 medium, 199 medium, and F12 medium.
  • the method for introducing each gene or its gene product into a somatic cell is not particularly limited and can be appropriately selected depending on the purpose.
  • a method using a vector, a method using synthesized mRNA, a recombinant protein The method used, etc. are mentioned.
  • a viral vector for example, a viral vector, an artificial chromosome vector, a plasmid vector, an episomal vector etc. are mentioned.
  • the viral vectors include retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, silvis Examples thereof include viral vectors, rhabdovirus vectors, paramyxovirus vectors, orthomyxovirus vectors, and the like.
  • the artificial chromosome vector examples include a YAC (Yeast Artificial Chromome) vector, a BAC (Bacterial Artificial Chromosome) vector, a PAC (P1-derived Artificial Chromome) vector, and the like.
  • the method for introducing the vector into the somatic cell is not particularly limited and may be appropriately selected depending on the purpose. For example, lipofection method, microinjection method, DEAE dextran method, gene gun method, electroporation method , Calcium phosphate method, and the like.
  • virus particles obtained using packaging cells may be used.
  • the packaging cell is a cell into which a gene encoding a structural protein of a virus has been introduced.
  • a recombinant virus vector incorporating a target gene is introduced into the cell, a recombinant virus particle incorporating the target gene is produced.
  • the packaging cell is not particularly limited and may be appropriately selected according to the purpose.
  • packaging cells based on human kidney-derived HEK293 cells or mouse fibroblast-derived NIH3T3 cells Ecotropic virus PLAT-E cells designed to express derived envelope glycoproteins (hereinafter sometimes referred to as “PLAT-E cells”), PLAT-A cells designed to express envelope glycoproteins derived from Amphotropic virus And PLAT-GP cells designed to express vesicular stomatitis virus-derived envelope glycoproteins (hereinafter sometimes referred to as “PLAT-GP cells”).
  • PLAT-A cells and PLAT-GP cells are preferable from the viewpoint of host orientation when a recombinant viral vector is introduced into human somatic cells.
  • the method for introducing the viral vector into the packaging cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a lipofection method, an electroporation method, and a calcium phosphate method.
  • the method for infecting the somatic cells with the obtained virus particles is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a polybrene method.
  • the vector may contain a marker gene for confirming the introduction of each gene.
  • the marker gene refers to a gene that enables selection and selection of cells by introducing the marker gene into cells.
  • Specific examples of the marker gene include a drug resistance gene, a fluorescent protein gene, a luminescent enzyme gene, a chromogenic enzyme gene, and the like. These may be used individually by 1 type and may use 2 or more types together.
  • Specific examples of the drug resistance gene include a neomycin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a zeocin resistance gene, and a hygromycin resistance gene.
  • fluorescent protein gene examples include a GFP gene, a yellow fluorescent protein (YFP) gene, and a red fluorescent protein (RFP) gene.
  • luminescent enzyme gene examples include luciferase gene.
  • chromogenic enzyme gene examples include ⁇ -galactosidase gene, ⁇ -glucuronidase gene, alkaline phosphatase gene, and the like.
  • one gene may be incorporated into one vector, or two or more genes may be incorporated.
  • the two or more genes can be expressed simultaneously (hereinafter sometimes referred to as “co-expression”).
  • the c-Myc gene and the Jarid2 mutant gene are preferably introduced so as to be coexpressed.
  • the method for incorporating two or more genes into the one vector is not particularly limited and may be appropriately selected depending on the intended purpose. However, it is preferable to incorporate the two or more genes through a linking sequence.
  • the linking sequence is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a gene sequence encoding 2A peptide derived from foot-and-mouth disease virus (Picornaviridae Aphthovirus), IRES (internal ribosome entry sites), and the like. It is done.
  • the number of introduction of each gene or its gene product into a somatic cell may be one time or two or more times. There is no particular limitation on the introduction timing of each gene or its gene product into a somatic cell, and it can be appropriately selected according to the purpose. Even if all the genes or their gene products are introduced simultaneously. It may be introduced at different times.
  • or its gene product may be used individually by 1 type, and may use 2 or more types together.
  • an embodiment using only a gene or an embodiment using only a gene product may be used, and among each of the above genes, a gene product may be used and another gene may be a gene. There may be.
  • each gene or its gene product As the introduction amount of each gene or its gene product into a somatic cell, all the genes or their gene products may be introduced in equal amounts or in different amounts.
  • the Oct3 / 4 gene is introduced in a large amount, for example, about 3 times the amount of the Sox2 gene, the Klf4 gene, or the c-Myc gene ( PNAS 106 (31): 12759-1276.2009, J. Biol. Chem. 287 (43): 36273-36282.201) are preferred.
  • genes or gene products other than the genes or gene products may be introduced as long as the effects of the present invention are not impaired.
  • the genes or gene products other than the genes or gene products thereof include, for example, the Oct family (Oct1A and Oct6) genes or their gene products, the Klf family (Klf1, Klf2, Klf4, and Klf5) genes or their gene products.
  • Myc family (N-Myc, and L-Myc) genes to their gene products
  • Sox family (Sox1, Sox3, Sox7, Sox15, Sox17, and Sox18) genes to their gene products
  • TERT genes to their gene products
  • SV40 Large T antigen gene or its gene product HPV16 E6 gene or its gene product, HPV16 E7 gene or its gene product, Bmil gene or its gene product, Fbx15 gene or its gene product , Nanog gene or its gene product, ERas gene or its gene product, ECAT15-2 gene or its gene product, Tcl1 gene or its gene product, ⁇ -catenin gene or its gene product, ECAT1 gene or its gene product, Esg1 gene or its Its gene product, Dnmt3L gene or its gene product, ECAT8 gene or its gene product, Gdf3 gene or its gene product, ECAT15-1 gene or its gene product, Fthl17 gene or its gene product, Rex1 gene or its gene product, UTF1 gene Or
  • Nr5a2 gene or its Gene product Rar family gene to its gene product (Proc. Natl. Acad. Sci. USA 108 (45): 18283-18288.2011); Utx gene to its gene product, Mdm2 gene to its gene product, Ring1b gene to Examples thereof include the gene product, the Wdr5 gene and the gene product thereof.
  • a low molecular compound may be administered as long as the effects of the present invention are not impaired.
  • the low molecular compound is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a GSK-3 inhibitor and an adenylate cyclase activator. Specific examples of the GSK-3 inhibitor include CHIR99021. Specific examples of the adenylate cyclase activator include Forskolin. There is no restriction
  • 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.
  • genes for culturing somatic cells into which each of the genes or gene products thereof has been introduced are introduced.
  • the gene product-introduced cell culture step is performed.
  • the gene or gene product-introduced cell culture step is a step of culturing somatic cells into which each gene or gene product has been introduced.
  • the culture conditions of the gene or its gene product-introduced cell are not particularly limited and can be appropriately selected according to the purpose.
  • the culture temperature is about 37 ° C.
  • the CO 2 concentration is about 2% to 5%
  • Etc is about 2% to 5%
  • the medium used for culturing the gene or its gene product-introduced cell is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the induced pluripotent stem cells produced by the method for producing induced pluripotent stem cells have differentiation pluripotency and self-renewal ability.
  • the term “pluripotency” means that all three germ layers can be differentiated.
  • the self-replicating ability means the ability to proliferate while maintaining an undifferentiated state.
  • the method for confirming whether or not the cell produced by the method for producing an induced pluripotent stem cell is an induced pluripotent stem cell is not particularly limited and can be appropriately selected depending on the purpose.
  • the somatic cell is a recombinant cell in which at least one of a reporter gene and a drug resistance gene is incorporated into a gene locus that is specifically highly expressed in differentiated pluripotent cells
  • This can be confirmed using the reporter gene and drug resistance gene.
  • a GFP gene is used as the reporter gene
  • a method of confirming GFP-positive cells with a flow cytometer can be mentioned, and a puromycin resistance gene can be used as the drug resistance gene. When used, it can be confirmed by administering puromycin to the cells.
  • the type of the induced pluripotent stem cell may be a prime type or a naive type, but a naive type is preferable in terms of excellent quality.
  • the method for confirming whether or not the artificial pluripotent stem cells are na ⁇ ve is not particularly limited and may be appropriately selected depending on the purpose. For example, the form of colonies, bFGF (basic fibroblast growth factor) Can be propagated in a single cell, whether it can be grown in a LIF-containing medium not containing LIF, or can be grown in a 2i-added medium (medium containing a MEK inhibitor and a GSK inhibitor) Whether or not it is used as an index can be confirmed.
  • bFGF basic fibroblast growth factor
  • the colony has a dome shape, can be grown in a LIF-containing medium not containing bFGF, can be grown in a 2i-added medium, and can be dispersed and passaged into single cells. If it is a cell that can be used, it can be determined to be naive.
  • the seed of the induced pluripotent stem cell is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably human.
  • composition for producing induced pluripotent stem cells is selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product. And at least one other component as necessary.
  • Jarid2 mutant gene or its gene product there is no particular limitation as at least one selected from the group consisting of the Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, and Sall4a gene or its gene product.
  • an embodiment including a Jarid2 mutant gene or its gene product (2) a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product
  • Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, and Sall4a gene or its gene product are preferred
  • Jarid2 mutation Gene or its gene product, PRDM14 gene or its gene product, Esrrb gene or its gene product and is more preferred embodiment including Sall4a gene or its gene product.
  • the more preferable embodiment is advantageous in that it is superior in the production efficiency of induced pluripotent stem cells, can be produced in a shorter period of time, and is superior in quality.
  • the Jarid2 mutant gene or its gene product is a gene encoding the N-terminal 1st to 551st amino acids of the Jarid2 protein or its gene product, as described in the method for producing induced pluripotent stem cells. It is.
  • the Jarid2 mutant gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
  • the mode of the Jarid2 mutant gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected according to the purpose.
  • the mode in which the gene is incorporated into a vector Synthetic mRNA embodiments, recombinant protein embodiments, and the like. Examples of the vector include those described in the method for producing induced pluripotent stem cells.
  • the synthetic mRNA and the recombinant protein can be produced by a known method.
  • Prdm14 gene and its gene product are the same as those described in the method for producing induced pluripotent stem cells.
  • the Prdm14 gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
  • the embodiment of the Prdm14 gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected according to the purpose. Examples include mRNA and recombinant protein. Examples of the vector include those described in the method for producing induced pluripotent stem cells.
  • the synthetic mRNA and the recombinant protein can be produced by a known method.
  • the Esrrb gene and its gene product are the same as those described in the method for producing induced pluripotent stem cells. Further, the Esrrb gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
  • the embodiment of the Esrrb gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected depending on the purpose.
  • Embodiment in which the gene is incorporated into a vector Examples include mRNA and recombinant protein. Examples of the vector include those described in the method for producing induced pluripotent stem cells.
  • the synthetic mRNA and the recombinant protein can be produced by a known method.
  • the Sall4a gene or its gene product is the same as that described in the method for producing induced pluripotent stem cells.
  • the Sall4a gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
  • the embodiment of the Sall4a gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected according to the purpose.
  • the embodiment in which the gene is incorporated into a vector synthesis Examples include mRNA and recombinant protein. Examples of the vector include those described in the method for producing induced pluripotent stem cells.
  • the synthetic mRNA and the recombinant protein can be produced by a known method.
  • the other configuration 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. Or a gene product thereof, and a c-Myc gene or a gene product thereof.
  • the Oct3 / 4 gene or its gene product, Sox2 gene or its gene product, Klf4 gene or its gene product, and c-Myc gene or its gene product are the same as those described in the method for producing induced pluripotent stem cells. belongs to. Further, each gene or gene product thereof may contain the same mutation as described in the method for producing induced pluripotent stem cells.
  • the Oct3 / 4 gene or its gene product, the Sox2 gene or its gene product, the Klf4 gene or its gene product, and the c-Myc gene or its gene product in the composition for producing induced pluripotent stem cells are particularly There is no restriction
  • the aspect by which the gene is integrated in the vector, the aspect of synthetic mRNA, the aspect of recombinant protein, etc. are mentioned.
  • the vector include those described in the method for producing induced pluripotent stem cells.
  • the synthetic mRNA and the recombinant protein can be produced by a known method.
  • the c-Myc gene and the Jarid2 mutant gene in the composition for producing induced pluripotent stem cells can be co-expressed.
  • the genes or gene products thereof may be divided into individual containers or may be combined into one container. Alternatively, any number may be collected in a container.
  • the amount of each gene or its gene product in the composition for producing induced pluripotent stem cells is not particularly limited, and all the genes or their gene products may be equal amounts or different amounts.
  • the composition for producing an induced pluripotent stem cell may contain a gene other than the gene or a gene product thereof or a gene product thereof.
  • a packaging cell is used. May be included. Examples of the genes or gene products other than the genes or gene products thereof and the packaging cells include those described in the method for producing induced pluripotent stem cells.
  • RNA was extracted from B6 mouse-derived embryonic stem cells (hereinafter sometimes referred to as “ESC”, obtained from the National University of Tsukuba Life Science Animal Resource Center), cDNA was synthesized, and represented by SEQ ID NOs: 3 and 4 below.
  • ESC B6 mouse-derived embryonic stem cells
  • SEQ ID NOs: 3 and 4 SEQ ID NOs: 3 and 4 below.
  • Amplification and recovery of the Jarid2 mutant gene (the gene encoding the 1st to 551st amino acids of the N-terminal of the Jarid2 protein (see SEQ ID NO: 12)) by PCR using the primers obtained, and the pMXs vector (National University) Incorporated into a multicloning site of the University of Tokyo Medical Science Institute), a Jard2 mutant gene-containing virus vector was obtained.
  • Jarid2 mutant gene-containing virus vector as a template, and using primers represented by SEQ ID NOs: 9 and 10 below as primers, a part of Picoraviridae Aphthovirus-derived 2A peptide and a Jarid2 mutant gene (Jarid2 protein)
  • a gene encoding the N-terminal 1st to 551st amino acids (hereinafter sometimes referred to as “2A_Jarid2 ⁇ C gene”) was amplified and recovered.
  • the c-Myc_2A gene and the 2A_Jarid2 ⁇ C gene are incorporated into a multi-cloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science).
  • RNA was extracted from B6 mouse-derived embryonic stem cells (obtained from the National University of Tsukuba Life Science Animal Resource Center), and cDNA was synthesized. Then, using the primers represented by SEQ ID NOs: 1 and 2 below, Jarid2 A gene (a gene encoding the full length of the Jarid2 protein (see SEQ ID NO: 11)) is amplified, recovered, incorporated into a multicloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science), and a Jarid2 gene-containing viral vector Got.
  • pMXs vector obtained from the National University Corporation, Tokyo Institute of Medical Science
  • Jarid2 gene-containing viral vector as a template and using primers represented by the following SEQ ID NOs: 7 and 8, a part of Picoraviridae Aphthovirus-derived 2A peptide and the Jarid2 gene (encoding the full length of the Jarid2 protein)
  • the gene to be amplified (hereinafter sometimes referred to as “2A_Jarid2WT gene”) was amplified and recovered.
  • the c-Myc_2A gene and the 2A_Jarid2WT gene are incorporated into a multi-cloning site of a pMXs vector (obtained from the National University Corporation Tokyo Medical Research Institute).
  • RNA was extracted from B6 mouse-derived embryonic stem cells (obtained from the National University of Tsukuba Life Science Animal Resource Center), and cDNA was synthesized, followed by PCR using the primers represented by SEQ ID NOs: 13 and 14 below by Prdm14.
  • a gene (gene encoding the full length of Prdm14 protein (see SEQ ID NO: 19)) is amplified and recovered, incorporated into a multicloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science), and a Prdm14 gene-containing viral vector Got.
  • RNA was extracted from B6 mouse-derived embryonic stem cells (obtained from the University of Tsukuba Life Science Animal Resource Center), cDNA was synthesized, and Esrrb was synthesized by PCR using primers represented by SEQ ID NOs: 15 and 16 below.
  • a gene (a gene encoding the full length of the Esrrb protein (see SEQ ID NO: 20)) is amplified, recovered, incorporated into a multi-cloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science), and an Esrrb gene-containing viral vector Got.
  • a gene (a gene encoding the full length of the Sall4a protein (see SEQ ID NO: 21)) is amplified and recovered, incorporated into a multicloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Medical Science Institute), and a Sall4a gene-containing viral vector Got.
  • the PLAT-E cell culture medium was changed the day after the transfection, and the culture supernatant of the PLAT-E cell was collected the day after the medium was changed, and then filtered using a 0.45 ⁇ m filter, and the Oct3 / 4 gene-containing virus solution-1.
  • Production Example 2-6 Virus solution-1 containing c-Myc gene and Jarid2 mutant gene
  • the point that the Oct3 / 4 gene-containing virus vector obtained in Production Example 1-1 was used in Production Example 2-1 was that the c-Myc gene obtained in Production Example 1-6 and the Jarid2 mutant were used.
  • the c-Myc gene and Jarid2 mutant gene-containing virus solution-1 were obtained in the same manner as in Production Example 2-1, except that the gene-containing virus vector was used.
  • the PLAT-GP cell culture medium was changed the day after the transfection, and the culture supernatant of the PLAT-GP cell was collected the day after the medium was replaced, and then filtered using a 0.45 ⁇ m filter, and Oct3 / 4 gene-containing virus solution-2.
  • Production Example 3-4 c-Myc gene-containing virus solution-2)
  • the point that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used was replaced with the c-Myc gene-containing virus vector obtained in the production example 1-4.
  • a c-Myc gene-containing virus solution-2 was obtained in the same manner as in Production Example 3-1.
  • Production Example 3-5 Viral fluid-2 containing c-Myc gene and Jarid2 mutant gene
  • the Oct3 / 4 gene-containing virus vector obtained in Production Example 1-1 was used, except that the c-Myc gene obtained in Production Example 1-6 and the Jarid2 mutant were used.
  • a c-Myc gene and a Jarid2 mutant gene-containing virus solution-2 were obtained in the same manner as in Production Example 3-1, except that the gene-containing virus vector was used.
  • NG-MEF mouse fetal fibroblasts
  • STOCK Tg Nanog gene promoter-dependent manner
  • STOCK Tg Nanog-GFP, Puro 1Yam (No. RBRC02290) was obtained, mated, and isolated and prepared from trypsin on day 13.5).
  • the NG-MEF was cultured using DMEM containing 10% serum at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
  • the virus solutions prepared by adding polybrene having a final concentration of 4 ⁇ g / mL to the virus solutions of (1) to (3) below were each infected with the NG-MEF.
  • the day after the infection the supernatant was transferred to LIF (LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • LIF LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and Jarid2 gene-containing virus solution- 1 600 ⁇ L (3) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and Jarid2 mutant gene-containing virus Liquid-1 600 ⁇ L
  • the ratio of the induced pluripotent stem cells to the total cells in any of the 6th day and 8th day after the virus infection is the above (1) And it was higher than in the case of (2), and it was shown that the production efficiency of induced pluripotent stem cells can be increased by using the Jarid2 mutant gene or its gene product.
  • Test Example 1-1 (Test Example 1-2: Production efficiency test-2) -cell- As the cells, NG-MEF was used as in Test Example 1-1.
  • Virus solution (1) 300 ⁇ L of Oct3 / 4 gene-containing virus solution-1 300 ⁇ L, Sox2 gene-containing virus solution-1 300 ⁇ L, Klf4 gene-containing virus solution-1 300 ⁇ L, and c-Myc gene-containing virus solution-1 300 ⁇ L (2) Oct3 / 4 gene-containing virus solution-1 300 ⁇ L, Sox2 gene-containing virus solution-1 300 ⁇ L, Klf4 gene-containing virus solution-1 300 ⁇ L, and c-Myc gene and Jarid2 gene-containing virus solution-1 300 ⁇ L (3) Oct3 / 4 gene-containing virus solution-1 300 ⁇ L, Sox2 gene-containing virus solution-1 300 ⁇ L, Klf4 gene-containing virus solution-1 300 ⁇ L, and c-Myc gene and Jarid2 gene-containing virus solution-1 300 ⁇ L (3) Oct3 / 4 gene-containing virus solution-1 300 ⁇ L, Sox2 gene-containing virus solution-1 300 ⁇ L, Klf4 gene-containing virus solution-1 300 ⁇ L, and c-
  • Test Example 1-1 (Test Example 1-3: Production efficiency test-3) -cell- As the cells, NG-MEF was used as in Test Example 1-1.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene, and Jarid2 mutant gene-containing virus solution-1 200 ⁇ L, and Foreign gene-free virus solution-1 600 ⁇ L (3) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, K
  • Fig. 1-3 On the 8th day after the virus infection, the NG-MEF was detached with trypsin, and the number of GFP positive cells per 100,000 cells was measured with a flow cytometer (BD FACSCalibur (registered trademark), manufactured by Nippon Becton Dickinson). .
  • the results are shown in Fig. 1-3.
  • (1) to (3) show the results when the virus solutions (1) to (3) were used, respectively. From the results shown in FIGS. 1-3, in the cases (2) and (3), the ratio of the induced pluripotent stem cells to the whole cells is higher than that in the case (1).
  • NG-MEF expressing GFP and puromycin resistance gene in a Nanog gene promoter-dependent manner (transgenic mouse STOCK Tg (Nanog-GFP, Puro) 1Yam (No. RBRC02290) deposited at RIKEN BioResource Center) was obtained, mated, and isolated from 13.5 day embryos using trypsin).
  • the NG-MEF was cultured using DMEM containing 10% serum at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
  • the virus solutions prepared by adding polybrene having a final concentration of 4 ⁇ g / mL to the virus solutions of (1) to (8) below were each infected with the NG-MEF.
  • the day after the infection the supernatant was transferred to LIF (LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • LIF LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene containing virus solution-1 200 ⁇ L, Sox2 gene containing virus solution-1 200 ⁇ L, Klf4 gene containing virus solution-1 200 ⁇ L, c-Myc gene containing virus solution-1 200 ⁇ L, Prdm14 gene containing virus solution-1 200 ⁇ L and foreign gene-free virus solution-1 400 ⁇ L (3) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, Esrrb gene-containing virus solution-1 200 ⁇ L and foreign gene-free virus solution-1
  • the number of iPS colonies is higher than in the cases of (1) to (7), and the Prdm14 gene to its gene product, the Sall4a gene to its gene product, It has been shown that the production efficiency of induced pluripotent stem cells can be increased by using the Esrrb gene or its gene product.
  • Test Example 1-5 Production efficiency test -5
  • NG-MEF was used in the same manner as in Test Example 1-4.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene containing virus solution-1 200 ⁇ L, Sox2 gene containing virus solution-1 200 ⁇ L, Klf4 gene containing virus solution-1 200 ⁇ L, c-Myc gene containing virus solution-1 200 ⁇ L, Prdm14 gene containing virus solution-1 200 ⁇ L, Sall4a gene-containing virus solution-1 200 ⁇ L, and Esrrb gene-containing virus solution-1 200 ⁇ L
  • FIGS. 1-5 The results are shown in FIGS. 1-5.
  • (1) and (2) show the results when the virus solutions (1) and (2) were used, respectively. From the result of FIG.
  • the ratio of induced pluripotent stem cells to the whole cells is higher than in the case of (1), and the Prdm14 gene or its gene product, the Sall4a gene or its It was shown that the production efficiency of induced pluripotent stem cells can be increased by using the gene product and the Esrrb gene or the gene product thereof.
  • HDF human dermal fibroblasts
  • a human skin fibroblast basic medium manufactured by Toyobo Co., Ltd.
  • Test Example 1-4 the procedure was the same as in Test Example 1-4 except that the cells were changed from NG-MEF to HDF and the virus solution was replaced with one of the virus solutions (1) and (2) below. Then, the HDF was infected with a virus.
  • Virus solution (1) Oct3 / 4 gene containing virus solution-2 200 ⁇ L, Sox2 gene containing virus solution-2 200 ⁇ L, Klf4 gene containing virus solution-2 200 ⁇ L, c-Myc gene containing virus solution-2 200 ⁇ L, and foreign gene-free virus solution -2 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-2 200 ⁇ L, Sox2 gene-containing virus solution-2 200 ⁇ L, Klf4 gene-containing virus solution-2 200 ⁇ L, c-Myc gene-containing virus solution-2 200 ⁇ L, Prdm14 gene-containing virus solution-2 200 ⁇ L, Sall4a gene-containing virus solution-2 200 ⁇ L, and Esrrb gene-containing virus solution-2 200 ⁇ L
  • FIGS. 1-6 show the results when the virus solutions (1) and (2) were used, respectively. From the results of FIG. 1-6, in the case of (2), the number of TRA-1-60 positive colonies is larger than in the case of (1), and the Prdm14 gene or its gene product, the Sall4a gene or its gene product. It has been shown that the production efficiency of induced pluripotent stem cells can be increased by using the Esrrb gene or its gene product.
  • Test Example 2-1 Production period test -1 -cell- As the cells, NG-MEF was used as in Test Example 1-1.
  • virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene, and Jarid2 mutant gene-containing virus solution-1 200 ⁇ L, and Foreign gene-free virus solution-1 600 ⁇ L
  • Test Example 2-2 Production period test-2
  • NG-MEF was used as in Test Example 1-1.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene, and Jarid2 mutant gene-containing virus solution-1 200 ⁇ L, Prdm14 Gene-containing virus solution-1 200 ⁇ L, Esrrb gene-containing virus solution-1 200 ⁇ L, and Sall4a gene-containing virus solution-1 200 ⁇
  • induced pluripotent stem cells were stably confirmed in three experiments ( In the case of (1) above, the number of induced pluripotent stem cell colonies (average 3) is stable in 3 experiments for the first time when puromycin is exposed on the 6th day after virus infection. Pluripotent stem cells were confirmed (number of induced pluripotent stem cell colonies: 8 on average). Therefore, by using the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product, an artificial pluripotent stem cell can be produced in a shorter period of time. It was shown that you can.
  • Test Example 2-3 Production period test-3 -cell- As the cell, NG-MEF was used in the same manner as in Test Example 1-4.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, 200 ⁇ L of Sox2 gene-containing virus solution-1 200 ⁇ L, and c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus Liquid-1 600 ⁇ L (2) Oct3 / 4 gene containing virus solution-1 200 ⁇ L, Sox2 gene containing virus solution-1 200 ⁇ L, Klf4 gene containing virus solution-1 200 ⁇ L, c-Myc gene containing virus solution-1 200 ⁇ L, Prdm14 gene containing virus solution-1 200 ⁇ L, Sall4a gene-containing virus solution-1 200 ⁇ L, and Esrrb gene-containing virus solution-1 200 ⁇ L
  • Fig. 2-3 D1 to D6 respectively indicate the start date of puromycin exposure (from day 1 to day 6 after virus infection), and “ ⁇ ” indicates the case where the virus solution of (1) was used. The result shows the result, and “ ⁇ ” shows the result when the virus solution of (2) was used. As shown in FIG. 2-3, D1 to D6 respectively indicate the start date of puromycin exposure (from day 1 to day 6 after virus infection), and “ ⁇ ” indicates the case where the virus solution of (1) was used. The result shows the result, and “ ⁇ ” shows the result when the virus solution of (2) was used. As shown in FIG.
  • Test Example 3-1 Quality test-1) -cell- HDF (Toyobo Co., Ltd.) was used as the cell.
  • a human skin fibroblast basic medium manufactured by Toyobo Co., Ltd. was used and cultured at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
  • -Virus infection- Virus solutions prepared by adding polybrene having a final concentration of 4 ⁇ g / mL to the virus solutions of (1) and (2) below were each infected with the HDF.
  • LIF LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • 20% KSR manufactured by GIBCO
  • 1 ⁇ NEAA manufactured by GIBCO
  • 2-mercaptoethanol manufactured by GIBCO
  • GlutaMax manufactured by GIBCO
  • Virus solution (1) Oct3 / 4 gene containing virus solution-2 200 ⁇ L, Sox2 gene containing virus solution-2 200 ⁇ L, Klf4 gene containing virus solution-2 200 ⁇ L, c-Myc gene containing virus solution-2 200 ⁇ L, and foreign gene-free virus solution -2 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-2 200 ⁇ L, Sox2 gene-containing virus solution-2 200 ⁇ L, Klf4 gene-containing virus solution-2 200 ⁇ L, c-Myc gene, and Jarid2 mutant gene-containing virus solution-2 200 ⁇ L, Prdm14 Gene-containing virus solution-2 200 ⁇ L, Esrrb gene-containing virus solution-2 200 ⁇ L, and Sall4a gene-containing virus solution-2 200 ⁇ L
  • FIGS. 3A to 3D show the results of photographing the cells 18 days after the virus infection and the cloned cells with an inverted microscope (Zeiss Axiovert 200M).
  • 3A and 3B are examples of photographs when the virus solution of (1) is used
  • FIGS. 3C and 3D are examples of photographs when the virus solution of (2) is used.
  • the scale bar represents 200 ⁇ m.
  • the induced pluripotent stem cells obtained using the virus solution of (1) above have a flat colony, can be grown on a bFGF-added medium, cannot grow on a 2i-added medium, and are single cells.
  • the induced pluripotent stem cells obtained using the virus solution of (2) above have a dome-like colony shape and can be grown in a LIF-added medium not containing bFGF, and can be grown in a 2i-added medium. It was found to be a naive-type induced pluripotent stem cell that can be dispersed and passaged into a single cell. Therefore, high-quality induced pluripotent stem cells are produced by using the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product. It was shown that it can.
  • Test Example 3-2 Quality test-2
  • -cell- HDF Toyobo Co., Ltd.
  • a human skin fibroblast basic medium manufactured by Toyobo Co., Ltd.
  • -Virus infection- Virus solutions prepared by adding polybrene having a final concentration of 4 ⁇ g / mL to the virus solutions of (1) and (2) below were each infected with the HDF.
  • LIF LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • 20% KSR manufactured by GIBCO
  • 1 ⁇ NEAA manufactured by GIBCO
  • 2-mercaptoethanol manufactured by GIBCO
  • GlutaMax manufactured by GIBCO
  • Virus solution (1) Oct3 / 4 gene containing virus solution-2 200 ⁇ L, Sox2 gene containing virus solution-2 200 ⁇ L, Klf4 gene containing virus solution-2 200 ⁇ L, c-Myc gene containing virus solution-2 200 ⁇ L, and foreign gene-free virus solution -2 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-2 200 ⁇ L, Sox2 gene-containing virus solution-2 200 ⁇ L, Klf4 gene-containing virus solution-2 200 ⁇ L, c-Myc gene-containing virus solution-2 200 ⁇ L, Prdm14 gene-containing virus solution-1 200 ⁇ L, Esrrb gene-containing virus solution-2 200 ⁇ L, and Sall4a gene-containing virus solution-2 200 ⁇ L
  • FIGS. 3E to 3H show the results of photographing the cells 18 days after the virus infection and the cloned cells with an inverted microscope (Zeiss Axiovert 200M).
  • 3E and 3F are examples of photographs when the virus solution of (1) is used
  • FIGS. 3G and 3H are examples of photographs when the virus solution of (2) is used.
  • the scale bar represents 200 ⁇ m.
  • the induced pluripotent stem cells obtained using the virus solution of (1) above have a flat colony, can be grown on a bFGF-added medium, cannot grow on a 2i-added medium, and are single cells.
  • the induced pluripotent stem cells obtained using the virus solution of (2) above have a dome-like colony shape and can be grown in a LIF-added medium not containing bFGF, and can be grown in a 2i-added medium. It was found to be a naive-type induced pluripotent stem cell that can be dispersed and passaged into a single cell. Therefore, it was shown that high-quality induced pluripotent stem cells can be produced by using the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product.
  • an induced pluripotent stem cell can be produced with high efficiency, so that an induced pluripotent stem cell can be established from a small number of somatic cells, It is thought that the amount of rare cells and tissues collected can be reduced. Furthermore, since it is possible to suppress proliferation of non-induced pluripotent stem cells and selectively increase induced pluripotent stem cells, it is considered that establishment of induced pluripotent stem cell clones is facilitated. In addition, according to the method for producing induced pluripotent stem cells of the present invention, since induced pluripotent stem cells can be produced in a short period of time, self-derived to diseases such as spinal cord injury requiring early cell transplantation.
  • induced pluripotent stem cells Enables the use of induced pluripotent stem cells, reduces the risk of genome damage due to the long-term induced pluripotent stem cell induction process, and facilitates the method of creating induced pluripotent stem cells by transient gene transfer It is considered possible to produce safe induced pluripotent stem cells. Furthermore, according to the method for producing induced pluripotent stem cells of the present invention, induced pluripotent stem cells can be produced efficiently and in a short period of time, so that the cost can be reduced as compared with conventional methods. Further, according to the method for producing induced pluripotent stem cells of the present invention, naive induced pluripotent stem cells can be produced even in the case of human cells, so that handling of induced pluripotent stem cells is simplified. It is considered that differentiation induction efficiency can be improved.
  • Examples of the aspect of the present invention include the following. ⁇ 1> (A) Oct3 / 4 gene or its gene product; (B) the Sox2 gene or its gene product; (C) the Klf4 gene or its gene product; (D) the c-Myc gene or its gene product; (E) introducing at least one selected from the group consisting of the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product into a somatic cell.
  • a method for producing an induced pluripotent stem cell wherein the Jarid2 mutant gene or a gene product thereof is a gene encoding the N-terminal amino acids 1 to 551 of the Jarid2 protein or a gene product thereof.
  • the Jarid2 mutant gene or a gene product thereof is a gene encoding the N-terminal amino acids 1 to 551 of the Jarid2 protein or a gene product thereof.
  • At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product is a Jarid2 mutation
  • ⁇ 3> At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product,
  • the method for producing an induced pluripotent stem cell according to the above ⁇ 2> comprising the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product.
  • ⁇ 4> The method for producing induced pluripotent stem cells according to any one of ⁇ 2> to ⁇ 3>, wherein the c-Myc gene and the Jarid2 mutant gene are coexpressed.
  • ⁇ 5> At least one selected from the group consisting of the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product is the Prdm14 gene Or the gene product, the Esrrb gene or the gene product thereof, and the Sall4a gene or the gene product thereof, or the method for producing an induced pluripotent stem cell according to ⁇ 1>.
  • ⁇ 6> The method for producing an induced pluripotent stem cell according to any one of ⁇ 1> to ⁇ 5>, wherein the induced pluripotent stem cell is a naive type.
  • ⁇ 7> The method for producing an induced pluripotent stem cell according to any one of ⁇ 1> to ⁇ 6>, wherein the induced pluripotent stem cell is a human induced pluripotent stem cell.
  • ⁇ 8> comprising at least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product, A composition for producing induced pluripotent stem cells, wherein the Jarid2 mutant gene or its gene product is a gene encoding the N-terminal 1st to 551st amino acids of the Jarid2 protein or its gene product. .
  • At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product is a Jarid2 mutant gene or The composition for producing induced pluripotent stem cells according to ⁇ 8>, including the gene product.
  • At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product is further added to the Prdm14 gene or its gene product.
  • the composition for producing induced pluripotent stem cells according to ⁇ 9> including the gene product, Esrrb gene or gene product, and Sall4a gene or gene product.
  • At least one selected from the group consisting of a Jard2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, or a Sall4a gene or its gene product is a Prdm14 gene or its gene
  • the composition for producing induced pluripotent stem cells according to ⁇ 8> comprising a product, an Esrrb gene or a gene product thereof, and a Sall4a gene or a gene product thereof.
  • ⁇ 12> Any one of ⁇ 8> to ⁇ 11>, further including Oct3 / 4 gene or gene product, Sox2 gene or gene product, Klf4 gene or gene product, and c-Myc gene or gene product
  • ⁇ 13> The composition for producing induced pluripotent stem cells according to ⁇ 12>, wherein the c-Myc gene and the Jarid2 mutant gene are coexpressed.
  • an induced pluripotent stem cell of the present invention it is possible to produce an induced pluripotent stem cell that is excellent in production efficiency, can be produced in a short period of time, and is excellent in quality. It can be suitably used in a method for producing induced pluripotent stem cells used as a regenerative medicine such as treatment, drug screening, and a tool for elucidating the cause of a disease.

Abstract

A method for manufacturing induced pluripotent stem cells comprising a process for introducing (A) Oct3/4 gene or a gene product thereof, (B) Sox2 gene or a gene product thereof, (C) Klf4 gene or a gene product thereof, (D) c-Myc gene or a gene product thereof, and (E) at least one selected from a group consisting of a Jarid2 mutant gene and gene products thereof, Prdm14 gene and gene products thereof, Esrrb gene and gene products thereof, and Sall4a gene and gene products thereof into a somatic cell. The Jarid2 mutant gene or gene product thereof is a gene or gene product that codes for the first to the 551st N-terminal amino acid of the Jarid2 protein.

Description

人工多能性幹細胞製造用組成物、及び人工多能性幹細胞の製造方法Composition for producing induced pluripotent stem cells and method for producing induced pluripotent stem cells
　本発明は、人工多能性幹細胞製造用組成物、及び人工多能性幹細胞の製造方法に関する。 The present invention relates to a composition for producing induced pluripotent stem cells and a method for producing induced pluripotent stem cells.
　人工多能性幹細胞（以下、「ｉＰＳ（ｉｎｄｕｃｅｄ　ｐｌｕｒｉｐｏｔｅｎｔ　ｓｔｅｍ）細胞」、又は「誘導多能性幹細胞」と称することがある）は、多能性、自己複製、及び高い増殖能といった性質を有しており、分化を誘導することにより、心筋細胞、血液細胞、網膜色素上皮細胞、神経細胞などに分化できることが知られている。そのため、例えば、細胞移植治療などの再生医療、薬剤スクリーニング、疾患の原因解明のためのツールなどとして用いることが期待されている。 Artificial pluripotent stem cells (hereinafter sometimes referred to as “iPS (induced pluripotent stem) cells” or “induced pluripotent stem cells”) have properties such as pluripotency, self-replication, and high proliferation ability. It is known that differentiation can be differentiated into cardiomyocytes, blood cells, retinal pigment epithelial cells, nerve cells and the like by inducing differentiation. Therefore, for example, it is expected to be used as a regenerative medicine such as cell transplantation treatment, drug screening, and a tool for elucidating the cause of a disease.
　前記人工多能性幹細胞の製造方法としては、例えば、Ｏｃｔ３／４遺伝子、Ｓｏｘ２遺伝子、Ｋｌｆ４遺伝子、及びｃ－Ｍｙｃ遺伝子を体細胞に導入することにより製造する方法が提案されている（例えば、特許文献１参照）。前記提案の方法によれば、人工多能性幹細胞を製造することができるものの、（１）製造効率が低い、（２）製造期間が長い、（３）ヒト人工多能性幹細胞では、Ｌｅｕｋｅｍｉａ　ｉｎｈｉｂｉｔｏｒｙ　ｆａｃｔｏｒ（以下、「ＬＩＦ」と称することがある）依存性を有するナイーブ型の人工多能性幹細胞が得られず、品質面で不十分である、という大きな問題がある。 As a method for producing the induced pluripotent stem cell, for example, a method for producing by introducing Oct3 / 4 gene, Sox2 gene, Klf4 gene, and c-Myc gene into somatic cells has been proposed (for example, patents). Reference 1). According to the proposed method, although induced pluripotent stem cells can be produced, (1) production efficiency is low, (2) production period is long, and (3) human induced pluripotent stem cells are Leukemia inhibitory. There is a big problem that naive-type induced pluripotent stem cells having a factor (hereinafter, sometimes referred to as “LIF”) dependency are not obtained and the quality is insufficient.
　したがって、製造効率に優れ、短期間で製造することができ、品質面にも優れた人工多能性幹細胞の製造方法の速やかな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 prompt development of a method for producing induced pluripotent stem cells that are excellent in production efficiency, can be produced in a short period of time, and are excellent in quality.
特許第４１８３７４２号公報Japanese Patent No. 4183742
　本発明は、前記従来における諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、製造効率に優れ、短期間で製造することができ、品質面にも優れた人工多能性幹細胞の製造方法、及び人工多能性幹細胞製造用組成物を提供することを目的とする。 This invention makes it a subject to solve the said conventional problems and to achieve the following objectives. That is, the present invention provides a method for producing an induced pluripotent stem cell that is excellent in production efficiency, can be produced in a short period of time, and is excellent in quality, and a composition for producing induced pluripotent stem cells. Objective.
　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＞　（Ａ）Ｏｃｔ３／４遺伝子乃至その遺伝子産物と、
　（Ｂ）Ｓｏｘ２遺伝子乃至その遺伝子産物と、
　（Ｃ）Ｋｌｆ４遺伝子乃至その遺伝子産物と、
　（Ｄ）ｃ－Ｍｙｃ遺伝子乃至その遺伝子産物と、
　（Ｅ）Ｊａｒｉｄ２（Ｊｕｍｏｎｊｉ，　ＡＴ　ｒｉｃｈ　ｉｎｔｅｒａｃｔｉｖｅ　ｄｏｍａｉｎ　２）変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４（ＰＲ　ｄｏｍａｉｎ　ｃｏｎｔａｉｎｉｎｇ　１４）遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ（Ｅｓｔｒｏｇｅｎ－ｒｅｌａｔｅｄ　ｒｅｃｅｐｔｏｒ　ｂｅｔａ）遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ（Ｓａｌ－ｌｉｋｅ　４、ｔｒａｎｓｃｒｉｐｔ　ｖａｒｉａｎｔ　ａ）遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種とを体細胞に導入する工程を含み、
　前記Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物が、Ｊａｒｉｄ２タンパク質のＮ末端の１番目から５５１番目のアミノ酸をコードする遺伝子乃至その遺伝子産物であることを特徴とする人工多能性幹細胞の製造方法である。
　＜２＞　Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種を含み、
　前記Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物が、Ｊａｒｉｄ２タンパク質のＮ末端の１番目から５５１番目のアミノ酸をコードする遺伝子乃至その遺伝子産物であることを特徴とする人工多能性幹細胞製造用組成物である。 Means for solving the problems are as follows. That is,
<1> (A) Oct3 / 4 gene or its gene product;
(B) the Sox2 gene or its gene product;
(C) the Klf4 gene or its gene product;
(D) the c-Myc gene or its gene product;
(E) Jarid2 (Jumonji, AT rich interactive domain 2) mutant gene or its gene product, Prdm14 (PR domain containing 14) gene or its gene product, Esrrb (Estrogen-related receptor gene, or its gene) (Sal-like 4, transscript variant a) introducing into a somatic cell at least one selected from the group consisting of a gene or a gene product thereof,
A method for producing an induced pluripotent stem cell, wherein the Jarid2 mutant gene or a gene product thereof is a gene encoding the N-terminal amino acids 1 to 551 of the Jarid2 protein or a gene product thereof.
<2> comprising at least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product,
A composition for producing induced pluripotent stem cells, wherein the Jarid2 mutant gene or its gene product is a gene encoding the N-terminal 1st to 551st amino acids of the Jarid2 protein or its gene product. .
　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、製造効率に優れ、短期間で製造することができ、品質面にも優れた人工多能性幹細胞の製造方法、及び人工多能性幹細胞製造用組成物を提供することができる。 According to the present invention, an artificial pluripotent stem cell that can solve the above-mentioned problems and can achieve the above-mentioned object, is excellent in production efficiency, can be produced in a short period of time, and is excellent in quality. A production method and a composition for producing induced pluripotent stem cells can be provided.
図１－１は、試験例１－１の結果を示すグラフである。FIG. 1-1 is a graph showing the results of Test Example 1-1. 図１－２は、試験例１－２の結果を示すグラフである。FIG. 1-2 is a graph showing the results of Test Example 1-2. 図１－３は、試験例１－３の結果を示すグラフである。FIG. 1-3 is a graph showing the results of Test Example 1-3. 図１－４は、試験例１－４の結果を示すグラフである。FIG. 1-4 is a graph showing the results of Test Example 1-4. 図１－５は、試験例１－５の結果を示すグラフである。FIG. 1-5 is a graph showing the results of Test Example 1-5. 図１－６は、試験例１－６の結果を示すグラフである。FIG. 1-6 is a graph showing the results of Test Example 1-6. 図２－１は、試験例２－１の結果を示すグラフである。FIG. 2-1 is a graph showing the results of Test Example 2-1. 図２－２は、試験例２－２の結果を示すグラフである。FIG. 2-2 is a graph showing the results of Test Example 2-2. 図２－３は、試験例２－３の結果を示すグラフである。FIG. 2-3 is a graph showing the results of Test Example 2-3. 図３Ａは、試験例３－１における（１）のウイルス液を用いた場合の結果の一例を示す写真である。FIG. 3A is a photograph showing an example of the results obtained when the virus solution of (1) in Test Example 3-1 was used. 図３Ｂは、試験例３－１における（１）のウイルス液を用いた場合の結果の他の一例を示す写真である。FIG. 3B is a photograph showing another example of the result obtained when the virus solution of (1) in Test Example 3-1 was used. 図３Ｃは、試験例３－１における（２）のウイルス液を用いた場合の結果の一例を示す写真である。FIG. 3C is a photograph showing an example of the results obtained when the virus solution of (2) in Test Example 3-1 was used. 図３Ｄは、試験例３－１における（２）のウイルス液を用いた場合の結果の他の一例を示す写真である。FIG. 3D is a photograph showing another example of the results obtained when the virus solution of (2) in Test Example 3-1 was used. 図３Ｅは、試験例３－２における（１）のウイルス液を用いた場合の結果の一例を示す写真である。FIG. 3E is a photograph showing an example of the results obtained when the virus solution of (1) in Test Example 3-2 was used. 図３Ｆは、試験例３－２における（１）のウイルス液を用いた場合の結果の他の一例を示す写真である。FIG. 3F is a photograph showing another example of the results obtained when the virus solution of (1) in Test Example 3-2 was used. 図３Ｇは、試験例３－２における（２）のウイルス液を用いた場合の結果の一例を示す写真である。FIG. 3G is a photograph showing an example of the results obtained when the virus solution of (2) in Test Example 3-2 was used. 図３Ｈは、試験例３－２における（２）のウイルス液を用いた場合の結果の他の一例を示す写真である。FIG. 3H is a photograph showing another example of the results obtained when the virus solution of (2) in Test Example 3-2 was used.
（人工多能性幹細胞の製造方法）
　本発明の人工多能性幹細胞の製造方法は、（Ａ）Ｏｃｔ３／４遺伝子乃至その遺伝子産物と、（Ｂ）Ｓｏｘ２遺伝子乃至その遺伝子産物と、（Ｃ）Ｋｌｆ４遺伝子乃至その遺伝子産物と、（Ｄ）ｃ－Ｍｙｃ遺伝子乃至その遺伝子産物と、（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種とを体細胞に導入する工程（以下、「遺伝子乃至その遺伝子産物導入工程」と称することがある）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 producing induced pluripotent stem cells)
The method for producing induced pluripotent stem cells of the present invention comprises (A) Oct3 / 4 gene or its gene product, (B) Sox2 gene or its gene product, (C) Klf4 gene or its gene product, (D Selected from the group consisting of :) c-Myc gene or its gene product, (E) Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, or Sall4a gene or its gene product At least one of the above-described processes into a somatic cell (hereinafter sometimes referred to as a “gene or gene product introduction process”), and further includes other processes as necessary.
＜遺伝子乃至その遺伝子産物導入工程＞
　前記遺伝子乃至その遺伝子産物導入工程は、少なくとも（Ａ）Ｏｃｔ３／４遺伝子乃至その遺伝子産物と、（Ｂ）Ｓｏｘ２遺伝子乃至その遺伝子産物と、（Ｃ）Ｋｌｆ４遺伝子乃至その遺伝子産物と、（Ｄ）ｃ－Ｍｙｃ遺伝子乃至その遺伝子産物と、（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種とを体細胞に導入する工程である。
　前記各遺伝子乃至その遺伝子産物を体細胞に導入することにより、体細胞から人工多能性幹細胞を製造することができる。
　前記遺伝子産物とは、遺伝子から転写されるｍＲＮＡ（メッセンジャーＲＮＡ）、前記ｍＲＮＡから翻訳されるタンパク質をいう。 <Gene or gene product introduction process>
The gene or its gene product introduction step includes at least (A) Oct3 / 4 gene or its gene product, (B) Sox2 gene or its gene product, (C) Klf4 gene or its gene product, and (D) c At least selected from the group consisting of the Myc gene or its gene product, and (E) the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product This is a step of introducing one species into somatic cells.
By introducing each gene or gene product thereof into a somatic cell, an induced pluripotent stem cell can be produced from the somatic cell.
The gene product refers to mRNA (messenger RNA) transcribed from a gene and protein translated from the mRNA.
　前記（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１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が、（１）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物を含む態様、（２）Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む態様、（３）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む態様が好ましく、（３）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む態様がより好ましい。
　前記より好ましい態様であると、人工多能性幹細胞の製造効率により優れ、かつ、より短期間で製造することができ、また、品質面にも優れる点で、有利である。 As at least one selected from the group consisting of (E) Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, and Sall4a gene or its gene product, there is a particular limitation. However, (1) an embodiment including a Jarid2 mutant gene or its gene product, (2) a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or An embodiment including the gene product, (3) an embodiment including the Jarid2 mutant gene through the gene product, the Prdm14 gene through the gene product, the Esrrb gene through the gene product, and the Sall4a gene through the gene product are preferred, and (3) Jarid Variant gene or its gene product, PRDM14 gene or its gene product, Esrrb gene or its gene product, and is more preferred embodiment including Sall4a gene or its gene product.
The more preferable embodiment is advantageous in that it is superior in the production efficiency of induced pluripotent stem cells, can be produced in a shorter period of time, and is superior in quality.
－Ｏｃｔ３／４遺伝子－
　前記Ｏｃｔ３／４遺伝子の由来としては、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、マウス、などが挙げられる。
　前記Ｏｃｔ３／４遺伝子の配列情報は、公知のデータベースから得ることができ、例えば、ＮＣＢＩでは、アクセッション番号ＮＭ＿００２７０１（ヒト）、ＮＭ＿０１３６３３（マウス）で入手することができる。 -Oct3 / 4 gene-
The origin of the Oct3 / 4 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
The sequence information of the Oct3 / 4 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_002701 (human) and NM_013633 (mouse).
－Ｓｏｘ２遺伝子－
　前記Ｓｏｘ２遺伝子の由来としては、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、マウス、などが挙げられる。
　前記Ｓｏｘ２遺伝子の配列情報は、公知のデータベースから得ることができ、例えば、ＮＣＢＩでは、アクセッション番号ＮＭ＿００３１０６（ヒト）、ＮＭ＿０１１４４３（マウス）で入手することができる。 -Sox2 gene-
The origin of the Sox2 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
The sequence information of the Sox2 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_003106 (human) and NM_011443 (mouse).
－Ｋｌｆ４遺伝子－
　前記Ｋｌｆ４遺伝子の由来としては、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、マウス、などが挙げられる。
　前記Ｋｌｆ４遺伝子の配列情報は、公知のデータベースから得ることができ、例えば、ＮＣＢＩでは、アクセッション番号ＮＭ＿００４２３５（ヒト）、ＮＭ＿０１０６３７（マウス）で入手することができる。 -Klf4 gene-
The origin of the Klf4 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
The sequence information of the Klf4 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_004235 (human) and NM_0103737 (mouse).
－ｃ－Ｍｙｃ遺伝子－
　前記ｃ－Ｍｙｃ遺伝子の由来としては、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、マウス、などが挙げられる。
　前記ｃ－Ｍｙｃ遺伝子の配列情報は、公知のデータベースから得ることができ、例えば、ＮＣＢＩでは、アクセッション番号ＮＭ＿００２４６７（ヒト）、ＮＭ＿０１０８４９（マウス）で入手することができる。 -C-Myc gene-
The origin of the c-Myc gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
The sequence information of the c-Myc gene can be obtained from a known database. For example, NCBI can obtain the accession numbers NM_002467 (human) and NM_010849 (mouse).
－Ｊａｒｉｄ２変異体遺伝子－
　前記Ｊａｒｉｄ２変異体遺伝子は、Ｊａｒｉｄ２タンパク質のＮ末端の１番目から５５１番目のアミノ酸をコードする遺伝子（配列番号１２参照）である。
　前記Ｊａｒｉｄ２遺伝子の由来としては、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、マウス、などが挙げられる。
　前記Ｊａｒｉｄ２遺伝子の配列情報は、公知のデータベースから得ることができ、例えば、ＮＣＢＩでは、アクセッション番号ＮＭ＿００４９７３（ヒト）、ＮＭ＿０２１８７８（マウス）で入手することができる。 -Jarid2 mutant gene-
The Jarid2 mutant gene is a gene (see SEQ ID NO: 12) encoding the 1st to 551st amino acids at the N-terminus of the Jarid2 protein.
The origin of the Jarid2 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
The sequence information of the Jarid2 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_004973 (human) and NM_021878 (mouse).
－Ｐｒｄｍ１４遺伝子－
　前記Ｐｒｄｍ１４遺伝子の由来としては、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、マウス、などが挙げられる。
　前記Ｐｒｄｍ１４遺伝子の配列情報は、公知のデータベースから得ることができ、例えば、ＮＣＢＩでは、アクセッション番号ＮＭ＿０２４５０４（ヒト）、ＮＭ＿００１０８１２０９（マウス、配列番号１９参照）で入手することができる。 -Prdm14 gene-
The origin of the Prdm14 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
The sequence information of the Prdm14 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_024504 (human) and NM_001081209 (mouse, see SEQ ID NO: 19).
－Ｅｓｒｒｂ遺伝子－
　前記Ｅｓｒｒｂ遺伝子の由来としては、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、マウス、などが挙げられる。
　前記Ｅｓｒｒｂ遺伝子の配列情報は、公知のデータベースから得ることができ、例えば、ＮＣＢＩでは、アクセッション番号ＮＭ＿００４４５２（ヒト）、ＮＭ＿０１１９３４（マウス、配列番号２０参照）で入手することができる。 -Esrrb gene-
There is no restriction | limiting in particular as origin of the said Esrrb gene, According to the objective, it can select suitably, For example, a human, a mouse | mouth etc. are mentioned.
The sequence information of the Esrrb gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_004452 (human) and NM_011934 (mouse, see SEQ ID NO: 20).
－Ｓａｌｌ４ａ遺伝子－
　前記Ｓａｌｌ４ａ遺伝子の由来としては、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、マウス、などが挙げられる。
　前記Ｓａｌｌ４ａ遺伝子の配列情報は、公知のデータベースから得ることができ、例えば、ＮＣＢＩでは、アクセッション番号ＮＭ＿０２０４３６（ヒト）、ＮＭ＿１７５３０３（マウス、配列番号２１参照）で入手することができる。 -Sall4a gene-
The origin of the Sall4a gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
The sequence information of the Sall4a gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_020436 (human) and NM_175303 (mouse, see SEQ ID NO: 21).
　前記Ｏｃｔ３／４遺伝子乃至その遺伝子産物、Ｓｏｘ２遺伝子乃至その遺伝子産物、Ｋｌｆ４遺伝子乃至その遺伝子産物、ｃ－Ｍｙｃ遺伝子乃至その遺伝子産物、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物の配列は、本発明の効果を損なわない限り特に制限はなく、前記各遺伝子の配列のうち、タンパク質に翻訳される部分のみであってもよいし、それ以外の部分を含んでもよい。また、前記各遺伝子乃至その遺伝子産物の配列は、変異が含まれていてもよい。
　前記変異としては、例えば、前記各遺伝子のタンパク質のアミノ酸配列に影響を与えない変異、前記各遺伝子のタンパク質のアミノ酸配列において、１個又は数個（２個～５個）のアミノ酸が欠失、置換、挿入、又は付加される変異、などが挙げられる。
　前記各遺伝子乃至その遺伝子産物が変異を有する場合の野生型との相同性としては、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、タンパク質に翻訳される部分の塩基配列において、７０％以上が好ましく、８０％以上がより好ましく、９０％以上が特に好ましい。 Oct3 / 4 gene to its gene product, Sox2 gene to its gene product, Klf4 gene to its gene product, c-Myc gene to its gene product, Jarid2 mutant gene to its gene product, Prdm14 gene to its gene product, Esrrb The sequence of the gene or its gene product, and the sequence of the Sall4a gene or its gene product are not particularly limited as long as the effects of the present invention are not impaired, and may be only the portion of the sequence of each gene that is translated into a protein. However, other parts may be included. Further, the sequence of each gene or gene product thereof may contain a mutation.
Examples of the mutation include a mutation that does not affect the amino acid sequence of the protein of each gene, and one or several (2 to 5) amino acids are deleted from the amino acid sequence of the protein of each gene. Substitution, insertion, or mutation to be added.
The homology with the wild-type when each gene or its gene product has a mutation is not particularly limited and can be appropriately selected according to the purpose. 70% or more is preferable, 80% or more is more preferable, and 90% or more is particularly preferable.
－体細胞－
　前記体細胞としては、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、胎児期の体細胞、成熟した体細胞、などが挙げられる。
　前記成熟した体細胞の具体例としては、脂肪組織由来間質（幹）細胞、神経幹細胞、造血幹細胞、間葉系幹細胞、***幹細胞等の組織幹細胞（体性幹細胞）；組織前駆細胞；リンパ球、上皮細胞、筋肉細胞、線維芽細胞等の既に分化した細胞、などが挙げられる。 -Somatic cells-
There is no restriction | limiting in particular as said somatic cell, According to the objective, it can select suitably, For example, a fetal somatic cell, a mature somatic cell, etc. are mentioned.
Specific examples of the mature somatic cells include tissue stem cells (somatic stem cells) such as adipose tissue-derived stromal (stem) cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, sperm stem cells; tissue precursor cells; lymphocytes And already differentiated cells such as epithelial cells, muscle cells, fibroblasts, and the like.
　前記体細胞は、人工多能性幹細胞の選択を容易にするために、組み換えたものであってもよい。
　前記組換え体細胞の具体例としては、分化多能性細胞において特異的に高発現する遺伝子の遺伝子座に、レポーター遺伝子、及び薬剤耐性遺伝子の少なくともいずれかを組み込んだ組換え体細胞が挙げられる。
　前記分化多能性細胞において特異的に高発現する遺伝子としては、例えば、Ｆｂｘ１５遺伝子、Ｎａｎｏｇ遺伝子、Ｏｃｔ３／４遺伝子、などが挙げられる。
　前記レポーター遺伝子としては、例えば、緑色蛍光タンパク質（ＧＦＰ）遺伝子、β－ガラクトシダーゼ遺伝子、などが挙げられる。
　前記薬剤耐性遺伝子としては、ピューロマイシン耐性遺伝子、ネオマイシン耐性遺伝子、などが挙げられる。 The somatic cells may be recombinant so as to facilitate selection of induced pluripotent stem cells.
Specific examples of the recombinant cell include a recombinant cell in which at least one of a reporter gene and a drug resistance gene is incorporated into a gene locus that is specifically highly expressed in a pluripotent cell. .
Examples of the gene that is specifically highly expressed in the pluripotent cells include Fbx15 gene, Nanog gene, Oct3 / 4 gene, and the like.
Examples of the reporter gene include a green fluorescent protein (GFP) gene and a β-galactosidase gene.
Examples of the drug resistance gene include a puromycin resistance gene and a neomycin resistance gene.
　前記体細胞を採取する個体としては、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、得られる人工多能性幹細胞を再生医療用途に用いる場合には、拒絶反応の観点から、個体自身、又はＭＨＣの型が同一若しくは実質的に同一の他個体が好ましい。
　ここで、前記ＭＨＣの型が実質的に同一とは、免疫抑制剤などの使用により、前記体細胞由来の人工多能性幹細胞から分化誘導して得られた細胞を個体に移植した場合に移植細胞が生着可能な程度にＭＨＣの型が一致していることをいう。 The individual that collects the somatic cells is not particularly limited and can be appropriately selected according to the purpose.However, when the obtained induced pluripotent stem cells are used for regenerative medicine, from the viewpoint of rejection, The individual itself or another individual with the same or substantially the same type of MHC is preferred.
Here, the type of the MHC is substantially the same when the cells obtained by inducing differentiation from the somatic cell-derived induced pluripotent stem cells are transplanted into an individual by using an immunosuppressant or the like. It means that the MHC types match to such an extent that cells can be engrafted.
　前記体細胞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、例えば、培養温度は約３７℃、ＣＯ２濃度は約２％～５％、などが挙げられる。
　前記体細胞の培養に用いる培地としては、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、５質量％～２０質量％の血清を含む、最小必須培地（ＭＥＭ）、ダルベッコ改変培地（ＤＭＥＭ）、ＲＰＭＩ１６４０培地、１９９培地、Ｆ１２培地、などが挙げられる。 The somatic cell culture conditions are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a culture temperature of about 37 ° C. and a CO 2 concentration of about 2% to 5%.
The medium used for culturing the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a medium (DMEM), RPMI 1640 medium, 199 medium, and F12 medium.
－導入方法－
　前記各遺伝子乃至その遺伝子産物を体細胞に導入方法としては、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、ベクターを用いる方法、合成したｍＲＮＡを用いる方法、組換えタンパク質を用いる方法、などが挙げられる。 -Introduction method-
The method for introducing each gene or its gene product into a somatic cell is not particularly limited and can be appropriately selected depending on the purpose. For example, a method using a vector, a method using synthesized mRNA, a recombinant protein The method used, etc. are mentioned.
－－ベクター－－
　前記ベクターとしては、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、ウイルスベクター、人工染色体ベクター、プラスミドベクター、エピソーマルベクター、などが挙げられる。
　前記ウイルスベクターの具体例としては、レトロウイルス（レンチウイルスを含む）ベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター、センダイウイルスベクター、ヘルペスウイルスベクター、ワクシニアウイルスベクター、ポックスウイルスベクター、ポリオウイルスベクター、シルビスウイルスベクター、ラブドウイルスベクター、パラミクソウイルスベクター、オルソミクソウイルスベクター、などが挙げられる。
　前記人工染色体ベクターの具体例としては、ＹＡＣ（Ｙｅａｓｔ　ａｒｔｉｆｉｃｉａｌ　ｃｈｒｏｍｏｓｏｍｅ）ベクター、ＢＡＣ（Ｂａｃｔｅｒｉａｌ　ａｒｔｉｆｉｃｉａｌ　ｃｈｒｏｍｏｓｏｍｅ）ベクター、ＰＡＣ（Ｐ１－ｄｅｒｉｖｅｄ　ａｒｔｉｆｉｃｉａｌ　ｃｈｒｏｍｏｓｏｍｅ）ベクター、などが挙げられる。
　前記ベクターを前記体細胞に導入する方法としては、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、リポフェクション法、マイクロインジェクション法、ＤＥＡＥデキストラン法、遺伝子銃法、エレクトロポレーション法、リン酸カルシウム法、などが挙げられる。 --vector--
There is no restriction | limiting in particular as said vector, According to the objective, it can select suitably, For example, a viral vector, an artificial chromosome vector, a plasmid vector, an episomal vector etc. are mentioned.
Specific examples of the viral vectors include retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, silvis Examples thereof include viral vectors, rhabdovirus vectors, paramyxovirus vectors, orthomyxovirus vectors, and the like.
Specific examples of the artificial chromosome vector include a YAC (Yeast Artificial Chromome) vector, a BAC (Bacterial Artificial Chromosome) vector, a PAC (P1-derived Artificial Chromome) vector, and the like.
The method for introducing the vector into the somatic cell is not particularly limited and may be appropriately selected depending on the purpose. For example, lipofection method, microinjection method, DEAE dextran method, gene gun method, electroporation method , Calcium phosphate method, and the like.
　前記ウイルスベクターを用いる場合には、パッケージング細胞を用いて得られたウイルス粒子を用いてもよい。
　前記パッケージング細胞は、ウイルスの構造タンパク質をコードする遺伝子を導入した細胞であり、該細胞に目的遺伝子を組み込んだ組換えウイルスベクターを導入すると、該目的遺伝子を組み込んだ組換えウイルス粒子を産生する。
　前記パッケージング細胞としては、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、ヒト腎臓由来のＨＥＫ２９３細胞やマウス繊維芽細胞由来のＮＩＨ３Ｔ３細胞をベースとしたパッケージング細胞、Ｅｃｏｔｒｏｐｉｃ　ｖｉｒｕｓ由来エンベロープ糖タンパク質を発現するよう設計されているＰＬＡＴ－Ｅ細胞（以下、「ＰＬＡＴ－Ｅ細胞」と称することがある）、Ａｍｐｈｏｔｒｏｐｉｃ　ｖｉｒｕｓ由来エンベロープ糖タンパク質を発現するよう設計されているＰＬＡＴ－Ａ細胞、水疱性口内炎ウイルス由来エンベロープ糖タンパク質を発現するよう設計されているＰＬＡＴ－ＧＰ細胞（以下、「ＰＬＡＴ－ＧＰ細胞」と称することがある）、などが挙げられる。これらの中でも、ヒト体細胞に対して組換えウイルスベクターを導入する場合にはＰＬＡＴ－Ａ細胞、ＰＬＡＴ－ＧＰ細胞が、宿主指向性の点で、好ましい。
　前記パッケージング細胞へのウイルスベクターの導入方法としては、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、リポフェクション法、エレクトロポレーション法、リン酸カルシウム法、などが挙げられる。
　前記得られたウイルス粒子を前記体細胞へ感染させる方法としては、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、ポリブレン法が挙げられる。 When the virus vector is used, virus particles obtained using packaging cells may be used.
The packaging cell is a cell into which a gene encoding a structural protein of a virus has been introduced. When a recombinant virus vector incorporating a target gene is introduced into the cell, a recombinant virus particle incorporating the target gene is produced. .
The packaging cell is not particularly limited and may be appropriately selected according to the purpose. For example, packaging cells based on human kidney-derived HEK293 cells or mouse fibroblast-derived NIH3T3 cells, Ecotropic virus PLAT-E cells designed to express derived envelope glycoproteins (hereinafter sometimes referred to as “PLAT-E cells”), PLAT-A cells designed to express envelope glycoproteins derived from Amphotropic virus And PLAT-GP cells designed to express vesicular stomatitis virus-derived envelope glycoproteins (hereinafter sometimes referred to as “PLAT-GP cells”). Among these, PLAT-A cells and PLAT-GP cells are preferable from the viewpoint of host orientation when a recombinant viral vector is introduced into human somatic cells.
The method for introducing the viral vector into the packaging cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a lipofection method, an electroporation method, and a calcium phosphate method.
The method for infecting the somatic cells with the obtained virus particles is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a polybrene method.
　前記ベクターは、前記各遺伝子の導入を確認するためのマーカー遺伝子を含んでいてもよい。
　前記マーカー遺伝子とは、該マーカー遺伝子を細胞に導入することにより、細胞の選別や選択を可能とするような遺伝子をいう。前記マーカー遺伝子の具体例としては、薬剤耐性遺伝子、蛍光タンパク質遺伝子、発光酵素遺伝子、発色酵素遺伝子、などが挙げられる。これらは、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。
　前記薬剤耐性遺伝子の具体例としては、ネオマイシン耐性遺伝子、テトラサイクリン耐性遺伝子、カナマイシン耐性遺伝子、ゼオシン耐性遺伝子、ハイグロマイシン耐性遺伝子、などが挙げられる。
　前記蛍光タンパク質遺伝子の具体例としては、ＧＦＰ遺伝子、黄色蛍光タンパク質（ＹＦＰ）遺伝子、赤色蛍光タンパク質（ＲＦＰ）遺伝子、などが挙げられる。
　前記発光酵素遺伝子の具体例としては、ルシフェラーゼ遺伝子、などが挙げられる。
　前記発色酵素遺伝子の具体例としては、βガラクトシターゼ遺伝子、βグルクロニダーゼ遺伝子、アルカリフォスファターゼ遺伝子、などが挙げられる。 The vector may contain a marker gene for confirming the introduction of each gene.
The marker gene refers to a gene that enables selection and selection of cells by introducing the marker gene into cells. Specific examples of the marker gene include a drug resistance gene, a fluorescent protein gene, a luminescent enzyme gene, a chromogenic enzyme gene, and the like. These may be used individually by 1 type and may use 2 or more types together.
Specific examples of the drug resistance gene include a neomycin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a zeocin resistance gene, and a hygromycin resistance gene.
Specific examples of the fluorescent protein gene include a GFP gene, a yellow fluorescent protein (YFP) gene, and a red fluorescent protein (RFP) gene.
Specific examples of the luminescent enzyme gene include luciferase gene.
Specific examples of the chromogenic enzyme gene include β-galactosidase gene, β-glucuronidase gene, alkaline phosphatase gene, and the like.
　前記ベクターを用いて前記各遺伝子を体細胞に導入する方法では、１つのベクターに１つの遺伝子を組み込んでもよいし、２つ以上の遺伝子を組み込んでもよい。前記１つのベクターに２つ以上の遺伝子を組み込むことにより、該２つ以上の遺伝子を同時に発現（以下、「共発現」と称することがある）させることができる。
　本発明の人工多能性幹細胞の製造方法では、前記ｃ－Ｍｙｃ遺伝子と、前記Ｊａｒｉｄ２変異体遺伝子とが共発現されるように導入することが好ましい。 In the method of introducing each gene into a somatic cell using the vector, one gene may be incorporated into one vector, or two or more genes may be incorporated. By incorporating two or more genes into the one vector, the two or more genes can be expressed simultaneously (hereinafter sometimes referred to as “co-expression”).
In the method for producing induced pluripotent stem cells of the present invention, the c-Myc gene and the Jarid2 mutant gene are preferably introduced so as to be coexpressed.
　前記１つのベクターに２つ以上の遺伝子を組み込む方法としては、特に制限はなく、目的に応じて適宜選択することができるが、前記２つ以上の遺伝子を、連結配列を通じて組み込むことが好ましい。
　前記連結配列としては、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、***ウイルス（Ｐｉｃｏｒｎａｖｉｒｉｄａｅ　Ａｐｈｔｈｏｖｉｒｕｓ）由来２Ａペプチドをコードする遺伝子配列、ＩＲＥＳ（ｉｎｔｅｒｎａｌ　ｒｉｂｏｓｏｍｅ　ｅｎｔｒｙ　ｓｉｔｅｓ）、などが挙げられる。 The method for incorporating two or more genes into the one vector is not particularly limited and may be appropriately selected depending on the intended purpose. However, it is preferable to incorporate the two or more genes through a linking sequence.
The linking sequence is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a gene sequence encoding 2A peptide derived from foot-and-mouth disease virus (Picornaviridae Aphthovirus), IRES (internal ribosome entry sites), and the like. It is done.
　前記ｍＲＮＡを前記体細胞に導入する方法としては、特に制限はなく、公知の方法を適宜選択して用いることができる。
　前記組換えタンパク質を前記体細胞に導入する方法としては、特に制限はなく、公知の方法を適宜選択して用いることができる。 There is no restriction | limiting in particular as a method of introduce | transducing the said mRNA into the said somatic cell, A well-known method can be selected suitably and can be used.
There is no restriction | limiting in particular as a method of introduce | transducing the said recombinant protein into the said somatic cell, A well-known method can be selected suitably and can be used.
　前記各遺伝子乃至その遺伝子産物の体細胞への導入回数は、１回であってもよいし、２回以上であってもよい。
　前記各遺伝子乃至その遺伝子産物の体細胞への導入時期としては、特に制限はなく、目的に応じて適宜選択することができ、前記各遺伝子乃至その遺伝子産物の全てを同時期に導入してもよいし、異なる時期に導入してもよい。
　前記遺伝子乃至その遺伝子産物は、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。また、遺伝子のみを用いる態様であってもよいし、遺伝子産物のみを用いる態様であってもよいし、前記各遺伝子のうち、ある遺伝子は遺伝子産物を用い、他の遺伝子は遺伝子を用いる態様であってもよい。 The number of introduction of each gene or its gene product into a somatic cell may be one time or two or more times.
There is no particular limitation on the introduction timing of each gene or its gene product into a somatic cell, and it can be appropriately selected according to the purpose. Even if all the genes or their gene products are introduced simultaneously. It may be introduced at different times.
The said gene thru | or its gene product may be used individually by 1 type, and may use 2 or more types together. In addition, an embodiment using only a gene or an embodiment using only a gene product may be used, and among each of the above genes, a gene product may be used and another gene may be a gene. There may be.
　前記各遺伝子乃至その遺伝子産物の体細胞への導入量としては、全ての遺伝子乃至その遺伝子産物を等量ずつ導入してもよいし、異なる量で導入してもよい。前記各遺伝子乃至その遺伝子産物として、遺伝子を用いる場合の例としては、Ｏｃｔ３／４遺伝子がＳｏｘ２遺伝子、Ｋｌｆ４遺伝子、又はｃ－Ｍｙｃ遺伝子に対して多量、例えば約３倍量、導入するのが（ＰＮＡＳ　１０６（３１）：１２７５９－１２７６４．２００９、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２８７（４３）：３６２７３－３６２８２．２０１２）好ましい。 As the introduction amount of each gene or its gene product into a somatic cell, all the genes or their gene products may be introduced in equal amounts or in different amounts. As an example of using a gene as each gene or its gene product, the Oct3 / 4 gene is introduced in a large amount, for example, about 3 times the amount of the Sox2 gene, the Klf4 gene, or the c-Myc gene ( PNAS 106 (31): 12759-1276.2009, J. Biol. Chem. 287 (43): 36273-36282.201) are preferred.
　前記遺伝子乃至その遺伝子産物導入工程では、本発明の効果を損なわない限り、前記各遺伝子乃至その遺伝子産物以外の遺伝子乃至その遺伝子産物を導入してもよい。
　前記各遺伝子乃至その遺伝子産物以外の遺伝子乃至その遺伝子産物としては、例えば、Ｏｃｔファミリー（Ｏｃｔ１Ａ、及びＯｃｔ６）遺伝子乃至その遺伝子産物、Ｋｌｆファミリー（Ｋｌｆ１、Ｋｌｆ２、Ｋｌｆ４、及びＫｌｆ５）遺伝子乃至その遺伝子産物、Ｍｙｃファミリー（Ｎ－Ｍｙｃ、及びＬ－Ｍｙｃ）遺伝子乃至その遺伝子産物、Ｓｏｘファミリー（Ｓｏｘ１、Ｓｏｘ３、Ｓｏｘ７、Ｓｏｘ１５、Ｓｏｘ１７、及びＳｏｘ１８）遺伝子乃至その遺伝子産物、ＴＥＲＴ遺伝子乃至その遺伝子産物、ＳＶ４０　Ｌａｒｇｅ　Ｔ　ａｎｔｉｇｅｎ遺伝子乃至その遺伝子産物、ＨＰＶ１６　Ｅ６遺伝子乃至その遺伝子産物、ＨＰＶ１６　Ｅ７遺伝子乃至その遺伝子産物、Ｂｍｉｌ遺伝子乃至その遺伝子産物、Ｆｂｘ１５遺伝子乃至その遺伝子産物、Ｎａｎｏｇ遺伝子乃至その遺伝子産物、ＥＲａｓ遺伝子乃至その遺伝子産物、ＥＣＡＴ１５－２遺伝子乃至その遺伝子産物、Ｔｃｌ１遺伝子乃至その遺伝子産物、β－ｃａｔｅｎｉｎ遺伝子乃至その遺伝子産物、ＥＣＡＴ１遺伝子乃至その遺伝子産物、Ｅｓｇ１遺伝子乃至その遺伝子産物、Ｄｎｍｔ３Ｌ遺伝子乃至その遺伝子産物、ＥＣＡＴ８遺伝子乃至その遺伝子産物、Ｇｄｆ３遺伝子乃至その遺伝子産物、ＥＣＡＴ１５－１遺伝子乃至その遺伝子産物、Ｆｔｈｌ１７遺伝子乃至その遺伝子産物、Ｒｅｘ１遺伝子乃至その遺伝子産物、ＵＴＦ１遺伝子乃至その遺伝子産物、Ｓｔｅｌｌａ遺伝子乃至その遺伝子産物、Ｓｔａｔ３遺伝子乃至その遺伝子産物、Ｇｒｂ２遺伝子乃至その遺伝子産物（特開２０１１－１８８８６０号公報参照）；ＧＬＩＳ１遺伝子乃至その遺伝子産物（Ｎａｔｕｒｅ　４７４（７３５０）：２２５－２２９．２０１１）；
Ｔｂｘ３遺伝子乃至その遺伝子産物（Ｎａｔｕｒｅ　４６３（７２８４）：１０９６－１００．２０１０）；ＴＥＴ（Ｔｅｎ－ｅｌｅｖｅｎ　ｔｒａｎｓｌｏｃａｔｉｏｎ）ファミリー（ＴＥＴ１、ＴＥＴ２、及びＴＥＴ３）遺伝子乃至その遺伝子産物（Ｎａｔｕｒｅ　４８８（７４１３）：６５２－６５５．２０１２、Ｎａｔｕｒｅ　４９５（７４４１）：３７０－３７４．２０１３）；Ｐａｒｐ１遺伝子乃至その遺伝子産物（Ｎａｔｕｒｅ　４８８（７４１３）：６５２－６５５．２０１２）；Ｋｄｍ２ａ遺伝子乃至その遺伝子産物、Ｋｄｍ２ｂ遺伝子乃至その遺伝子産物（Ｃｅｌｌ　Ｓｔｅｍ　Ｃｅｌｌ　９（６）：５７５－５８７．２０１１、Ｎａｔ．Ｃｅｌｌ　Ｂｉｏｌ．１４（５）：４５７－４６６．２０１２）；Ｎｒ５ａ２遺伝子乃至その遺伝子産物、Ｒａｒファミリー遺伝子乃至その遺伝子産物（Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ　１０８（４５）：１８２８３－１８２８８．２０１１）；Ｕｔｘ遺伝子乃至その遺伝子産物、Ｍｄｍ２遺伝子乃至その遺伝子産物、Ｒｉｎｇ１ｂ遺伝子乃至その遺伝子産物、Ｗｄｒ５遺伝子乃至その遺伝子産物、などが挙げられる。 In the gene or gene product introduction step, genes or gene products other than the genes or gene products may be introduced as long as the effects of the present invention are not impaired.
Examples of the genes or gene products other than the genes or gene products thereof include, for example, the Oct family (Oct1A and Oct6) genes or their gene products, the Klf family (Klf1, Klf2, Klf4, and Klf5) genes or their gene products. , Myc family (N-Myc, and L-Myc) genes to their gene products, Sox family (Sox1, Sox3, Sox7, Sox15, Sox17, and Sox18) genes to their gene products, TERT genes to their gene products, SV40 Large T antigen gene or its gene product, HPV16 E6 gene or its gene product, HPV16 E7 gene or its gene product, Bmil gene or its gene product, Fbx15 gene or its gene product , Nanog gene or its gene product, ERas gene or its gene product, ECAT15-2 gene or its gene product, Tcl1 gene or its gene product, β-catenin gene or its gene product, ECAT1 gene or its gene product, Esg1 gene or its Its gene product, Dnmt3L gene or its gene product, ECAT8 gene or its gene product, Gdf3 gene or its gene product, ECAT15-1 gene or its gene product, Fthl17 gene or its gene product, Rex1 gene or its gene product, UTF1 gene Or its gene product, Stella gene or its gene product, Stat3 gene or its gene product, Grb2 gene or its gene product (see JP 2011-188860 A); LIS1 gene or its gene product (Nature 474 (7350): 225-229.2011);
Tbx3 gene or its gene product (Nature 463 (7284): 1096-100.2010); TET (Ten-eleven transcription) family (TET1, TET2, and TET3) gene or its gene product (Nature 488 (7413): 652- 655.2012, Nature 495 (7441): 370-374.2013); Parp1 gene or its gene product (Nature 488 (7413): 652-655.2012); Kdm2a gene or its gene product, Kdm2b gene or its gene product (Cell Stem Cell 9 (6): 575-587.2011, Nat. Cell Biol.14 (5): 457-466.2012); Nr5a2 gene or its Gene product, Rar family gene to its gene product (Proc. Natl. Acad. Sci. USA 108 (45): 18283-18288.2011); Utx gene to its gene product, Mdm2 gene to its gene product, Ring1b gene to Examples thereof include the gene product, the Wdr5 gene and the gene product thereof.
　前記遺伝子乃至その遺伝子産物導入工程では、本発明の効果を損なわない限り、低分子化合物を投与してもよい。
　前記低分子化合物としては、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、ＧＳＫ－３阻害剤、アデニル酸シクラーゼ活性化剤、などが挙げられる。
　前記ＧＳＫ－３阻害剤の具体例としては、ＣＨＩＲ９９０２１などが挙げられる。
　前記アデニル酸シクラーゼ活性化剤の具体例としては、Ｆｏｒｓｋｏｌｉｎなどが挙げられる。
　前記低分子化合物の投与量としては、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。 In the gene or gene product introduction step, a low molecular compound may be administered as long as the effects of the present invention are not impaired.
The low molecular compound is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a GSK-3 inhibitor and an adenylate cyclase activator.
Specific examples of the GSK-3 inhibitor include CHIR99021.
Specific examples of the adenylate cyclase activator include Forskolin.
There is no restriction | limiting in particular as a dosage amount of the said low molecular compound, According to the objective, it can select suitably.
＜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、前記各遺伝子乃至その遺伝子産物が導入された体細胞を培養する遺伝子乃至その遺伝子産物導入細胞培養工程、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. For example, genes for culturing somatic cells into which each of the genes or gene products thereof has been introduced. Or the gene product-introduced cell culture step.
－遺伝子乃至その遺伝子産物導入細胞培養工程－
　前記遺伝子乃至その遺伝子産物導入細胞培養工程は、前記各遺伝子乃至その遺伝子産物が導入された体細胞を培養する工程である。
　前記遺伝子乃至その遺伝子産物導入細胞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、例えば、培養温度は約３７℃、ＣＯ２濃度は約２％～５％、などが挙げられる。
　前記遺伝子乃至その遺伝子産物導入細胞の培養に用いる培地としては、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、ＬＩＦ、２０％　Ｋｎｏｃｋｏｕｔ　Ｓｅｒｕｍ　Ｒｅｐｌａｃｅｍｅｎｔ（以下、「ＫＳＲ」と称することがある）、１×ＮＥＡＡ、２－メルカプトエタノール、ＧｌｕｔａＭａｘを含むＤＭＥＭ、などが挙げられる。
　前記遺伝子乃至その遺伝子産物導入細胞培養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る。 -Gene or gene product introduction cell culture process-
The gene or gene product-introduced cell culture step is a step of culturing somatic cells into which each gene or gene product has been introduced.
The culture conditions of the gene or its gene product-introduced cell are not particularly limited and can be appropriately selected according to the purpose. For example, the culture temperature is about 37 ° C., the CO 2 concentration is about 2% to 5%, Etc.
The medium used for culturing the gene or its gene product-introduced cell is not particularly limited and may be appropriately selected depending on the intended purpose. For example, LIF, 20% Knockout Serum Replacement (hereinafter referred to as “KSR”) And 1 × NEAA, 2-mercaptoethanol, DMEM containing GlutaMax, and the like.
There is no restriction | limiting in particular as a period of the said gene thru | or gene product introduction | transduction cell culture process, According to the objective, it can select suitably.
＜人工多能性幹細胞＞
　前記人工多能性幹細胞の製造方法により製造される人工多能性幹細胞は、分化多能性、及び自己複製能を有する。前記分化多能性とは、三胚葉系列すべてに分化できることを意味する。また、前記自己複製能とは、未分化状態を保持したまま増殖できる能力を意味する。 <Artificial pluripotent stem cells>
The induced pluripotent stem cells produced by the method for producing induced pluripotent stem cells have differentiation pluripotency and self-renewal ability. The term “pluripotency” means that all three germ layers can be differentiated. The self-replicating ability means the ability to proliferate while maintaining an undifferentiated state.
　前記人工多能性幹細胞の製造方法により製造された細胞が人工多能性幹細胞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る。
　例えば、前記体細胞として、分化多能性細胞において特異的に高発現する遺伝子の遺伝子座に、レポーター遺伝子、及び薬剤耐性遺伝子の少なくともいずれかを組み込んだ組換え体細胞を用いた場合には、前記レポーター遺伝子、及び薬剤耐性遺伝子を利用して確認することができる。具体的には、前記レポーター遺伝子として、ＧＦＰ遺伝子を用いた場合には、例えば、フローサイトメーターによりＧＦＰ陽性の細胞を確認する方法が挙げられる、また、前記薬剤耐性遺伝子として、ピューロマイシン耐性遺伝子を用いた場合には、細胞にピューロマイシンを投与することにより確認することができる。 The method for confirming whether or not the cell produced by the method for producing an induced pluripotent stem cell is an induced pluripotent stem cell is not particularly limited and can be appropriately selected depending on the purpose.
For example, when the somatic cell is a recombinant cell in which at least one of a reporter gene and a drug resistance gene is incorporated into a gene locus that is specifically highly expressed in differentiated pluripotent cells, This can be confirmed using the reporter gene and drug resistance gene. Specifically, when a GFP gene is used as the reporter gene, for example, a method of confirming GFP-positive cells with a flow cytometer can be mentioned, and a puromycin resistance gene can be used as the drug resistance gene. When used, it can be confirmed by administering puromycin to the cells.
　前記人工多能性幹細胞の型としては、プライム型であってもよいし、ナイーブ型であってもよいが、品質に優れる点で、ナイーブ型が好ましい。
　前記人工多能性幹細胞がナイーブ型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、コロニーの形態、ｂＦＧＦ（ｂａｓｉｃ　ｆｉｂｒｏｂｌａｓｔ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ）を含まないＬＩＦ添加培地で増殖できるか否か、２ｉ添加培地（ＭＥＫ阻害剤とＧＳＫ阻害剤とを添加した培地）で増殖できるか否か、単一細胞に分散して継代することができるか否かを指標とすることにより確認することができる。具体的には、コロニーの形態がドーム状であり、ｂＦＧＦを含まないＬＩＦ添加培地で増殖することができ、２ｉ添加培地で増殖することができ、単一細胞に分散して継代することができる細胞である場合には、ナイーブ型であると判断することができる。 The type of the induced pluripotent stem cell may be a prime type or a naive type, but a naive type is preferable in terms of excellent quality.
The method for confirming whether or not the artificial pluripotent stem cells are naïve is not particularly limited and may be appropriately selected depending on the purpose. For example, the form of colonies, bFGF (basic fibroblast growth factor) Can be propagated in a single cell, whether it can be grown in a LIF-containing medium not containing LIF, or can be grown in a 2i-added medium (medium containing a MEK inhibitor and a GSK inhibitor) Whether or not it is used as an index can be confirmed. Specifically, the colony has a dome shape, can be grown in a LIF-containing medium not containing bFGF, can be grown in a 2i-added medium, and can be dispersed and passaged into single cells. If it is a cell that can be used, it can be determined to be naive.
　前記人工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が、ヒトが好ましい。 The seed of the induced pluripotent stem cell is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably human.
（人工多能性幹細胞製造用組成物）
　本発明の人工多能性幹細胞製造用組成物は、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物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構成を含む。 (Composition for producing induced pluripotent stem cells)
The composition for producing induced pluripotent stem cells of the present invention is selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product. And at least one other component as necessary.
　前記Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１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が、（１）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物を含む態様、（２）Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む態様、（３）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む態様が好ましく、（３）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む態様がより好ましい。
　前記より好ましい態様であると、人工多能性幹細胞の製造効率により優れ、かつ、より短期間で製造することができ、また、品質面にも優れる点で、有利である。 There is no particular limitation as at least one selected from the group consisting of the Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, and Sall4a gene or its gene product. (1) an embodiment including a Jarid2 mutant gene or its gene product, (2) a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product (3) Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, and Sall4a gene or its gene product are preferred, (3) Jarid2 mutation Gene or its gene product, PRDM14 gene or its gene product, Esrrb gene or its gene product, and is more preferred embodiment including Sall4a gene or its gene product.
The more preferable embodiment is advantageous in that it is superior in the production efficiency of induced pluripotent stem cells, can be produced in a shorter period of time, and is superior in quality.
＜Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物＞
　前記Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物は、Ｊａｒｉｄ２タンパク質のＮ末端の１番目から５５１番目のアミノ酸をコードする遺伝子乃至その遺伝子産物であり、前記人工多能性幹細胞の製造方法で記載したものと同様である。また、前記Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物は、前記人工多能性幹細胞の製造方法で記載したものと同様の変異が含まれていてもよい。
　前記人工多能性幹細胞製造用組成物における前記Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物の態様としては、特に制限はなく、目的に応じて適宜選択することができ、遺伝子がベクターに組み込まれている態様、合成ｍＲＮＡの態様、組換えタンパク質の態様、などが挙げられる。
　前記ベクターとしては、前記人工多能性幹細胞の製造方法で記載したものと同様のものが挙げられる。
　前記合成ｍＲＮＡ、前記組換えタンパク質は、公知の方法により製造することができる。 <Jarid2 mutant gene or its gene product>
The Jarid2 mutant gene or its gene product is a gene encoding the N-terminal 1st to 551st amino acids of the Jarid2 protein or its gene product, as described in the method for producing induced pluripotent stem cells. It is. In addition, the Jarid2 mutant gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
The mode of the Jarid2 mutant gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected according to the purpose. The mode in which the gene is incorporated into a vector , Synthetic mRNA embodiments, recombinant protein embodiments, and the like.
Examples of the vector include those described in the method for producing induced pluripotent stem cells.
The synthetic mRNA and the recombinant protein can be produced by a known method.
＜Ｐｒｄｍ１４遺伝子乃至その遺伝子産物＞
　前記Ｐｒｄｍ１４遺伝子乃至その遺伝子産物は、前記人工多能性幹細胞の製造方法で記載したものと同様である。また、前記Ｐｒｄｍ１４遺伝子乃至その遺伝子産物は、前記人工多能性幹細胞の製造方法で記載したものと同様の変異が含まれていてもよい。
　前記人工多能性幹細胞製造用組成物における前記Ｐｒｄｍ１４遺伝子乃至その遺伝子産物の態様としては、特に制限はなく、目的に応じて適宜選択することができ、遺伝子がベクターに組み込まれている態様、合成ｍＲＮＡの態様、組換えタンパク質の態様、などが挙げられる。
　前記ベクターとしては、前記人工多能性幹細胞の製造方法で記載したものと同様のものが挙げられる。
　前記合成ｍＲＮＡ、前記組換えタンパク質は、公知の方法により製造することができる。 <Prdm14 gene or its gene product>
The Prdm14 gene and its gene product are the same as those described in the method for producing induced pluripotent stem cells. In addition, the Prdm14 gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
The embodiment of the Prdm14 gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected according to the purpose. Examples include mRNA and recombinant protein.
Examples of the vector include those described in the method for producing induced pluripotent stem cells.
The synthetic mRNA and the recombinant protein can be produced by a known method.
＜Ｅｓｒｒｂ遺伝子乃至その遺伝子産物＞
　前記Ｅｓｒｒｂ遺伝子乃至その遺伝子産物は、前記人工多能性幹細胞の製造方法で記載したものと同様である。また、前記Ｅｓｒｒｂ遺伝子乃至その遺伝子産物は、前記人工多能性幹細胞の製造方法で記載したものと同様の変異が含まれていてもよい。
　前記人工多能性幹細胞製造用組成物における前記Ｅｓｒｒｂ遺伝子乃至その遺伝子産物の態様としては、特に制限はなく、目的に応じて適宜選択することができ、遺伝子がベクターに組み込まれている態様、合成ｍＲＮＡの態様、組換えタンパク質の態様、などが挙げられる。
　前記ベクターとしては、前記人工多能性幹細胞の製造方法で記載したものと同様のものが挙げられる。
　前記合成ｍＲＮＡ、前記組換えタンパク質は、公知の方法により製造することができる。 <Esrb gene or its gene product>
The Esrrb gene and its gene product are the same as those described in the method for producing induced pluripotent stem cells. Further, the Esrrb gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
The embodiment of the Esrrb gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected depending on the purpose. Embodiment in which the gene is incorporated into a vector Examples include mRNA and recombinant protein.
Examples of the vector include those described in the method for producing induced pluripotent stem cells.
The synthetic mRNA and the recombinant protein can be produced by a known method.
＜Ｓａｌｌ４ａ遺伝子乃至その遺伝子産物＞
　前記Ｓａｌｌ４ａ遺伝子乃至その遺伝子産物は、前記人工多能性幹細胞の製造方法で記載したものと同様である。また、前記Ｓａｌｌ４ａ遺伝子乃至その遺伝子産物は、前記人工多能性幹細胞の製造方法で記載したものと同様の変異が含まれていてもよい。
　前記人工多能性幹細胞製造用組成物における前記Ｓａｌｌ４ａ遺伝子乃至その遺伝子産物の態様としては、特に制限はなく、目的に応じて適宜選択することができ、遺伝子がベクターに組み込まれている態様、合成ｍＲＮＡの態様、組換えタンパク質の態様、などが挙げられる。
　前記ベクターとしては、前記人工多能性幹細胞の製造方法で記載したものと同様のものが挙げられる。
　前記合成ｍＲＮＡ、前記組換えタンパク質は、公知の方法により製造することができる。 <Sall4a gene or its gene product>
The Sall4a gene or its gene product is the same as that described in the method for producing induced pluripotent stem cells. In addition, the Sall4a gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
The embodiment of the Sall4a gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected according to the purpose. The embodiment in which the gene is incorporated into a vector, synthesis Examples include mRNA and recombinant protein.
Examples of the vector include those described in the method for producing induced pluripotent stem cells.
The synthetic mRNA and the recombinant protein can be produced by a known method.
＜その他の構成＞
　前記その他の構成としては、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、Ｏｃｔ３／４遺伝子乃至その遺伝子産物、Ｓｏｘ２遺伝子乃至その遺伝子産物、Ｋｌｆ４遺伝子乃至その遺伝子産物、及びｃ－Ｍｙｃ遺伝子乃至その遺伝子産物を含むことが好ましい。 <Other configurations>
The other configuration 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. Or a gene product thereof, and a c-Myc gene or a gene product thereof.
　前記Ｏｃｔ３／４遺伝子乃至その遺伝子産物、Ｓｏｘ２遺伝子乃至その遺伝子産物、Ｋｌｆ４遺伝子乃至その遺伝子産物、及びｃ－Ｍｙｃ遺伝子乃至その遺伝子産物は、前記人工多能性幹細胞の製造方法で記載したものと同様のものである。また、前記各遺伝子乃至その遺伝子産物は、前記人工多能性幹細胞の製造方法で記載したものと同様の変異が含まれていてもよい。
　前記人工多能性幹細胞製造用組成物における前記Ｏｃｔ３／４遺伝子乃至その遺伝子産物、Ｓｏｘ２遺伝子乃至その遺伝子産物、Ｋｌｆ４遺伝子乃至その遺伝子産物、及びｃ－Ｍｙｃ遺伝子乃至その遺伝子産物の態様としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、遺伝子がベクターに組み込まれている態様、合成ｍＲＮＡの態様、組換えタンパク質の態様、などが挙げられる。
　前記ベクターとしては、前記人工多能性幹細胞の製造方法で記載したものと同様のものが挙げられる。
　前記合成ｍＲＮＡ、前記組換えタンパク質は、公知の方法により製造することができる。 The Oct3 / 4 gene or its gene product, Sox2 gene or its gene product, Klf4 gene or its gene product, and c-Myc gene or its gene product are the same as those described in the method for producing induced pluripotent stem cells. belongs to. Further, each gene or gene product thereof may contain the same mutation as described in the method for producing induced pluripotent stem cells.
The Oct3 / 4 gene or its gene product, the Sox2 gene or its gene product, the Klf4 gene or its gene product, and the c-Myc gene or its gene product in the composition for producing induced pluripotent stem cells are particularly There is no restriction | limiting, According to the objective, it can select suitably, For example, the aspect by which the gene is integrated in the vector, the aspect of synthetic mRNA, the aspect of recombinant protein, etc. are mentioned.
Examples of the vector include those described in the method for producing induced pluripotent stem cells.
The synthetic mRNA and the recombinant protein can be produced by a known method.
　前記人工多能性幹細胞製造用組成物における前記ｃ－Ｍｙｃ遺伝子と、前記Ｊａｒｉｄ２変異体遺伝子とは共発現できる態様であることが好ましい。前記共発現できる態様としては、前記人工多能性幹細胞の製造方法で記載したように前記連結配列を通じて、前記ｃ－Ｍｙｃ遺伝子と、前記Ｊａｒｉｄ２変異体遺伝子とを連結することが好ましい。 It is preferable that the c-Myc gene and the Jarid2 mutant gene in the composition for producing induced pluripotent stem cells can be co-expressed. As an aspect capable of co-expression, it is preferable to link the c-Myc gene and the Jarid2 mutant gene through the linking sequence as described in the method for producing induced pluripotent stem cells.
　前記人工多能性幹細胞製造用組成物は、前記各遺伝子乃至その遺伝子産物が、個別の容器に分けられているものであってもよいし、１つの容器にまとめられているものであってもよいし、任意の数ごとに容器にまとめられているものであってもよい。
　前記人工多能性幹細胞製造用組成物における前記各遺伝子乃至その遺伝子産物の量としては、特に制限はなく、全ての遺伝子乃至その遺伝子産物を等量としてもよいし、異なる量としてもよい。 In the composition for producing induced pluripotent stem cells, the genes or gene products thereof may be divided into individual containers or may be combined into one container. Alternatively, any number may be collected in a container.
The amount of each gene or its gene product in the composition for producing induced pluripotent stem cells is not particularly limited, and all the genes or their gene products may be equal amounts or different amounts.
　前記人工多能性幹細胞製造用組成物は、前記各遺伝子乃至その遺伝子産物以外の遺伝子乃至その遺伝子産物を含んでいてもよく、また、前記ウイルスベクターを用いる場合には、例えば、パッケージング細胞を含んでいてもよい。
　前記各遺伝子乃至その遺伝子産物以外の遺伝子乃至その遺伝子産物、前記パッケージング細胞としては、前記人工多能性幹細胞の製造方法で記載したものと同様のものが挙げられる。 The composition for producing an induced pluripotent stem cell may contain a gene other than the gene or a gene product thereof or a gene product thereof. When the viral vector is used, for example, a packaging cell is used. May be included.
Examples of the genes or gene products other than the genes or gene products thereof and the packaging cells include those described in the method for producing induced pluripotent stem cells.
　以下、製造例、試験例を挙げて、本発明を説明するが、本発明は、以下の製造例、試験例に何ら限定されるものではない。 Hereinafter, the present invention will be described with reference to production examples and test examples, but the present invention is not limited to the following production examples and test examples.
（製造例１－１：Ｏｃｔ３／４遺伝子含有ウイルスベクター）
　マウス由来野生型Ｏｃｔ３／４タンパク質の全長をコードする遺伝子がｐＭＸｓ－ｇｗのマルチクローニングサイトに組み込まれている、Ｏｃｔ３／４遺伝子含有ウイルスベクターを使用した（国立大学法人京都大学ｉＰＳ細胞研究所より入手、Ｃｅｌｌ　１２６（４）：６６３－６７６．２００６）。 (Production Example 1-1: Virus vector containing Oct3 / 4 gene)
An Oct3 / 4 gene-containing viral vector in which the gene encoding the full length of the mouse-derived wild-type Oct3 / 4 protein was incorporated into the multi-cloning site of pMXs-gw was used (obtained from Kyoto University iPS Cell Laboratory) Cell 126 (4): 663-676.2006).
（製造例１－２：Ｓｏｘ２遺伝子含有ウイルスベクター）
　マウス由来野生型Ｓｏｘ２タンパク質の全長をコードする遺伝子がｐＭＸｓ－ｇｗのマルチクローニングサイトに組み込まれている、Ｓｏｘ２遺伝子含有ウイルスベクターを使用した（国立大学法人京都大学ｉＰＳ細胞研究所より入手、Ｃｅｌｌ　１２６（４）：６６３－６７６．２００６）。 (Production Example 1-2: Virus vector containing Sox2 gene)
A Sox2 gene-containing viral vector in which the gene encoding the full-length mouse-derived wild-type Sox2 protein was incorporated into the multi-cloning site of pMXs-gw was used (obtained from the National University Corporation Kyoto University iPS Cell Research Institute, Cell 126 ( 4): 663-676.2006).
（製造例１－３：Ｋｌｆ４遺伝子含有ウイルスベクター）
　マウス由来野生型Ｋｌｆ４タンパク質の全長をコードする遺伝子がｐＭＸｓ－ｇｗのマルチクローニングサイトに組み込まれている、Ｋｌｆ４遺伝子含有ウイルスベクターを使用した（国立大学法人京都大学ｉＰＳ細胞研究所より入手、Ｃｅｌｌ　１２６（４）：６６３－６７６．２００６）。 (Production Example 1-3: Virus vector containing Klf4 gene)
A Klf4 gene-containing viral vector in which the gene encoding the full length of the mouse-derived wild-type Klf4 protein was incorporated into the multicloning site of pMXs-gw was used (obtained from the National University Corporation Kyoto University iPS Cell Research Institute, Cell 126 ( 4): 663-676.2006).
（製造例１－４：ｃ－Ｍｙｃ遺伝子含有ウイルスベクター）
　マウス由来野生型ｃ－Ｍｙｃタンパク質の全長をコードする遺伝子がｐＭＸｓ－ｇｗのマルチクローニングサイトに組み込まれている、ｃ－Ｍｙｃ遺伝子含有ウイルスベクターを使用した（国立大学法人京都大学ｉＰＳ細胞研究所より入手、Ｃｅｌｌ　１２６（４）：６６３－６７６．２００６）。 (Production Example 1-4: Virus vector containing c-Myc gene)
A c-Myc gene-containing virus vector in which the gene encoding the full-length mouse-derived wild-type c-Myc protein is incorporated into the multicloning site of pMXs-gw was used (obtained from the National Institute for Kyoto University iPS Cell Research Institute). 126 (4): 663-676.2006).
（製造例１－５：Ｊａｒｉｄ２変異体遺伝子含有ウイルスベクター）
　Ｂ６マウス由来胚性幹細胞（以下、「ＥＳＣ」と称することがある、国立大学法人筑波大学生命科学動物資源センターより入手）よりＲＮＡを抽出し、ｃＤＮＡを合成後、下記配列番号３及び４で表されるプライマーを用い、ＰＣＲ法にてＪａｒｉｄ２変異体遺伝子（Ｊａｒｉｄ２タンパク質のＮ末端の１番目から５５１番目のアミノ酸をコードする遺伝子（配列番号１２参照））を増幅、回収し、ｐＭＸｓベクター（国立大学法人東京大学医科学研究所より入手）のマルチクローニングサイトに組み込み、Ｊａｒｉｄ２変異体遺伝子含有ウイルスベクターを得た。
＜プライマー＞
（１）５’→３’：ＡＧＴＴＡＡＴＴＡＡＧＧＡＴＣＣＡＣＣＡＴＧＡＧＣＡＡＧＧＡＡＡＧＡＣＣＣＡＡＧ（配列番号３）
（２）５’→３’：ＴＴＡＴＴＴＴＡＴＣＧＴＣＧＡＣＴＣＡＣＧＣＴＧＣＣＣＡＣＣ（配列番号４） (Production Example 1-5: Virus vector containing Jarid2 mutant gene)
RNA was extracted from B6 mouse-derived embryonic stem cells (hereinafter sometimes referred to as “ESC”, obtained from the National University of Tsukuba Life Science Animal Resource Center), cDNA was synthesized, and represented by SEQ ID NOs: 3 and 4 below. Amplification and recovery of the Jarid2 mutant gene (the gene encoding the 1st to 551st amino acids of the N-terminal of the Jarid2 protein (see SEQ ID NO: 12)) by PCR using the primers obtained, and the pMXs vector (National University) Incorporated into a multicloning site of the University of Tokyo Medical Science Institute), a Jard2 mutant gene-containing virus vector was obtained.
<Primer>
(1) 5 ′ → 3 ′: AGTTAATTAAGGATCCCACCATGAGCAAGGAAAGACCCAAG (SEQ ID NO: 3)
(2) 5 ′ → 3 ′: TTATTTTATCGGTCGACTCACGCTGCCCACC (SEQ ID NO: 4)
（製造例１－６：ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルスベクター）
　鋳型として、前記ｃ－Ｍｙｃ遺伝子含有ウイルスベクターを用い、プライマーとして下記配列番号５及び６で表されるプライマーを用いて、ｃ－Ｍｙｃ遺伝子と、Ｐｉｃｏｒｎａｖｉｒｉｄａｅ　Ａｐｈｔｈｏｖｉｒｕｓ由来２Ａペプチドの一部とをコードする遺伝子（以下、「ｃ－Ｍｙｃ＿２Ａ遺伝子」と称することがある）を増幅、回収した。
　また、鋳型として、前記Ｊａｒｉｄ２変異体遺伝子含有ウイルスベクターを用い、プライマーとして下記配列番号９及び１０で表されるプライマーを用いて、Ｐｉｃｏｒｎａｖｉｒｉｄａｅ　Ａｐｈｔｈｏｖｉｒｕｓ由来２Ａペプチドの一部と、Ｊａｒｉｄ２変異体遺伝子（Ｊａｒｉｄ２タンパク質のＮ末端の１番目から５５１番目のアミノ酸をコードする遺伝子（以下、「２Ａ＿Ｊａｒｉｄ２ΔＣ遺伝子」と称することがある））を増幅、回収した。
　前記ｃ－Ｍｙｃ＿２Ａ遺伝子と、前記２Ａ＿Ｊａｒｉｄ２ΔＣ遺伝子とをｐＭＸｓベクター（国立大学法人東京大学医科学研究所より入手）のマルチクローニングサイトに組み込み、ｃ－Ｍｙｃ遺伝子と、Ｊａｒｉｄ２変異体遺伝子とが、Ｐｉｃｏｒｎａｖｉｒｉｄａｅ　Ａｐｈｔｈｏｖｉｒｕｓ由来２Ａペプチドをコードする遺伝子で連結されたｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルスベクターを得た。
＜プライマー＞
（１）５’→３’：ＧＧＴＧＧＴＡＣＧＧＧＡＡＴＴＣＣＡＣＣＡＴＧＣＣＣＣＴＣＡＡＣＧＴＧ（配列番号５）
（２）５’→３’：ＧＴＴＣＡＧＧＡＧＣＴＧＣＴＴＣＡＣＡＧＧＧＧＣＣＡＣＧＡＴＣＴＴＣＴＧＣＴＴＧＴＧＧＣＣＧＣＴＧＣＣＴＣＣＧＣＣＧＣＣＴＧＣＡＣＣＡＧＡＧＴＴＴＣＧＡＡＧ（配列番号６）
（３）５’→３’：ＡＡＧＣＡＧＣＴＣＣＴＧＡＡＣＴＴＣＧＡＣＣＴＧＣＴＣＡＡＧＣＴＧＧＣＣＧＧＣＧＡＣＧＴＧＧＡＧＣＣＴＡＡＣＣＣＴＧＧＣＣＣＴＡＴＧＡＧＣＡＡＧＧＡＡＡＧＡＣＣＣＡＡＧ（配列番号９）
（４）５’→３’：ＴＴＡＴＴＴＴＡＴＣＧＴＣＧＡＣＴＣＡＣＧＣＴＧＣＣＣＡＣＣ（配列番号１０）
　前記配列番号６中、「１番目から５１番目（ＧＴＴＣＡＧＧＡＧＣＴＧＣＴＴＣＡＣＡＧＧＧＧＣＣＡＣＧＡＴＣＴＴＣＴＧＣＴＴＧＴＧＧＣＣＧＣＴＧＣＣ）」は、前記２Ａペプチドをコードする遺伝子に対応し、「５２番目から６０番目（ＴＣＣＧＣＣＧＣＣ）」は、リンカー（ＧＧＧ）に対応する。
　前記配列番号９中、「１番目から６３番目（ＡＡＧＣＡＧＣＴＣＣＴＧＡＡＣＴＴＣＧＡＣＣＴＧＣＴＣＡＡＧＣＴＧＧＣＣＧＧＣＧＡＣＧＴＧＧＡＧＣＣＴＡＡＣＣＣＴＧＧＣＣＣＴ）」は、前記２Ａペプチドをコードする遺伝子に対応する。 (Production Example 1-6: Viral vector containing c-Myc gene and Jarid2 mutant gene)
A gene encoding the c-Myc gene and part of the 2A peptide derived from Picoraviridae Aphthovirus using the c-Myc gene-containing virus vector as a template and the primers represented by SEQ ID NOs: 5 and 6 below as primers (Hereinafter sometimes referred to as “c-Myc_2A gene”) was amplified and recovered.
Further, using the above-mentioned Jarid2 mutant gene-containing virus vector as a template, and using primers represented by SEQ ID NOs: 9 and 10 below as primers, a part of Picoraviridae Aphthovirus-derived 2A peptide and a Jarid2 mutant gene (Jarid2 protein) A gene encoding the N-terminal 1st to 551st amino acids (hereinafter sometimes referred to as “2A_Jarid2ΔC gene”) was amplified and recovered.
The c-Myc_2A gene and the 2A_Jarid2ΔC gene are incorporated into a multi-cloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science). A virus vector containing a c-Myc gene linked with a gene encoding 2A peptide and a Jarid2 mutant gene was obtained.
<Primer>
(1) 5 ′ → 3 ′: GGTGGTACGGGAATTCCACCATGCCCCTCAACGTG (SEQ ID NO: 5)
(2) 5 ′ → 3 ′: GTTCAGGAGCTGCTTCACAGGGGCCCACGATCTTCTGCTTGTGGCCGCTGCCTCCCGCCGCCTGCACCAGAGTTTCGAAG (SEQ ID NO: 6)
(3) 5 ′ → 3 ′: AAGCAGCTCCCTGAACTTCGACCTGCTCAAGCTGGCCGGCGACGTGGAGCCTAACCCTGGCCCTATAGCAAGGAAAGAACCCAAG (SEQ ID NO: 9)
(4) 5 ′ → 3 ′: TTATTTTATCGGTCGACTCACGCTGCCCACC (SEQ ID NO: 10)
In SEQ ID NO: 6, “1st to 51st (GTTCAGGAGCTGCTTCACAGGGGCCACGATCTTCTGCTTGTGGCCCGCTGCC)” corresponds to the gene encoding the 2A peptide, and “52th to 60th (TCCGCCGCCC)” corresponds to the linker (GGG).
In SEQ ID NO: 9, “1st to 63rd (AAGCAGCTCTCGAACTTCCGACCTGCTCAAGCTGGCCGGGCAGCGTGGAGCTAACCCTGGCCCT)” corresponds to the gene encoding the 2A peptide.
（比較製造例１－１：Ｊａｒｉｄ２遺伝子含有ウイルスベクター）
　Ｂ６マウス由来胚性幹細胞（国立大学法人筑波大学生命科学動物資源センターより入手）よりＲＮＡを抽出し、ｃＤＮＡを合成後、下記配列番号１及び２で表されるプライマーを用い、ＰＣＲ法にてＪａｒｉｄ２遺伝子（Ｊａｒｉｄ２タンパク質の全長をコードする遺伝子（配列番号１１参照））を増幅、回収し、ｐＭＸｓベクター（国立大学法人東京大学医科学研究所より入手）のマルチクローニングサイトに組み込み、Ｊａｒｉｄ２遺伝子含有ウイルスベクターを得た。
＜プライマー＞
（１）５’→３’：ＧＴＴＡＡＴＴＡＡＧＧＡＴＣＣＡＣＣＡＴＧＡＧＣＡＡＧＧＡＡＡＧＡＣＣＣＡＡＧ（配列番号１）
（２）５’→３’：ＴＴＡＴＴＴＴＡＴＣＧＴＣＧＡＣＴＣＡＴＧＡＧＧＡＴＧＧＧＡＧＣＣＧＡＧ（配列番号２） (Comparative Production Example 1-1: Jarid2 gene-containing virus vector)
RNA was extracted from B6 mouse-derived embryonic stem cells (obtained from the National University of Tsukuba Life Science Animal Resource Center), and cDNA was synthesized. Then, using the primers represented by SEQ ID NOs: 1 and 2 below, Jarid2 A gene (a gene encoding the full length of the Jarid2 protein (see SEQ ID NO: 11)) is amplified, recovered, incorporated into a multicloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science), and a Jarid2 gene-containing viral vector Got.
<Primer>
(1) 5 ′ → 3 ′: GTTAATTAAGGATCCCACCATGAGCAAGGAAAGACCCAAG (SEQ ID NO: 1)
(2) 5 ′ → 3 ′: TTATTTTATTCGTCGAACTCATGAGGATGGGAGCCGAG (SEQ ID NO: 2)
（比較製造例１－２：ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２遺伝子含有ウイルスベクター）
　鋳型として、前記ｃ－Ｍｙｃ遺伝子含有ウイルスベクターを用い、プライマーとして下記配列番号５及び６で表されるプライマーを用いて、ｃ－Ｍｙｃ＿２Ａ遺伝子を増幅、回収した。
　また、鋳型として、前記Ｊａｒｉｄ２遺伝子含有ウイルスベクターを用い、プライマーとして下記配列番号７及び８で表されるプライマーを用いて、Ｐｉｃｏｒｎａｖｉｒｉｄａｅ　Ａｐｈｔｈｏｖｉｒｕｓ由来２Ａペプチドの一部と、Ｊａｒｉｄ２遺伝子（Ｊａｒｉｄ２タンパク質の全長をコードする遺伝子（以下、「２Ａ＿Ｊａｒｉｄ２ＷＴ遺伝子」と称することがある））を増幅、回収した。
　前記ｃ－Ｍｙｃ＿２Ａ遺伝子と、前記２Ａ＿Ｊａｒｉｄ２ＷＴ遺伝子とをｐＭＸｓベクター（国立大学法人東京大学医科学研究所より入手）のマルチクローニングサイトに組み込み、ｃ－Ｍｙｃ遺伝子と、Ｊａｒｉｄ２遺伝子とが、Ｐｉｃｏｒｎａｖｉｒｉｄａｅ　Ａｐｈｔｈｏｖｉｒｕｓ由来２Ａペプチドをコードする遺伝子で連結されたｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２遺伝子含有ウイルスベクターを得た。
＜プライマー＞
（１）５’→３’：ＧＧＴＧＧＴＡＣＧＧＧＡＡＴＴＣＣＡＣＣＡＴＧＣＣＣＣＴＣＡＡＣＧＴＧ（配列番号５）
（２）５’→３’：ＧＴＴＣＡＧＧＡＧＣＴＧＣＴＴＣＡＣＡＧＧＧＧＣＣＡＣＧＡＴＣＴＴＣＴＧＣＴＴＧＴＧＧＣＣＧＣＴＧＣＣＴＣＣＧＣＣＧＣＣＴＧＣＡＣＣＡＧＡＧＴＴＴＣＧＡＡＧ（配列番号６）
（３）５’→３’：ＡＡＧＣＡＧＣＴＣＣＴＧＡＡＣＴＴＣＧＡＣＣＴＧＣＴＣＡＡＧＣＴＧＧＣＣＧＧＣＧＡＣＧＴＧＧＡＧＣＣＴＡＡＣＣＣＴＧＧＣＣＣＴＡＴＧＡＧＣＡＡＧＧＡＡＡＧＡＣＣＣＡＡＧ（配列番号７）
（４）５’→３’：ＴＴＡＴＴＴＴＡＴＣＧＴＣＧＡＣＴＣＡＴＧＡＧＧＡＴＧＧＧＡＧＣＣＧＡＧ（配列番号８）
　前記配列番号６中、「１番目から５１番目（ＧＴＴＣＡＧＧＡＧＣＴＧＣＴＴＣＡＣＡＧＧＧＧＣＣＡＣＧＡＴＣＴＴＣＴＧＣＴＴＧＴＧＧＣＣＧＣＴＧＣＣ）」は、前記２Ａペプチドをコードする遺伝子に対応し、「５２番目から６０番目（ＴＣＣＧＣＣＧＣＣ）」は、リンカー（ＧＧＧ）に対応する。
　前記配列番号７中、「１番目から６３番目（ＡＡＧＣＡＧＣＴＣＣＴＧＡＡＣＴＴＣＧＡＣＣＴＧＣＴＣＡＡＧＣＴＧＧＣＣＧＧＣＧＡＣＧＴＧＧＡＧＣＣＴＡＡＣＣＣＴＧＧＣＣＣＴ）」は、前記２Ａペプチドをコードする遺伝子に対応する。 (Comparative Production Example 1-2: Viral vector containing c-Myc gene and Jarid2 gene)
The c-Myc_2A gene was amplified and recovered using the c-Myc gene-containing virus vector as a template and the primers represented by the following SEQ ID NOs: 5 and 6 as primers.
In addition, using the above-mentioned Jarid2 gene-containing viral vector as a template and using primers represented by the following SEQ ID NOs: 7 and 8, a part of Picoraviridae Aphthovirus-derived 2A peptide and the Jarid2 gene (encoding the full length of the Jarid2 protein) The gene to be amplified (hereinafter sometimes referred to as “2A_Jarid2WT gene”) was amplified and recovered.
The c-Myc_2A gene and the 2A_Jarid2WT gene are incorporated into a multi-cloning site of a pMXs vector (obtained from the National University Corporation Tokyo Medical Research Institute). A viral vector containing a c-Myc gene linked with a gene encoding and a Jarid2 gene was obtained.
<Primer>
(1) 5 ′ → 3 ′: GGTGGTACGGGAATTCCACCATGCCCCTCAACGTG (SEQ ID NO: 5)
(2) 5 ′ → 3 ′: GTTCAGGAGCTGCTTCACAGGGGCCCACGATCTTCTGCTTGTGGCCGCTGCCTCCCGCCGCCTGCACCAGAGTTTCGAAG (SEQ ID NO: 6)
(3) 5 ′ → 3 ′: AAGCAGCTCCCTGAACTTCGACCTGCTCAAGCTGGCCGGCGACGTGGAGCCTAACCCTGGCCCTATAGCACAGGAAAAACCCAAG (SEQ ID NO: 7)
(4) 5 ′ → 3 ′: TTATTTTATCGTCGAACTCATGAGGATGGGAGCCGAG (SEQ ID NO: 8)
In SEQ ID NO: 6, “1st to 51st (GTTCAGGAGCTGCTTCACAGGGGCCACGATCTTCTGCTTGTGGCCCGCTGCC)” corresponds to the gene encoding the 2A peptide, and “52th to 60th (TCCGCCGCCC)” corresponds to the linker (GGG).
In SEQ ID NO: 7, “1st to 63rd (AAGCAGCTCTCGAACTTCCGACCTGCTCAAGCTGGCCGGGCAGCGTGGAGCTAACCCTGGCCCT)” corresponds to the gene encoding the 2A peptide.
（製造例１－７：Ｐｒｄｍ１４遺伝子含有ウイルスベクター）
　Ｂ６マウス由来胚性幹細胞（国立大学法人筑波大学生命科学動物資源センターより入手）よりＲＮＡを抽出し、ｃＤＮＡを合成後、下記配列番号１３及び１４で表されるプライマーを用い、ＰＣＲ法にてＰｒｄｍ１４遺伝子（Ｐｒｄｍ１４タンパク質の全長をコードする遺伝子（配列番号１９参照））を増幅、回収し、ｐＭＸｓベクター（国立大学法人東京大学医科学研究所より入手）のマルチクローニングサイトに組み込み、Ｐｒｄｍ１４遺伝子含有ウイルスベクターを得た。
＜プライマー＞
（１）５’→３’：ＡＧＴＴＡＡＴＴＡＡＧＧＡＴＣＣＡＣＣＡＴＧＧＣＣＴＴＡＣＣＧＣＣＣＴＣＴＧ（配列番号１３）
（２）５’→３’：ＴＴＡＴＴＴＴＡＴＣＧＴＣＧＡＣＣＴＡＧＣＡＧＧＴＴＴＴＡＴＧＡＡＧＣＣＴＣ（配列番号１４） (Production Example 1-7: Prdm14 gene-containing virus vector)
RNA was extracted from B6 mouse-derived embryonic stem cells (obtained from the National University of Tsukuba Life Science Animal Resource Center), and cDNA was synthesized, followed by PCR using the primers represented by SEQ ID NOs: 13 and 14 below by Prdm14. A gene (gene encoding the full length of Prdm14 protein (see SEQ ID NO: 19)) is amplified and recovered, incorporated into a multicloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science), and a Prdm14 gene-containing viral vector Got.
<Primer>
(1) 5 ′ → 3 ′: AGTTAATTAAGGATCCCACCATGGCCCTACCGCCCCTTG (SEQ ID NO: 13)
(2) 5 ′ → 3 ′: TTATTTTATCGTCGCACCTAGCAGGTTTTATGAAGCCTC (SEQ ID NO: 14)
（製造例１－８：Ｅｓｒｒｂ遺伝子含有ウイルスベクター）
　Ｂ６マウス由来胚性幹細胞（国立大学法人筑波大学生命科学動物資源センターより入手）よりＲＮＡを抽出し、ｃＤＮＡを合成後、下記配列番号１５及び１６で表されるプライマーを用い、ＰＣＲ法にてＥｓｒｒｂ遺伝子（Ｅｓｒｒｂタンパク質の全長をコードする遺伝子（配列番号２０参照））を増幅、回収し、ｐＭＸｓベクター（国立大学法人東京大学医科学研究所より入手）のマルチクローニングサイトに組み込み、Ｅｓｒｒｂ遺伝子含有ウイルスベクターを得た。
＜プライマー＞
（１）５’→３’：ＡＧＴＴＡＡＴＴＡＡＧＧＡＴＣＣＡＣＣＡＴＧＧＡＣＧＴＧＴＣＣＧＡＡＣＴＣＴＧ（配列番号１５）
（２）５’→３’：ＴＴＡＴＴＴＴＡＴＣＧＴＣＧＡＣＴＣＡＣＡＣＣＴＴＧＧＣＣＴＣＣＡＧＣＡ（配列番号１６） (Production Example 1-8: Virus vector containing Esrrb gene)
RNA was extracted from B6 mouse-derived embryonic stem cells (obtained from the University of Tsukuba Life Science Animal Resource Center), cDNA was synthesized, and Esrrb was synthesized by PCR using primers represented by SEQ ID NOs: 15 and 16 below. A gene (a gene encoding the full length of the Esrrb protein (see SEQ ID NO: 20)) is amplified, recovered, incorporated into a multi-cloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science), and an Esrrb gene-containing viral vector Got.
<Primer>
(1) 5 ′ → 3 ′: AGTTAATTAAGGATCCCACCATGACGGTGTCCGAACTCTG (SEQ ID NO: 15)
(2) 5 ′ → 3 ′: TTATTTTATCGTCCGACTCACACTTGGCCTCCAGCA (SEQ ID NO: 16)
（製造例１－９：Ｓａｌｌ４ａ遺伝子含有ウイルスベクター）
　Ｂ６マウス由来胚性幹細胞（国立大学法人筑波大学生命科学動物資源センターより入手）よりＲＮＡを抽出し、ｃＤＮＡを合成後、下記配列番号１７及び１８で表されるプライマーを用い、ＰＣＲ法にてＳａｌｌ４ａ遺伝子（Ｓａｌｌ４ａタンパク質の全長をコードする遺伝子（配列番号２１参照））を増幅、回収し、ｐＭＸｓベクター（国立大学法人東京大学医科学研究所より入手）のマルチクローニングサイトに組み込み、Ｓａｌｌ４ａ遺伝子含有ウイルスベクターを得た。
＜プライマー＞
（１）５’→３’：ＡＧＴＴＡＡＴＴＡＡＧＧＡＴＣＣＡＣＣＡＴＧＴＣＧＡＧＧＣＧＣＡＡＧＣＡＧＧ（配列番号１７）
（２）５’→３’：ＴＴＡＴＴＴＴＡＴＣＧＴＣＧＡＣＴＡＧＣＴＧＡＣＡＧＣＡＡＴＣＴＴＡＴＴＴＴＣ（配列番号１８） (Production Example 1-9: Virus vector containing Sall4a gene)
RNA was extracted from B6 mouse-derived embryonic stem cells (obtained from the National University of Tsukuba Life Science Animal Resource Center), cDNA was synthesized, and then PCR was performed using the primers represented by SEQ ID NOs: 17 and 18 below by PCR. A gene (a gene encoding the full length of the Sall4a protein (see SEQ ID NO: 21)) is amplified and recovered, incorporated into a multicloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Medical Science Institute), and a Sall4a gene-containing viral vector Got.
<Primer>
(1) 5 ′ → 3 ′: AGTTAATTAAGGATCCCACCATGTCGAGGGCGCAAGCAGG (SEQ ID NO: 17)
(2) 5 ′ → 3 ′: TTATTTTATTCGTCGACTAGCTGACAGCAATTTTTTTTC (SEQ ID NO: 18)
（製造例２－１：Ｏｃｔ３／４遺伝子含有ウイルス液－１）
　トランスフェクション前日にパッケージング細胞ＰＬＡＴ－Ｅ（国立大学法人東京大学医科学研究所より入手）を１０ｃｍのゼラチンコートディッシュ１枚あたり３．６×１０６個播種した。なお、培地としては、１０％血清を含むＤＭＥＭを用い、培養温度３７℃、ＣＯ２濃度５％で培養した。
　前記ＰＬＡＴ－Ｅ細胞に、ＦｕＧｅｎｅ　６（プロメガ社製）を用いて前記製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターをトランスフェクションした。
　前記トランスフェクション翌日に前記ＰＬＡＴ－Ｅ細胞の培地を交換し、前記培地を交換した翌日に、前記ＰＬＡＴ－Ｅ細胞の培養上清を回収し、次いで、０．４５μｍフィルターを用いて濾過し、Ｏｃｔ３／４遺伝子含有ウイルス液－１とした。 (Production Example 2-1: Oct3 / 4 gene-containing virus solution-1)
On the day before transfection, 3.6 × 10 6 packaging cells PLAT-E (obtained from the Institute of Medical Science, The University of Tokyo) were seeded per 10 cm gelatin-coated dish. As a medium, DMEM containing 10% serum was used and cultured at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
The PLAT-E cells were transfected with the Oct3 / 4 gene-containing virus vector obtained in Production Example 1-1 using FuGene 6 (Promega).
The PLAT-E cell culture medium was changed the day after the transfection, and the culture supernatant of the PLAT-E cell was collected the day after the medium was changed, and then filtered using a 0.45 μm filter, and the Oct3 / 4 gene-containing virus solution-1.
（製造例２－２：Ｓｏｘ２遺伝子含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－２で得られたＳｏｘ２遺伝子含有ウイルスベクターに代えた以外は、製造例２－１と同様にして、Ｓｏｘ２遺伝子含有ウイルス液－１を得た。 (Production Example 2-2: Sox2 gene-containing virus solution-1)
In the production example 2-1, except that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used, except that the Sox2 gene-containing virus vector obtained in the production example 1-2 was used. In the same manner as in Production Example 2-1, a virus solution-1 containing Sox2 gene was obtained.
（製造例２－３：Ｋｌｆ４遺伝子含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－３で得られたＫｌｆ４遺伝子含有ウイルスベクターに代えた以外は、製造例２－１と同様にして、Ｋｌｆ４遺伝子含有ウイルス液－１を得た。 (Production Example 2-3: Klf4 gene-containing virus solution-1)
In the production example 2-1, except that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used, except that the Klf4 gene-containing virus vector obtained in the production example 1-3 was used. In the same manner as in Production Example 2-1, a Klf4 gene-containing virus solution-1 was obtained.
（製造例２－４：ｃ－Ｍｙｃ遺伝子含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－４で得られたｃ－Ｍｙｃ遺伝子含有ウイルスベクターに代えた以外は、製造例２－１と同様にして、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１を得た。 (Production Example 2-4: Virus solution containing c-Myc gene-1)
In the production example 2-1, the point that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used was replaced with the c-Myc gene-containing virus vector obtained in the production example 1-4. Except for the above, c-Myc gene-containing virus solution-1 was obtained in the same manner as in Production Example 2-1.
（製造例２－５：Ｊａｒｉｄ２変異体遺伝子含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－５で得られたＪａｒｉｄ２変異体遺伝子含有ウイルスベクターに代えた以外は、製造例２－１と同様にして、Ｊａｒｉｄ２変異体遺伝子含有ウイルス液－１を得た。 (Production Example 2-5: Virus solution-1 containing Jarid2 mutant gene)
In the production example 2-1, the point that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used was replaced with the Jarid2 mutant gene-containing virus vector obtained in the production example 1-5. Except for the above, a Jarid2 mutant gene-containing virus solution-1 was obtained in the same manner as in Production Example 2-1.
（製造例２－６：ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－６で得られたｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルスベクターに代えた以外は、製造例２－１と同様にして、ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－１を得た。 (Production Example 2-6: Virus solution-1 containing c-Myc gene and Jarid2 mutant gene)
The point that the Oct3 / 4 gene-containing virus vector obtained in Production Example 1-1 was used in Production Example 2-1 was that the c-Myc gene obtained in Production Example 1-6 and the Jarid2 mutant were used. The c-Myc gene and Jarid2 mutant gene-containing virus solution-1 were obtained in the same manner as in Production Example 2-1, except that the gene-containing virus vector was used.
（比較製造例２－１：Ｊａｒｉｄ２遺伝子含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記比較製造例１－１で得られたＪａｒｉｄ２遺伝子含有ウイルスベクターに代えた以外は、製造例２－１と同様にして、Ｊａｒｉｄ２遺伝子含有ウイルス液－１を得た。 (Comparative Production Example 2-1: Jarid2 gene-containing virus solution-1)
In the production example 2-1, the point that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used was replaced with the Jarid2 gene-containing virus vector obtained in the comparative production example 1-1. Except for the above, a Jarid2 gene-containing virus solution-1 was obtained in the same manner as in Production Example 2-1.
（比較製造例２－２：ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２遺伝子含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記比較製造例１－２で得られたｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２遺伝子含有ウイルスベクターに代えた以外は、製造例２－１と同様にして、ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２遺伝子含有ウイルス液－１を得た。 (Comparative Production Example 2-2: Virus solution-1 containing c-Myc gene and Jarid2 gene)
In the production example 2-1, the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used. The c-Myc gene and the Jarid2 gene obtained in the comparative production example 1-2 The c-Myc gene and Jarid2 gene-containing virus solution-1 were obtained in the same manner as in Production Example 2-1, except that the virus vector was replaced.
（製造例２－７：Ｐｒｄｍ１４遺伝子含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－７で得られたＰｒｄｍ１４遺伝子含有ウイルスベクターに代えた以外は、製造例２－１と同様にして、Ｐｒｄｍ１４遺伝子含有ウイルス液－１を得た。 (Production Example 2-7: Prdm14 gene-containing virus solution-1)
In the production example 2-1, except that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used, except that the Prdm14 gene-containing virus vector obtained in the production example 1-7 was used. Produced Prdm14 gene-containing virus solution-1 in the same manner as in Production Example 2-1.
（製造例２－８：Ｅｓｒｒｂ遺伝子含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－８で得られたＥｓｒｒｂ遺伝子含有ウイルスベクターに代えた以外は、製造例２－１と同様にして、Ｅｓｒｒｂ遺伝子含有ウイルス液－１を得た。 (Production Example 2-8: Virus solution containing Esrrb gene-1)
In the production example 2-1, except that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used, except that the Esrrb gene-containing virus vector obtained in the production example 1-8 was used. Obtained Esrrb gene-containing virus solution-1 in the same manner as in Production Example 2-1.
（製造例２－９：Ｓａｌｌ４ａ遺伝子含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－９で得られたＳａｌｌ４ａ遺伝子含有ウイルスベクターに代えた以外は、製造例２－１と同様にして、Ｓａｌｌ４ａ遺伝子含有ウイルス液－１を得た。 (Production Example 2-9: Sall4a gene-containing virus solution-1)
In the production example 2-1, except that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used, except that the Sall4a gene-containing virus vector obtained in the production example 1-9 was used. In the same manner as in Production Example 2-1, a Sall4a gene-containing virus solution-1 was obtained.
（比較製造例２－３：外来遺伝子不含有ウイルス液－１）
　前記製造例２－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記ｐＭＸｓベクターのみに代えた以外は、製造例２－１と同様にして、外来遺伝子不含有ウイルス液－１を得た。 (Comparative Production Example 2-3: Virus solution 1 containing no foreign gene)
In Production Example 2-1, except that the Oct3 / 4 gene-containing virus vector obtained in Production Example 1-1 was used, except that only the pMXs vector was used, the same as Production Example 2-1. A foreign solution-free virus solution-1 was obtained.
（製造例３－１：Ｏｃｔ３／４遺伝子含有ウイルス液－２）
　トランスフェクション前日にパッケージング細胞ＰＬＡＴ－ＧＰ（国立大学法人東京大学医科学研究所より入手）を１０ｃｍのゼラチンコートディッシュ１枚あたり３．６×１０６個播種した。なお、培地としては、１０％血清を含むＤＭＥＭを用い、培養温度３７℃、ＣＯ２濃度５％で培養した。
　前記ＰＬＡＴ－ＧＰ細胞に、ＦｕＧｅｎｅ　６（プロメガ社製）を用いて前記製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを、ＶＳＶ－Ｇ発現ベクター（ＧＩＢＣＯ社製）と共にトランスフェクションした。
　前記トランスフェクション翌日に前記ＰＬＡＴ－ＧＰ細胞の培地を交換し、前記培地を交換した翌日に、前記ＰＬＡＴ－ＧＰ細胞の培養上清を回収し、次いで、０．４５μｍフィルターを用いて濾過し、Ｏｃｔ３／４遺伝子含有ウイルス液－２とした。 (Production Example 3-1: Oct3 / 4 gene-containing virus solution-2)
On the day before transfection, 3.6 × 10 6 packaging cells PLAT-GP (obtained from the Institute of Medical Science, The University of Tokyo) were seeded per 10 cm gelatin-coated dish. As a medium, DMEM containing 10% serum was used and cultured at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
The PLAT-GP cells were transfected with the Oct3 / 4 gene-containing viral vector obtained in Production Example 1-1 together with the VSV-G expression vector (GIBCO) using FuGene 6 (Promega). .
The PLAT-GP cell culture medium was changed the day after the transfection, and the culture supernatant of the PLAT-GP cell was collected the day after the medium was replaced, and then filtered using a 0.45 μm filter, and Oct3 / 4 gene-containing virus solution-2.
（製造例３－２：Ｓｏｘ２遺伝子含有ウイルス液－２）
　前記製造例３－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－２で得られたＳｏｘ２遺伝子含有ウイルスベクターに代えた以外は、製造例３－１と同様にして、Ｓｏｘ２遺伝子含有ウイルス液－２を得た。 (Production Example 3-2: Virus solution containing Sox2 gene-2)
In the production example 3-1, except that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used, except that the Sox2 gene-containing virus vector obtained in the production example 1-2 was used. In the same manner as in Production Example 3-1, a virus solution containing Sox2 gene-2 was obtained.
（製造例３－３：Ｋｌｆ４遺伝子含有ウイルス液－２）
　前記製造例３－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－３で得られたＫｌｆ４遺伝子含有ウイルスベクターに代えた以外は、製造例３－１と同様にして、Ｋｌｆ４遺伝子含有ウイルス液－２を得た。 (Production Example 3-3: Klf4 gene-containing virus solution-2)
In the production example 3-1, except that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used, except that the Klf4 gene-containing virus vector obtained in the production example 1-3 was used. In the same manner as in Production Example 3-1, a Klf4 gene-containing virus solution-2 was obtained.
（製造例３－４：ｃ－Ｍｙｃ遺伝子含有ウイルス液－２）
　前記製造例３－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－４で得られたｃ－Ｍｙｃ遺伝子含有ウイルスベクターに代えた以外は、製造例３－１と同様にして、ｃ－Ｍｙｃ遺伝子含有ウイルス液－２を得た。 (Production Example 3-4: c-Myc gene-containing virus solution-2)
In the production example 3-1, the point that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used was replaced with the c-Myc gene-containing virus vector obtained in the production example 1-4. A c-Myc gene-containing virus solution-2 was obtained in the same manner as in Production Example 3-1.
（製造例３－５：ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－２）
　前記製造例３－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－６で得られたｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルスベクターに代えた以外は、製造例３－１と同様にして、ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－２を得た。 (Production Example 3-5: Viral fluid-2 containing c-Myc gene and Jarid2 mutant gene)
In Production Example 3-1, the Oct3 / 4 gene-containing virus vector obtained in Production Example 1-1 was used, except that the c-Myc gene obtained in Production Example 1-6 and the Jarid2 mutant were used. A c-Myc gene and a Jarid2 mutant gene-containing virus solution-2 were obtained in the same manner as in Production Example 3-1, except that the gene-containing virus vector was used.
（製造例３－６：Ｐｒｄｍ１４遺伝子含有ウイルス液－２）
　前記製造例３－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－７で得られたＰｒｄｍ１４遺伝子含有ウイルスベクターに代えた以外は、製造例３－１と同様にして、Ｐｒｄｍ１４遺伝子含有ウイルス液－２を得た。 (Production Example 3-6: Prdm14 gene-containing virus solution-2)
In the production example 3-1, except that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used, except that the Prdm14 gene-containing virus vector obtained in the production example 1-7 was used. In the same manner as in Production Example 3-1, a Prdm14 gene-containing virus solution-2 was obtained.
（製造例３－７：Ｅｓｒｒｂ遺伝子含有ウイルス液－２）
　前記製造例３－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－８で得られたＥｓｒｒｂ遺伝子含有ウイルスベクターに代えた以外は、製造例３－１と同様にして、Ｅｓｒｒｂ遺伝子含有ウイルス液－２を得た。 (Production Example 3-7: Virus solution containing Esrrb gene-2)
In the production example 3-1, except that the Oct3 / 4 gene-containing viral vector obtained in the production example 1-1 was used, except that the Esrrb gene-containing viral vector obtained in the production example 1-8 was used. Obtained Esrrb gene-containing virus solution-2 in the same manner as in Production Example 3-1.
（製造例３－８：Ｓａｌｌ４ａ遺伝子含有ウイルス液－２）
　前記製造例３－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記製造例１－９で得られたＳａｌｌ４ａ遺伝子含有ウイルスベクターに代えた以外は、製造例３－１と同様にして、Ｓａｌｌ４ａ遺伝子含有ウイルス液－２を得た。 (Production Example 3-8: Sall4a gene-containing virus solution-2)
In the production example 3-1, except that the Oct3 / 4 gene-containing virus vector obtained in production example 1-1 was used, except that the Sall4a gene-containing virus vector obtained in the production example 1-9 was used. In the same manner as in Production Example 3-1, a Sall4a gene-containing virus solution-2 was obtained.
（比較製造例３－１：外来遺伝子不含有ウイルス液－２）
　前記製造例３－１において、製造例１－１で得られたＯｃｔ３／４遺伝子含有ウイルスベクターを用いていた点を、前記ｐＭＸｓベクターのみに代えた以外は、製造例３－１と同様にして、外来遺伝子不含有ウイルス液－２を得た。 (Comparative Production Example 3-1: Foreign gene-free virus solution-2)
In Production Example 3-1, the same method as in Production Example 3-1 was used except that the Oct3 / 4 gene-containing viral vector obtained in Production Example 1-1 was used instead of only the pMXs vector. A foreign gene-free virus solution-2 was obtained.
（試験例１－１：製造効率試験－１）
－細胞－
　細胞として、Ｎａｎｏｇ遺伝子プロモーター依存的にＧＦＰ及びピューロマイシン耐性遺伝子を発現するマウス胎仔線維芽細胞（以下、「ＮＧ－ＭＥＦ」と称することがある、理化学研究所バイオリソースセンターに寄託されているトランスジェニックマウスＳＴＯＣＫ　Ｔｇ（Ｎａｎｏｇ－ＧＦＰ，Ｐｕｒｏ）１Ｙａｍ（Ｎｏ．ＲＢＲＣ０２２９０）を入手、交配し、１３．５日胚からトリプシンを用いて単離、調製した）を用いた。前記ＮＧ－ＭＥＦの培養には、１０％血清を含むＤＭＥＭを用い、培養温度３７℃、ＣＯ２濃度５％で培養した。 (Test Example 1-1: Production efficiency test -1)
-cell-
Transgenic mice deposited at the RIKEN BioResource Center, which may be referred to as mouse fetal fibroblasts (hereinafter referred to as “NG-MEF”) that express GFP and puromycin resistance genes in a Nanog gene promoter-dependent manner STOCK Tg (Nanog-GFP, Puro) 1Yam (No. RBRC02290) was obtained, mated, and isolated and prepared from trypsin on day 13.5). The NG-MEF was cultured using DMEM containing 10% serum at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
－ウイルス感染－
　下記（１）から（３）のそれぞれのウイルス液に、最終濃度４μｇ／ｍＬのポリブレンを加えて調製したウイルス液を、それぞれ前記ＮＧ－ＭＥＦに感染させた。
　前記感染の翌日に、上清をＬＩＦ（ＬＩＦタンパク質を発現するベクターｐＣＡＧＧＳ－ＬＩＦ（理科学研究所　発生・再生科学より入手）をＣｏｓ－７細胞にリン酸カルシウム法により導入し、その培養上清を用いた）、２０％　ＫＳＲ（ＧＩＢＣＯ社製）、１×ＮＥＡＡ（ＧＩＢＣＯ社製）、２－メルカプトエタノール（ＧＩＢＣＯ社製）、ＧｌｕｔａＭａｘ（ＧＩＢＣＯ社製）を含むＤＭＥＭに置き換え、その後は、１日おきに培地交換を行った。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　６００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、及びＪａｒｉｄ２遺伝子含有ウイルス液－１　６００μＬ
（３）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－１　６００μＬ -Virus infection-
The virus solutions prepared by adding polybrene having a final concentration of 4 μg / mL to the virus solutions of (1) to (3) below were each infected with the NG-MEF.
The day after the infection, the supernatant was transferred to LIF (LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used. 20% KSR (manufactured by GIBCO), 1 × NEAA (manufactured by GIBCO), 2-mercaptoethanol (manufactured by GIBCO), GlutaMax (manufactured by GIBCO), and then replaced every other day. The medium was changed.
[Virus solution]
(1) 200 μL of Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, and foreign gene-free virus solution -1 600μL
(2) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, and Jarid2 gene-containing virus solution- 1 600μL
(3) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, and Jarid2 mutant gene-containing virus Liquid-1 600μL
－人工多能性幹細胞の割合－
　前記ウイルス感染後６日目（Ｄａｙ　６）、又は８日目（Ｄａｙ　８）に、前記ＮＧ－ＭＥＦをトリプシンで剥離し、フローサイトメーター（ＢＤ　ＦＡＣＳＣａｌｉｂｕｒ（登録商標）、日本ベクトン・ディッキンソン社製）で１０万個あたりのＧＦＰ陽性細胞数を計測した。結果を図１－１に示した。
　図１－１中、（１）から（３）は、それぞれ前記（１）から（３）のウイルス液を用いた場合の結果を示す。
　図１－１の結果から、前記（３）の場合には、ウイルス感染後６日目、及び８日目のいずれの場合にも全細胞に対する人工多能性幹細胞の割合が、前記（１）及び（２）の場合よりも高く、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物を用いることにより、人工多能性幹細胞の製造効率を高めることができることが示された。 -Proportion of induced pluripotent stem cells-
On the 6th day (Day 6) or 8th day (Day 8) after the virus infection, the NG-MEF was detached with trypsin, and a flow cytometer (BD FACSCalibur (registered trademark), manufactured by Nippon Becton Dickinson) The number of GFP positive cells per 100,000 cells was counted. The results are shown in FIG. 1-1.
1-1, (1) to (3) show the results when the virus solutions (1) to (3) were used, respectively.
From the result of FIG. 1-1, in the case of (3) above, the ratio of the induced pluripotent stem cells to the total cells in any of the 6th day and 8th day after the virus infection is the above (1) And it was higher than in the case of (2), and it was shown that the production efficiency of induced pluripotent stem cells can be increased by using the Jarid2 mutant gene or its gene product.
（試験例１－２：製造効率試験－２）
－細胞－
　細胞は、前記試験例１－１と同様に、ＮＧ－ＭＥＦを用いた。 (Test Example 1-2: Production efficiency test-2)
-cell-
As the cells, NG-MEF was used as in Test Example 1-1.
－ウイルス感染－
　前記試験例１－１において、ウイルス液を下記（１）から（３）のいずれかのウイルス液に代えた以外は、前記試験例１－１と同様にして、前記ＮＧ－ＭＥＦにウイルスを感染させた。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－１　３００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　３００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　３００μＬ、及びｃ－Ｍｙｃ遺伝子含有ウイルス液－１　３００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－１　３００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　３００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　３００μＬ、並びにｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２遺伝子含有ウイルス液－１　３００μＬ
（３）Ｏｃｔ３／４遺伝子含有ウイルス液－１　３００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　３００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　３００μＬ、並びにｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－１　３００μＬ -Virus infection-
In the test example 1-1, the virus solution was infected with the NG-MEF in the same manner as in the test example 1-1 except that the virus solution was replaced with any one of the virus solutions (1) to (3) below. I let you.
[Virus solution]
(1) 300 μL of Oct3 / 4 gene-containing virus solution-1 300 μL, Sox2 gene-containing virus solution-1 300 μL, Klf4 gene-containing virus solution-1 300 μL, and c-Myc gene-containing virus solution-1 300 μL
(2) Oct3 / 4 gene-containing virus solution-1 300 μL, Sox2 gene-containing virus solution-1 300 μL, Klf4 gene-containing virus solution-1 300 μL, and c-Myc gene and Jarid2 gene-containing virus solution-1 300 μL
(3) Oct3 / 4 gene-containing virus solution-1 300 μL, Sox2 gene-containing virus solution-1 300 μL, Klf4 gene-containing virus solution-1 300 μL, and c-Myc gene and Jarid2 mutant gene-containing virus solution-1 300 μL
－人工多能性幹細胞のコロニー数の計測－
　前記ウイルス感染後８日目から１２日目までピューロマイシン（最終濃度１．５μｇ／ｍＬ）に曝露し、ウイルス感染後１２日目に蛍光顕微鏡下でＧＦＰ陽性コロニー数を計測した。結果を図１－２に示した。
　図１－２中、（１）から（３）は、それぞれ前記（１）から（３）のウイルス液を用いた場合の結果を示す。
　図１－２の結果から、前記（３）の場合には、人工多能性幹細胞のコロニー数（「ｉＰＳコロニー数」と称することもある）が、前記（１）及び（２）の場合よりも高く、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物を用いることにより、人工多能性幹細胞の製造効率を高めることができることが示された。 -Counting the number of colonies of induced pluripotent stem cells-
It was exposed to puromycin (final concentration 1.5 μg / mL) from the 8th day to the 12th day after the virus infection, and the number of GFP positive colonies was counted under a fluorescence microscope on the 12th day after the virus infection. The results are shown in FIG.
In FIG. 1-2, (1) to (3) show the results when the virus solutions (1) to (3) were used, respectively.
From the results of FIG. 1-2, in the case of (3), the number of colonies of induced pluripotent stem cells (sometimes referred to as “iPS colony number”) is greater than in the cases of (1) and (2). It was also shown that the production efficiency of induced pluripotent stem cells can be increased by using the Jarid2 mutant gene or its gene product.
（試験例１－３：製造効率試験－３）
－細胞－
　細胞は、前記試験例１－１と同様に、ＮＧ－ＭＥＦを用いた。 (Test Example 1-3: Production efficiency test-3)
-cell-
As the cells, NG-MEF was used as in Test Example 1-1.
－ウイルス感染－
　前記試験例１－１において、ウイルス液を下記（１）から（３）のいずれかのウイルス液に代えた以外は、前記試験例１－１と同様にして、前記ＮＧ－ＭＥＦにウイルスを感染させた。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　６００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－１　２００μＬ、並びに外来遺伝子不含有ウイルス液－１　６００μＬ
（３）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－１　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－１　２００μＬ、Ｅｓｒｒｂ遺伝子含有ウイルス液－１　２００μＬ、並びにＳａｌｌ４ａ遺伝子含有ウイルス液－１　２００μＬ -Virus infection-
In the test example 1-1, the virus solution was infected with the NG-MEF in the same manner as in the test example 1-1 except that the virus solution was replaced with any one of the virus solutions (1) to (3) below. I let you.
[Virus solution]
(1) 200 μL of Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, and foreign gene-free virus solution -1 600μL
(2) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene, and Jarid2 mutant gene-containing virus solution-1 200 μL, and Foreign gene-free virus solution-1 600μL
(3) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene, and Jarid2 mutant gene-containing virus solution-1 200 μL, Prdm14 Gene-containing virus solution-1 200 μL, Esrrb gene-containing virus solution-1 200 μL, and Sall4a gene-containing virus solution-1 200 μL
－人工多能性幹細胞の割合－
　前記ウイルス感染後８日目に、前記ＮＧ－ＭＥＦをトリプシンで剥離し、フローサイトメーター（ＢＤ　ＦＡＣＳＣａｌｉｂｕｒ（登録商標）、日本ベクトン・ディッキンソン社製）で１０万個あたりのＧＦＰ陽性細胞数を計測した。結果を図１－３に示した。
　図１－３中、（１）から（３）は、それぞれ前記（１）から（３）のウイルス液を用いた場合の結果を示す。
　図１－３の結果から、前記（２）及び（３）の場合には、全細胞に対する人工多能性幹細胞の割合が、前記（１）の場合よりも高く、また、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物と、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物と用いた前記（３）の場合には、より人工多能性幹細胞の製造効率を高めることができることが示された。 -Proportion of induced pluripotent stem cells-
On the 8th day after the virus infection, the NG-MEF was detached with trypsin, and the number of GFP positive cells per 100,000 cells was measured with a flow cytometer (BD FACSCalibur (registered trademark), manufactured by Nippon Becton Dickinson). . The results are shown in Fig. 1-3.
In FIG. 1-3, (1) to (3) show the results when the virus solutions (1) to (3) were used, respectively.
From the results shown in FIGS. 1-3, in the cases (2) and (3), the ratio of the induced pluripotent stem cells to the whole cells is higher than that in the case (1). In the case of the above (3) using the gene product and the Prdm14 gene to the gene product, the Esrrb gene to the gene product, and the Sall4a gene to the gene product, the production efficiency of the induced pluripotent stem cell is further increased. It was shown that
（試験例１－４：製造効率試験－４）
－細胞－
　細胞として、Ｎａｎｏｇ遺伝子プロモーター依存的にＧＦＰ及びピューロマイシン耐性遺伝子を発現するＮＧ－ＭＥＦ（理化学研究所バイオリソースセンターに寄託されているトランスジェニックマウスＳＴＯＣＫ　Ｔｇ（Ｎａｎｏｇ－ＧＦＰ，Ｐｕｒｏ）１Ｙａｍ（Ｎｏ．ＲＢＲＣ０２２９０）を入手、交配し、１３．５日胚からトリプシンを用いて単離、調製した）を用いた。前記ＮＧ－ＭＥＦの培養には、１０％血清を含むＤＭＥＭを用い、培養温度３７℃、ＣＯ２濃度５％で培養した。 (Test Example 1-4: Production efficiency test-4)
-cell-
As a cell, NG-MEF expressing GFP and puromycin resistance gene in a Nanog gene promoter-dependent manner (transgenic mouse STOCK Tg (Nanog-GFP, Puro) 1Yam (No. RBRC02290) deposited at RIKEN BioResource Center) Was obtained, mated, and isolated from 13.5 day embryos using trypsin). The NG-MEF was cultured using DMEM containing 10% serum at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
－ウイルス感染－
　下記（１）から（８）のそれぞれのウイルス液に、最終濃度４μｇ／ｍＬのポリブレンを加えて調製したウイルス液を、それぞれ前記ＮＧ－ＭＥＦに感染させた。
　前記感染の翌日に、上清をＬＩＦ（ＬＩＦタンパク質を発現するベクターｐＣＡＧＧＳ－ＬＩＦ（理科学研究所　発生・再生科学より入手）をＣｏｓ－７細胞にリン酸カルシウム法により導入し、その培養上清を用いた）、２０％　ＫＳＲ（ＧＩＢＣＯ社製）、１×ＮＥＡＡ（ＧＩＢＣＯ社製）、２－メルカプトエタノール（ＧＩＢＣＯ社製）、ＧｌｕｔａＭａｘ（ＧＩＢＣＯ社製）を含むＤＭＥＭに置き換え、その後は、１日おきに培地交換を行った。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　６００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　４００μＬ
（３）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、Ｅｓｒｒｂ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　４００μＬ
（４）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、Ｓａｌｌ４ａ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　４００μＬ
（５）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－１　２００μＬ、Ｓａｌｌ４ａ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　２００μＬ
（６）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－１　２００μＬ、Ｅｓｒｒｂ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　２００μＬ
（７）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、Ｓａｌｌ４ａ遺伝子含有ウイルス液－１　２００μＬ、Ｅｓｒｒｂ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　２００μＬ
（８）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－１　２００μＬ、Ｓａｌｌ４ａ遺伝子含有ウイルス液－１　２００μＬ、及びＥｓｒｒｂ遺伝子含有ウイルス液－１　２００μＬ -Virus infection-
The virus solutions prepared by adding polybrene having a final concentration of 4 μg / mL to the virus solutions of (1) to (8) below were each infected with the NG-MEF.
The day after the infection, the supernatant was transferred to LIF (LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used. 20% KSR (manufactured by GIBCO), 1 × NEAA (manufactured by GIBCO), 2-mercaptoethanol (manufactured by GIBCO), GlutaMax (manufactured by GIBCO), and then replaced every other day. The medium was changed.
[Virus solution]
(1) 200 μL of Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, and foreign gene-free virus solution -1 600μL
(2) Oct3 / 4 gene containing virus solution-1 200 μL, Sox2 gene containing virus solution-1 200 μL, Klf4 gene containing virus solution-1 200 μL, c-Myc gene containing virus solution-1 200 μL, Prdm14 gene containing virus solution-1 200 μL and foreign gene-free virus solution-1 400 μL
(3) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, Esrrb gene-containing virus solution-1 200 μL and foreign gene-free virus solution-1 400 μL
(4) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, Sall4a gene-containing virus solution-1 200 μL and foreign gene-free virus solution-1 400 μL
(5) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, Prdm14 gene-containing virus solution-1 200 μL, Sall4a gene-containing virus solution-1 200 μL, and foreign gene-free virus solution-1 200 μL
(6) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, Prdm14 gene-containing virus solution-1 200 μL, Esrrb gene-containing virus solution-1 200 μL, and foreign gene-free virus solution-1 200 μL
(7) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, Sall4a gene-containing virus solution-1 200 μL, Esrrb gene-containing virus solution-1 200 μL, and foreign gene-free virus solution-1 200 μL
(8) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, Prdm14 gene-containing virus solution-1 200 μL, Sall4a gene-containing virus solution-1 200 μL, and Esrrb gene-containing virus solution-1 200 μL
－人工多能性幹細胞のコロニー数の計測－
　前記ウイルス感染後８日目から１２日目までピューロマイシン（最終濃度１．５μｇ／ｍＬ）に曝露し、ウイルス感染後１２日目に蛍光顕微鏡下でＧＦＰ陽性コロニー数を計測した。結果を図１－４に示した。
　図１－４中、（１）から（８）は、それぞれ前記（１）から（８）のウイルス液を用いた場合の結果を示す。
　図１－４の結果から、前記（８）の場合には、ｉＰＳコロニー数が、前記（１）から（７）の場合よりも高く、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｓａｌｌ４ａ遺伝子乃至その遺伝子産物、及びＥｓｒｒｂ遺伝子乃至その遺伝子産物を用いることにより、人工多能性幹細胞の製造効率を高めることができることが示された。 -Counting the number of colonies of induced pluripotent stem cells-
It was exposed to puromycin (final concentration 1.5 μg / mL) from the 8th day to the 12th day after the virus infection, and the number of GFP positive colonies was counted under a fluorescence microscope on the 12th day after the virus infection. The results are shown in FIGS. 1-4.
1-4, (1) to (8) show the results when the virus solutions (1) to (8) were used, respectively.
From the results of FIGS. 1-4, in the case of (8), the number of iPS colonies is higher than in the cases of (1) to (7), and the Prdm14 gene to its gene product, the Sall4a gene to its gene product, It has been shown that the production efficiency of induced pluripotent stem cells can be increased by using the Esrrb gene or its gene product.
（試験例１－５：製造効率試験－５）
－細胞－
　細胞は、前記試験例１－４と同様に、ＮＧ－ＭＥＦを用いた。 (Test Example 1-5: Production efficiency test -5)
-cell-
As the cell, NG-MEF was used in the same manner as in Test Example 1-4.
－ウイルス感染－
　前記試験例１－４において、ウイルス液を下記（１）及び（２）のいずれかのウイルス液に代えた以外は、前記試験例１－４と同様にして、前記ＮＧ－ＭＥＦにウイルスを感染させた。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　６００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－１　２００μＬ、Ｓａｌｌ４ａ遺伝子含有ウイルス液－１　２００μＬ、及びＥｓｒｒｂ遺伝子含有ウイルス液－１　２００μＬ -Virus infection-
In the test example 1-4, the virus solution was infected with the NG-MEF in the same manner as in the test example 1-4 except that the virus solution was replaced with one of the virus solutions (1) and (2) below. I let you.
[Virus solution]
(1) 200 μL of Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, and foreign gene-free virus solution -1 600μL
(2) Oct3 / 4 gene containing virus solution-1 200 μL, Sox2 gene containing virus solution-1 200 μL, Klf4 gene containing virus solution-1 200 μL, c-Myc gene containing virus solution-1 200 μL, Prdm14 gene containing virus solution-1 200 μL, Sall4a gene-containing virus solution-1 200 μL, and Esrrb gene-containing virus solution-1 200 μL
－人工多能性幹細胞の割合－
　前記ウイルス感染後８日目に、前記ＮＧ－ＭＥＦをトリプシンで剥離し、フローサイトメーター（ＢＤ　ＦＡＣＳＣａｌｉｂｕｒ（登録商標）、日本ベクトン・ディッキンソン社製）で１０万個あたりのＧＦＰ陽性細胞数を計測した。結果を図１－５に示した。
　図１－５中、（１）及び（２）は、それぞれ前記（１）及び（２）のウイルス液を用いた場合の結果を示す。
　図１－５の結果から、前記（２）の場合には、全細胞に対する人工多能性幹細胞の割合が、前記（１）の場合よりも高く、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｓａｌｌ４ａ遺伝子乃至その遺伝子産物、及びＥｓｒｒｂ遺伝子乃至その遺伝子産物を用いることにより、人工多能性幹細胞の製造効率を高めることができることが示された。 -Proportion of induced pluripotent stem cells-
On the 8th day after the virus infection, the NG-MEF was detached with trypsin, and the number of GFP positive cells per 100,000 cells was measured with a flow cytometer (BD FACSCalibur (registered trademark), manufactured by Nippon Becton Dickinson). . The results are shown in FIGS. 1-5.
In FIGS. 1-5, (1) and (2) show the results when the virus solutions (1) and (2) were used, respectively.
From the result of FIG. 1-5, in the case of (2), the ratio of induced pluripotent stem cells to the whole cells is higher than in the case of (1), and the Prdm14 gene or its gene product, the Sall4a gene or its It was shown that the production efficiency of induced pluripotent stem cells can be increased by using the gene product and the Esrrb gene or the gene product thereof.
（試験例１－６：製造効率試験－６）
－細胞－
　細胞として、ヒト皮膚線維芽細胞（以下、「ＨＤＦ」と称することがある、東洋紡株式会社製）を用いた。前記ＨＤＦの培養には、ヒト皮膚線維芽細胞基本培地（東洋紡株式会社製）を用い、培養温度３７℃、ＣＯ２濃度５％で培養した。 (Test Example 1-6: Production efficiency test-6)
-cell-
As the cells, human dermal fibroblasts (hereinafter, sometimes referred to as “HDF”, manufactured by Toyobo Co., Ltd.) were used. For culturing the HDF, a human skin fibroblast basic medium (manufactured by Toyobo Co., Ltd.) was used and cultured at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
－ウイルス感染－
　前記試験例１－４において、細胞をＮＧ－ＭＥＦから前記ＨＤＦに代え、ウイルス液を下記（１）及び（２）のいずれかのウイルス液に代えた以外は、前記試験例１－４と同様にして、前記ＨＤＦにウイルスを感染させた。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－２　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－２　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－２　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－２　２００μＬ、及び外来遺伝子不含有ウイルス液－２　６００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－２　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－２　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－２　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－２　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－２　２００μＬ、Ｓａｌｌ４ａ遺伝子含有ウイルス液－２　２００μＬ、及びＥｓｒｒｂ遺伝子含有ウイルス液－２　２００μＬ -Virus infection-
In Test Example 1-4, the procedure was the same as in Test Example 1-4 except that the cells were changed from NG-MEF to HDF and the virus solution was replaced with one of the virus solutions (1) and (2) below. Then, the HDF was infected with a virus.
[Virus solution]
(1) Oct3 / 4 gene containing virus solution-2 200 μL, Sox2 gene containing virus solution-2 200 μL, Klf4 gene containing virus solution-2 200 μL, c-Myc gene containing virus solution-2 200 μL, and foreign gene-free virus solution -2 600μL
(2) Oct3 / 4 gene-containing virus solution-2 200 μL, Sox2 gene-containing virus solution-2 200 μL, Klf4 gene-containing virus solution-2 200 μL, c-Myc gene-containing virus solution-2 200 μL, Prdm14 gene-containing virus solution-2 200 μL, Sall4a gene-containing virus solution-2 200 μL, and Esrrb gene-containing virus solution-2 200 μL
－人工多能性幹細胞のコロニー数の計測－
　前記ウイルス感染後１５日目にＡｌｅｘａ　Ｆｌｕｏｒ　４８８標識されたＴＲＡ－１－６０抗体（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社）を用いて染色し、蛍光顕微鏡下で陽性コロニー数を計測した。結果を図１－６に示した。
　図１－６中、（１）及び（２）は、それぞれ前記（１）及び（２）のウイルス液を用いた場合の結果を示す。
　図１－６の結果から、前記（２）の場合には、ＴＲＡ－１－６０陽性コロニー数が、前記（１）の場合よりも多く、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｓａｌｌ４ａ遺伝子乃至その遺伝子産物、及びＥｓｒｒｂ遺伝子乃至その遺伝子産物を用いることにより、人工多能性幹細胞の製造効率を高めることができることが示された。 -Counting the number of colonies of induced pluripotent stem cells-
On day 15 after the virus infection, staining was performed using Alexa Fluor 488-labeled TRA-1-60 antibody (BD Biosciences), and the number of positive colonies was counted under a fluorescence microscope. The results are shown in FIGS. 1-6.
In FIGS. 1-6, (1) and (2) show the results when the virus solutions (1) and (2) were used, respectively.
From the results of FIG. 1-6, in the case of (2), the number of TRA-1-60 positive colonies is larger than in the case of (1), and the Prdm14 gene or its gene product, the Sall4a gene or its gene product. It has been shown that the production efficiency of induced pluripotent stem cells can be increased by using the Esrrb gene or its gene product.
（試験例２－１：製造期間試験－１）
－細胞－
　細胞は、前記試験例１－１と同様に、ＮＧ－ＭＥＦを用いた。 (Test Example 2-1: Production period test -1)
-cell-
As the cells, NG-MEF was used as in Test Example 1-1.
－ウイルス感染－
　前記試験例１－１において、ウイルス液を下記（１）及び（２）のいずれかのウイルス液に代えた以外は、前記試験例１－１と同様にして、前記ＮＧ－ＭＥＦにウイルスを感染させた。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　６００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－１　２００μＬ、並びに外来遺伝子不含有ウイルス液－１　６００μＬ -Virus infection-
In the test example 1-1, the virus solution was infected with the NG-MEF in the same manner as in the test example 1-1 except that the virus solution was replaced with one of the virus solutions (1) and (2) below. I let you.
[Virus solution]
(1) 200 μL of Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, and foreign gene-free virus solution -1 600μL
(2) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene, and Jarid2 mutant gene-containing virus solution-1 200 μL, and Foreign gene-free virus solution-1 600μL
－人工多能性幹細胞のコロニー数の計測－
　前記ウイルス感染後１日目、２日目、３日目、４日目、５日目、又は６日目から１２日目までピューロマイシン（最終濃度１．５μｇ／ｍＬ）に曝露し、ウイルス感染後１２日目に蛍光顕微鏡下でＧＦＰ陽性コロニー数を計測した。結果を図２－１に示した。
　図２－１中、Ｄ１からＤ６は、それぞれピューロマイシンの曝露開始日（ウイルス感染後１日目から６日目）を示し、「◆」は、前記（１）のウイルス液を用いた場合の結果を示し、「■」は、前記（２）のウイルス液を用いた場合の結果を示す。
　図２－１に示すように、前記（２）の場合には、ウイルス感染後４日目にピューロマイシンを曝露した場合でも３回の実験で安定的に人工多能性幹細胞が確認された（人工多能性幹細胞のコロニー数：平均３個）のに対し、前記（１）の場合には、ウイルス感染後６日目にピューロマイシンを曝露した場合に初めて３回の実験で安定的に人工多能性幹細胞が確認された（人工多能性幹細胞のコロニー数：平均８個）。
　したがって、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物を用いることにより、人工多能性幹細胞を短期間で製造することができることが示された。 -Counting the number of colonies of induced pluripotent stem cells-
Exposure to puromycin (final concentration 1.5 μg / mL) from day 1, day 2, day 3, day 4, day 5, day 6 to day 12 after virus infection After 12 days, the number of GFP positive colonies was counted under a fluorescence microscope. The results are shown in FIG.
In FIG. 2-1, D1 to D6 indicate the start date of puromycin exposure (from day 1 to day 6 after virus infection), and “♦” indicates the case where the virus solution of (1) was used. The result shows the result, and “■” shows the result when the virus solution of (2) was used.
As shown in FIG. 2-1, in the case of (2) above, even when puromycin was exposed on the fourth day after virus infection, induced pluripotent stem cells were stably confirmed in three experiments ( In the case of (1) above, the number of induced pluripotent stem cell colonies (average: 3) is stable in 3 experiments for the first time when puromycin is exposed on the 6th day after virus infection. Pluripotent stem cells were confirmed (number of induced pluripotent stem cell colonies: 8 on average).
Therefore, it was shown that artificial pluripotent stem cells can be produced in a short period of time by using the Jarid2 mutant gene or its gene product.
（試験例２－２：製造期間試験－２）
－細胞－
　細胞は、前記試験例１－１と同様に、ＮＧ－ＭＥＦを用いた。 (Test Example 2-2: Production period test-2)
-cell-
As the cells, NG-MEF was used as in Test Example 1-1.
－ウイルス感染－
　前記試験例１－１において、ウイルス液を下記（１）及び（２）のいずれかのウイルス液に代えた以外は、前記試験例１－１と同様にして、前記ＮＧ－ＭＥＦにウイルスを感染させた。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　６００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－１　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－１　２００μＬ、Ｅｓｒｒｂ遺伝子含有ウイルス液－１　２００μＬ、並びにＳａｌｌ４ａ遺伝子含有ウイルス液－１　２００μＬ -Virus infection-
In the test example 1-1, the virus solution was infected with the NG-MEF in the same manner as in the test example 1-1 except that the virus solution was replaced with one of the virus solutions (1) and (2) below. I let you.
[Virus solution]
(1) 200 μL of Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene-containing virus solution-1 200 μL, and foreign gene-free virus solution -1 600μL
(2) Oct3 / 4 gene-containing virus solution-1 200 μL, Sox2 gene-containing virus solution-1 200 μL, Klf4 gene-containing virus solution-1 200 μL, c-Myc gene, and Jarid2 mutant gene-containing virus solution-1 200 μL, Prdm14 Gene-containing virus solution-1 200 μL, Esrrb gene-containing virus solution-1 200 μL, and Sall4a gene-containing virus solution-1 200 μL
－人工多能性幹細胞のコロニー数の計測－
　前記ウイルス感染後１日目、２日目、３日目、４日目、５日目、又は６日目から１２日目までピューロマイシン（最終濃度１．５μｇ／ｍＬ）に曝露し、ウイルス感染後１２日目に蛍光顕微鏡下でＧＦＰ陽性コロニー数を計測した。結果を図２－２に示した。
　図２－２中、Ｄ１からＤ６は、それぞれピューロマイシンの曝露開始日（ウイルス感染後１日目から６日目）を示し、「◆」は、前記（１）のウイルス液を用いた場合の結果を示し、「■」は、前記（２）のウイルス液を用いた場合の結果を示す。
　図２－２に示すように、前記（２）の場合には、ウイルス感染後２日目にピューロマイシンを曝露した場合でも３回の実験で安定的に人工多能性幹細胞が確認された（人工多能性幹細胞のコロニー数：平均３個）のに対し、前記（１）の場合には、ウイルス感染後６日目にピューロマイシンを曝露した場合に初めて３回の実験で安定的に人工多能性幹細胞が確認された（人工多能性幹細胞のコロニー数：平均８個）。
　したがって、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物と、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物とを用いることにより、人工多能性幹細胞をより短期間で製造することができることが示された。 -Counting the number of colonies of induced pluripotent stem cells-
Exposure to puromycin (final concentration 1.5 μg / mL) from day 1, day 2, day 3, day 4, day 5, day 6 to day 12 after virus infection After 12 days, the number of GFP positive colonies was counted under a fluorescence microscope. The results are shown in FIG.
In FIG. 2-2, D1 to D6 respectively indicate the start date of puromycin exposure (from day 1 to day 6 after virus infection), and “◆” indicates the case where the virus solution of (1) was used. The result shows the result, and “■” shows the result when the virus solution of (2) was used.
As shown in FIG. 2-2, in the case of (2) above, even when puromycin was exposed on the second day after virus infection, induced pluripotent stem cells were stably confirmed in three experiments ( In the case of (1) above, the number of induced pluripotent stem cell colonies (average 3) is stable in 3 experiments for the first time when puromycin is exposed on the 6th day after virus infection. Pluripotent stem cells were confirmed (number of induced pluripotent stem cell colonies: 8 on average).
Therefore, by using the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product, an artificial pluripotent stem cell can be produced in a shorter period of time. It was shown that you can.
（試験例２－３：製造期間試験－３）
－細胞－
　細胞は、前記試験例１－４と同様に、ＮＧ－ＭＥＦを用いた。 (Test Example 2-3: Production period test-3)
-cell-
As the cell, NG-MEF was used in the same manner as in Test Example 1-4.
－ウイルス感染－
　前記試験例１－４において、ウイルス液を下記（１）及び（２）のいずれかのウイルス液に代えた以外は、前記試験例１－４と同様にして、前記ＮＧ－ＭＥＦにウイルスを感染させた。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、及びｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、及び外来遺伝子不含有ウイルス液－１　６００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－１　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－１　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－１　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－１　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－１　２００μＬ、Ｓａｌｌ４ａ遺伝子含有ウイルス液－１　２００μＬ、及びＥｓｒｒｂ遺伝子含有ウイルス液－１　２００μＬ -Virus infection-
In the test example 1-4, the virus solution was infected with the NG-MEF in the same manner as in the test example 1-4 except that the virus solution was replaced with one of the virus solutions (1) and (2) below. I let you.
[Virus solution]
(1) 200 μL of Oct3 / 4 gene-containing virus solution-1 200 μL, 200 μL of Sox2 gene-containing virus solution-1 200 μL, and c-Myc gene-containing virus solution-1 200 μL, and foreign gene-free virus Liquid-1 600μL
(2) Oct3 / 4 gene containing virus solution-1 200 μL, Sox2 gene containing virus solution-1 200 μL, Klf4 gene containing virus solution-1 200 μL, c-Myc gene containing virus solution-1 200 μL, Prdm14 gene containing virus solution-1 200 μL, Sall4a gene-containing virus solution-1 200 μL, and Esrrb gene-containing virus solution-1 200 μL
－人工多能性幹細胞のコロニー数の計測－
　前記ウイルス感染後１日目、２日目、３日目、４日目、５日目、又は６日目から１２日目までピューロマイシン（最終濃度１．５μｇ／ｍＬ）に曝露し、ウイルス感染後１２日目に蛍光顕微鏡下でＧＦＰ陽性コロニー数を計測した。結果を図２－３に示した。
　図２－３中、Ｄ１からＤ６は、それぞれピューロマイシンの曝露開始日（ウイルス感染後１日目から６日目）を示し、「◆」は、前記（１）のウイルス液を用いた場合の結果を示し、「■」は、前記（２）のウイルス液を用いた場合の結果を示す。
　図２－３に示すように、前記（２）の場合には、ウイルス感染後２日目にピューロマイシンを曝露した場合でも３回の実験で安定的に人工多能性幹細胞が確認された（人工多能性幹細胞のコロニー数：平均９個）のに対し、前記（１）の場合には、ウイルス感染後６日目にピューロマイシンを曝露した場合に初めて３回の実験で安定的に人工多能性幹細胞が確認された（人工多能性幹細胞のコロニー数：平均８個）。
　したがって、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を用いることにより、人工多能性幹細胞を短期間で製造することができることが示された。 -Counting the number of colonies of induced pluripotent stem cells-
Exposure to puromycin (final concentration 1.5 μg / mL) from day 1, day 2, day 3, day 4, day 5, day 6 to day 12 after virus infection After 12 days, the number of GFP positive colonies was counted under a fluorescence microscope. The results are shown in Fig. 2-3.
In FIG. 2-3, D1 to D6 respectively indicate the start date of puromycin exposure (from day 1 to day 6 after virus infection), and “♦” indicates the case where the virus solution of (1) was used. The result shows the result, and “■” shows the result when the virus solution of (2) was used.
As shown in FIG. 2-3, in the case of (2) above, even when puromycin was exposed on the second day after virus infection, induced pluripotent stem cells were stably confirmed in three experiments ( In the case of (1) above, the number of induced pluripotent stem cell colonies (average: 9) is stable in 3 experiments for the first time when puromycin is exposed on the 6th day after virus infection. Pluripotent stem cells were confirmed (number of induced pluripotent stem cell colonies: 8 on average).
Therefore, it was shown that artificial pluripotent stem cells can be produced in a short period of time by using the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product.
（試験例３－１：品質試験－１）
－細胞－
　細胞として、ＨＤＦ（東洋紡株式会社製）を用いた。前記ＨＤＦの培養には、ヒト皮膚線維芽細胞基本培地（東洋紡株式会社製）を用い、培養温度３７℃、ＣＯ２濃度５％で培養した。 (Test Example 3-1: Quality test-1)
-cell-
HDF (Toyobo Co., Ltd.) was used as the cell. For culturing the HDF, a human skin fibroblast basic medium (manufactured by Toyobo Co., Ltd.) was used and cultured at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
－ウイルス感染－
　下記（１）及び（２）のそれぞれのウイルス液に、最終濃度４μｇ／ｍＬのポリブレンを加えて調製したウイルス液を、それぞれ前記ＨＤＦに感染させた。
　前記感染の翌日に、上清をＬＩＦ（ＬＩＦタンパク質を発現するベクターｐＣＡＧＧＳ－ＬＩＦ（理科学研究所　発生・再生科学より入手）をＣｏｓ－７細胞にリン酸カルシウム法により導入し、その培養上清を用いた）、２０％　ＫＳＲ（ＧＩＢＣＯ社製）、１×ＮＥＡＡ（ＧＩＢＣＯ社製）、２－メルカプトエタノール（ＧＩＢＣＯ社製）、ＧｌｕｔａＭａｘ（ＧＩＢＣＯ社製）を含むＤＭＥＭに置き換え、その後は、１日おきに培地交換を行った。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－２　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－２　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－２　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－２　２００μＬ、及び外来遺伝子不含有ウイルス液－２　６００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－２　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－２　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－２　２００μＬ、ｃ－Ｍｙｃ遺伝子、及びＪａｒｉｄ２変異体遺伝子含有ウイルス液－２　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－２　２００μＬ、Ｅｓｒｒｂ遺伝子含有ウイルス液－２　２００μＬ、並びにＳａｌｌ４ａ遺伝子含有ウイルス液－２　２００μＬ -Virus infection-
Virus solutions prepared by adding polybrene having a final concentration of 4 μg / mL to the virus solutions of (1) and (2) below were each infected with the HDF.
The day after the infection, the supernatant was transferred to LIF (LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used. 20% KSR (manufactured by GIBCO), 1 × NEAA (manufactured by GIBCO), 2-mercaptoethanol (manufactured by GIBCO), GlutaMax (manufactured by GIBCO), and then replaced every other day. The medium was changed.
[Virus solution]
(1) Oct3 / 4 gene containing virus solution-2 200 μL, Sox2 gene containing virus solution-2 200 μL, Klf4 gene containing virus solution-2 200 μL, c-Myc gene containing virus solution-2 200 μL, and foreign gene-free virus solution -2 600μL
(2) Oct3 / 4 gene-containing virus solution-2 200 μL, Sox2 gene-containing virus solution-2 200 μL, Klf4 gene-containing virus solution-2 200 μL, c-Myc gene, and Jarid2 mutant gene-containing virus solution-2 200 μL, Prdm14 Gene-containing virus solution-2 200 μL, Esrrb gene-containing virus solution-2 200 μL, and Sall4a gene-containing virus solution-2 200 μL
－人工多能性幹細胞－
　前記ウイルス感染後１８日目の細胞、及びクローン化した細胞を倒立顕微鏡（Ｚｅｉｓｓ　Ａｘｉｏｖｅｒｔ　２００Ｍ）で撮影した結果を図３Ａから３Ｄに示した。
　図３Ａ及び３Ｂは、前記（１）のウイルス液を用いた場合の写真の一例であり、図３Ｃ及び３Ｄは、前記（２）のウイルス液を用いた場合の写真の一例である。図３Ａから３Ｄ中、スケールバーは、２００μｍを表す。
　前記（１）のウイルス液を用いて得られた人工多能性幹細胞は、コロニーの形態が扁平状であり、ｂＦＧＦ添加培地で増殖することができ、２ｉ添加培地では増殖できず、単一細胞に分散すると死んでしまう細胞であり、プライム型人工多能性幹細胞であることがわかった。
　一方、前記（２）のウイルス液を用いて得られた人工多能性幹細胞は、コロニーの形態がドーム状であり、ｂＦＧＦを含まないＬＩＦ添加培地で増殖することができ、２ｉ添加培地で増殖することができ、単一細胞に分散して継代することができる細胞であり、ナイーブ型人工多能性幹細胞であることがわかった。
　したがって、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物と、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物とを用いることにより、高品質の人工多能性幹細胞を製造することができることが示された。 -Artificial pluripotent stem cells-
FIGS. 3A to 3D show the results of photographing the cells 18 days after the virus infection and the cloned cells with an inverted microscope (Zeiss Axiovert 200M).
3A and 3B are examples of photographs when the virus solution of (1) is used, and FIGS. 3C and 3D are examples of photographs when the virus solution of (2) is used. In FIGS. 3A to 3D, the scale bar represents 200 μm.
The induced pluripotent stem cells obtained using the virus solution of (1) above have a flat colony, can be grown on a bFGF-added medium, cannot grow on a 2i-added medium, and are single cells. It was found to be a prime-type induced pluripotent stem cell.
On the other hand, the induced pluripotent stem cells obtained using the virus solution of (2) above have a dome-like colony shape and can be grown in a LIF-added medium not containing bFGF, and can be grown in a 2i-added medium. It was found to be a naive-type induced pluripotent stem cell that can be dispersed and passaged into a single cell.
Therefore, high-quality induced pluripotent stem cells are produced by using the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product. It was shown that it can.
（試験例３－２：品質試験－２）
－細胞－
　細胞として、ＨＤＦ（東洋紡株式会社製）を用いた。前記ＨＤＦの培養には、ヒト皮膚線維芽細胞基本培地（東洋紡株式会社製）を用い、培養温度３７℃、ＣＯ２濃度５％で培養した。 (Test Example 3-2: Quality test-2)
-cell-
HDF (Toyobo Co., Ltd.) was used as the cell. For culturing the HDF, a human skin fibroblast basic medium (manufactured by Toyobo Co., Ltd.) was used and cultured at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
－ウイルス感染－
　下記（１）及び（２）のそれぞれのウイルス液に、最終濃度４μｇ／ｍＬのポリブレンを加えて調製したウイルス液を、それぞれ前記ＨＤＦに感染させた。
　前記感染の翌日に、上清をＬＩＦ（ＬＩＦタンパク質を発現するベクターｐＣＡＧＧＳ－ＬＩＦ（理科学研究所　発生・再生科学より入手）をＣｏｓ－７細胞にリン酸カルシウム法により導入し、その培養上清を用いた）、２０％　ＫＳＲ（ＧＩＢＣＯ社製）、１×ＮＥＡＡ（ＧＩＢＣＯ社製）、２－メルカプトエタノール（ＧＩＢＣＯ社製）、ＧｌｕｔａＭａｘ（ＧＩＢＣＯ社製）を含むＤＭＥＭに置き換え、その後は、１日おきに培地交換を行った。
〔ウイルス液〕
（１）Ｏｃｔ３／４遺伝子含有ウイルス液－２　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－２　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－２　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－２　２００μＬ、及び外来遺伝子不含有ウイルス液－２　６００μＬ
（２）Ｏｃｔ３／４遺伝子含有ウイルス液－２　２００μＬ、Ｓｏｘ２遺伝子含有ウイルス液－２　２００μＬ、Ｋｌｆ４遺伝子含有ウイルス液－２　２００μＬ、ｃ－Ｍｙｃ遺伝子含有ウイルス液－２　２００μＬ、Ｐｒｄｍ１４遺伝子含有ウイルス液－１　２００μＬ、Ｅｓｒｒｂ遺伝子含有ウイルス液－２　２００μＬ、及びＳａｌｌ４ａ遺伝子含有ウイルス液－２　２００μＬ -Virus infection-
Virus solutions prepared by adding polybrene having a final concentration of 4 μg / mL to the virus solutions of (1) and (2) below were each infected with the HDF.
The day after the infection, the supernatant was transferred to LIF (LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used. 20% KSR (manufactured by GIBCO), 1 × NEAA (manufactured by GIBCO), 2-mercaptoethanol (manufactured by GIBCO), GlutaMax (manufactured by GIBCO), and then replaced every other day. The medium was changed.
[Virus solution]
(1) Oct3 / 4 gene containing virus solution-2 200 μL, Sox2 gene containing virus solution-2 200 μL, Klf4 gene containing virus solution-2 200 μL, c-Myc gene containing virus solution-2 200 μL, and foreign gene-free virus solution -2 600μL
(2) Oct3 / 4 gene-containing virus solution-2 200 μL, Sox2 gene-containing virus solution-2 200 μL, Klf4 gene-containing virus solution-2 200 μL, c-Myc gene-containing virus solution-2 200 μL, Prdm14 gene-containing virus solution-1 200 μL, Esrrb gene-containing virus solution-2 200 μL, and Sall4a gene-containing virus solution-2 200 μL
－人工多能性幹細胞－
　前記ウイルス感染後１８日目の細胞、及びクローン化した細胞を倒立顕微鏡（Ｚｅｉｓｓ　Ａｘｉｏｖｅｒｔ　２００Ｍ）で撮影した結果を図３Ｅから３Ｈに示した。
　図３Ｅ及び３Ｆは、前記（１）のウイルス液を用いた場合の写真の一例であり、図３Ｇ及び３Ｈは、前記（２）のウイルス液を用いた場合の写真の一例である。図３Ｅから３Ｈ中、スケールバーは、２００μｍを表す。
　前記（１）のウイルス液を用いて得られた人工多能性幹細胞は、コロニーの形態が扁平状であり、ｂＦＧＦ添加培地で増殖することができ、２ｉ添加培地では増殖できず、単一細胞に分散すると死んでしまう細胞であり、プライム型人工多能性幹細胞であることがわかった。
　一方、前記（２）のウイルス液を用いて得られた人工多能性幹細胞は、コロニーの形態がドーム状であり、ｂＦＧＦを含まないＬＩＦ添加培地で増殖することができ、２ｉ添加培地で増殖することができ、単一細胞に分散して継代することができる細胞であり、ナイーブ型人工多能性幹細胞であることがわかった。
　したがって、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を用いることにより、高品質の人工多能性幹細胞を製造することができることが示された。 -Artificial pluripotent stem cells-
FIGS. 3E to 3H show the results of photographing the cells 18 days after the virus infection and the cloned cells with an inverted microscope (Zeiss Axiovert 200M).
3E and 3F are examples of photographs when the virus solution of (1) is used, and FIGS. 3G and 3H are examples of photographs when the virus solution of (2) is used. In FIGS. 3E to 3H, the scale bar represents 200 μm.
The induced pluripotent stem cells obtained using the virus solution of (1) above have a flat colony, can be grown on a bFGF-added medium, cannot grow on a 2i-added medium, and are single cells. It was found to be a prime-type induced pluripotent stem cell.
On the other hand, the induced pluripotent stem cells obtained using the virus solution of (2) above have a dome-like colony shape and can be grown in a LIF-added medium not containing bFGF, and can be grown in a 2i-added medium. It was found to be a naive-type induced pluripotent stem cell that can be dispersed and passaged into a single cell.
Therefore, it was shown that high-quality induced pluripotent stem cells can be produced by using the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product.
　本発明の人工多能性幹細胞の製造方法によれば、人工多能性幹細胞を高効率で製造することができるので、少数の体細胞からの人工多能性幹細胞の樹立が可能となり、また、希少な細胞や組織の採取量を軽減することができると考えられる。更に、非人工多能性幹細胞の増殖を抑え、人工多能性幹細胞を選択的に増やすことが可能であるため、人工多能性幹細胞クローンの樹立が容易になると考えられる。
　また、本発明の人工多能性幹細胞の製造方法によれば、人工多能性幹細胞を短期間で製造することができるので、早期に細胞移植が必要な脊髄損傷などの疾患への自己由来の人工多能性幹細胞の利用を可能とし、また、長期間の人工多能性幹細胞誘導過程によるゲノム損傷リスクを軽減したり、一過性の遺伝子導入による人工多能性幹細胞の作製方法を容易にしたりするなど、安全な人工多能性幹細胞の作製が可能となると考えられる。
　更に、本発明の人工多能性幹細胞の製造方法によれば、人工多能性幹細胞を効率良く短期間で製造することができるので、従来の方法と比べてコストを軽減することができる。
　また、本発明の人工多能性幹細胞の製造方法によれば、ヒト細胞の場合でもナイーブ型の人工多能性幹細胞を製造することができるので、人工多能性幹細胞の取扱いを簡便化することができ、分化誘導効率を向上させることができると考えられる。 According to the method for producing an induced pluripotent stem cell of the present invention, an induced pluripotent stem cell can be produced with high efficiency, so that an induced pluripotent stem cell can be established from a small number of somatic cells, It is thought that the amount of rare cells and tissues collected can be reduced. Furthermore, since it is possible to suppress proliferation of non-induced pluripotent stem cells and selectively increase induced pluripotent stem cells, it is considered that establishment of induced pluripotent stem cell clones is facilitated.
In addition, according to the method for producing induced pluripotent stem cells of the present invention, since induced pluripotent stem cells can be produced in a short period of time, self-derived to diseases such as spinal cord injury requiring early cell transplantation. Enables the use of induced pluripotent stem cells, reduces the risk of genome damage due to the long-term induced pluripotent stem cell induction process, and facilitates the method of creating induced pluripotent stem cells by transient gene transfer It is considered possible to produce safe induced pluripotent stem cells.
Furthermore, according to the method for producing induced pluripotent stem cells of the present invention, induced pluripotent stem cells can be produced efficiently and in a short period of time, so that the cost can be reduced as compared with conventional methods.
Further, according to the method for producing induced pluripotent stem cells of the present invention, naive induced pluripotent stem cells can be produced even in the case of human cells, so that handling of induced pluripotent stem cells is simplified. It is considered that differentiation induction efficiency can be improved.
　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＞　（Ａ）Ｏｃｔ３／４遺伝子乃至その遺伝子産物と、
　（Ｂ）Ｓｏｘ２遺伝子乃至その遺伝子産物と、
　（Ｃ）Ｋｌｆ４遺伝子乃至その遺伝子産物と、
　（Ｄ）ｃ－Ｍｙｃ遺伝子乃至その遺伝子産物と、
　（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種とを体細胞に導入する工程を含み、
　前記Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物が、Ｊａｒｉｄ２タンパク質のＮ末端の１番目から５５１番目のアミノ酸をコードする遺伝子乃至その遺伝子産物であることを特徴とする人工多能性幹細胞の製造方法である。
　＜２＞　（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物を含む前記＜１＞に記載の人工多能性幹細胞の製造方法である。
　＜３＞　（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、更に、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む前記＜２＞に記載の人工多能性幹細胞の製造方法である。
　＜４＞　ｃ－Ｍｙｃ遺伝子と、Ｊａｒｉｄ２変異体遺伝子とが共発現される前記＜２＞から＜３＞のいずれかに記載の人工多能性幹細胞の製造方法である。
　＜５＞　（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む前記＜１＞に記載の人工多能性幹細胞の製造方法である。
　＜６＞　人工多能性幹細胞が、ナイーブ型である前記＜１＞から＜５＞のいずれかに記載の人工多能性幹細胞の製造方法である。
　＜７＞　人工多能性幹細胞が、ヒト人工多能性幹細胞である前記＜１＞から＜６＞のいずれかに記載の人工多能性幹細胞の製造方法である。
　＜８＞　Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種を含み、
　前記Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物が、Ｊａｒｉｄ２タンパク質のＮ末端の１番目から５５１番目のアミノ酸をコードする遺伝子乃至その遺伝子産物であることを特徴とする人工多能性幹細胞製造用組成物である。
　＜９＞　Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物を含む前記＜８＞に記載の人工多能性幹細胞製造用組成物である。
　＜１０＞　Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、更に、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む前記＜９＞に記載の人工多能性幹細胞製造用組成物である。
　＜１１＞　Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む前記＜８＞に記載の人工多能性幹細胞製造用組成物である。
　＜１２＞　更に、Ｏｃｔ３／４遺伝子乃至その遺伝子産物、Ｓｏｘ２遺伝子乃至その遺伝子産物、Ｋｌｆ４遺伝子乃至その遺伝子産物、及びｃ－Ｍｙｃ遺伝子乃至その遺伝子産物を含む前記＜８＞から＜１１＞のいずれかに記載の人工多能性幹細胞製造用組成物である。
　＜１３＞　ｃ－Ｍｙｃ遺伝子と、Ｊａｒｉｄ２変異体遺伝子とが共発現される前記＜１２＞に記載の人工多能性幹細胞製造用組成物である。 Examples of the aspect of the present invention include the following.
<1> (A) Oct3 / 4 gene or its gene product;
(B) the Sox2 gene or its gene product;
(C) the Klf4 gene or its gene product;
(D) the c-Myc gene or its gene product;
(E) introducing at least one selected from the group consisting of the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product into a somatic cell. Including steps,
A method for producing an induced pluripotent stem cell, wherein the Jarid2 mutant gene or a gene product thereof is a gene encoding the N-terminal amino acids 1 to 551 of the Jarid2 protein or a gene product thereof.
<2> (E) At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product is a Jarid2 mutation The method for producing induced pluripotent stem cells according to <1> above, comprising a body gene or a gene product thereof.
<3> (E) At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product, The method for producing an induced pluripotent stem cell according to the above <2>, comprising the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product.
<4> The method for producing induced pluripotent stem cells according to any one of <2> to <3>, wherein the c-Myc gene and the Jarid2 mutant gene are coexpressed.
<5> (E) At least one selected from the group consisting of the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product is the Prdm14 gene Or the gene product, the Esrrb gene or the gene product thereof, and the Sall4a gene or the gene product thereof, or the method for producing an induced pluripotent stem cell according to <1>.
<6> The method for producing an induced pluripotent stem cell according to any one of <1> to <5>, wherein the induced pluripotent stem cell is a naive type.
<7> The method for producing an induced pluripotent stem cell according to any one of <1> to <6>, wherein the induced pluripotent stem cell is a human induced pluripotent stem cell.
<8> comprising at least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product,
A composition for producing induced pluripotent stem cells, wherein the Jarid2 mutant gene or its gene product is a gene encoding the N-terminal 1st to 551st amino acids of the Jarid2 protein or its gene product. .
<9> At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product is a Jarid2 mutant gene or The composition for producing induced pluripotent stem cells according to <8>, including the gene product.
<10> At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product is further added to the Prdm14 gene or its gene product. The composition for producing induced pluripotent stem cells according to <9>, including the gene product, Esrrb gene or gene product, and Sall4a gene or gene product.
<11> At least one selected from the group consisting of a Jard2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, or a Sall4a gene or its gene product is a Prdm14 gene or its gene The composition for producing induced pluripotent stem cells according to <8>, comprising a product, an Esrrb gene or a gene product thereof, and a Sall4a gene or a gene product thereof.
<12> Any one of <8> to <11>, further including Oct3 / 4 gene or gene product, Sox2 gene or gene product, Klf4 gene or gene product, and c-Myc gene or gene product The composition for producing induced pluripotent stem cells described in 1. above.
<13> The composition for producing induced pluripotent stem cells according to <12>, wherein the c-Myc gene and the Jarid2 mutant gene are coexpressed.
　本発明の人工多能性幹細胞の製造方法によれば、製造効率に優れ、短期間で製造することができ、品質面にも優れた人工多能性幹細胞を製造することができるので、細胞移植治療などの再生医療、薬剤スクリーニング、疾患の原因解明のためのツールなどとして用いる人工多能性幹細胞の製造方法に好適に利用可能である。 According to the method for producing an induced pluripotent stem cell of the present invention, it is possible to produce an induced pluripotent stem cell that is excellent in production efficiency, can be produced in a short period of time, and is excellent in quality. It can be suitably used in a method for producing induced pluripotent stem cells used as a regenerative medicine such as treatment, drug screening, and a tool for elucidating the cause of a disease.

Claims (13)

  1. 　（Ａ）Ｏｃｔ３／４遺伝子乃至その遺伝子産物と、
    　（Ｂ）Ｓｏｘ２遺伝子乃至その遺伝子産物と、
    　（Ｃ）Ｋｌｆ４遺伝子乃至その遺伝子産物と、
    　（Ｄ）ｃ－Ｍｙｃ遺伝子乃至その遺伝子産物と、
    　（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種とを体細胞に導入する工程を含み、
    　前記Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物が、Ｊａｒｉｄ２タンパク質のＮ末端の１番目から５５１番目のアミノ酸をコードする遺伝子乃至その遺伝子産物であることを特徴とする人工多能性幹細胞の製造方法。     (A) the Oct3 / 4 gene or its gene product;
    (B) the Sox2 gene or its gene product;
    (C) the Klf4 gene or its gene product;
    (D) the c-Myc gene or its gene product;
    (E) introducing at least one selected from the group consisting of the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product into a somatic cell. Including steps,
    A method for producing induced pluripotent stem cells, wherein the Jarid2 mutant gene or a gene product thereof is a gene encoding the N-terminal 1st to 551st amino acids of the Jarid2 protein or a gene product thereof.
  2. 　（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物を含む請求項１に記載の人工多能性幹細胞の製造方法。 (E) At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, or a Sall4a gene or its gene product, The method for producing induced pluripotent stem cells according to claim 1, comprising the gene product.
  3. 　（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、更に、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む請求項２に記載の人工多能性幹細胞の製造方法。 (E) At least one selected from the group consisting of the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product is further selected from the Prdm14 gene or its gene product. The method for producing induced pluripotent stem cells according to claim 2, comprising the gene product, the Esrrb gene or the gene product thereof, and the Sall4a gene or the gene product thereof.
  4. 　ｃ－Ｍｙｃ遺伝子と、Ｊａｒｉｄ２変異体遺伝子とが共発現される請求項２から３のいずれかに記載の人工多能性幹細胞の製造方法。 The method for producing induced pluripotent stem cells according to any one of claims 2 to 3, wherein the c-Myc gene and the Jarid2 mutant gene are co-expressed.
  5. 　（Ｅ）Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む請求項１に記載の人工多能性幹細胞の製造方法。 (E) At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, or a Sall4a gene or its gene product is a Prdm14 gene or its gene The method for producing induced pluripotent stem cells according to claim 1, comprising a product, an Esrrb gene or a gene product thereof, and a Sall4a gene or a gene product thereof.
  6. 　人工多能性幹細胞が、ナイーブ型である請求項１から５のいずれかに記載の人工多能性幹細胞の製造方法。 The method for producing induced pluripotent stem cells according to any one of claims 1 to 5, wherein the induced pluripotent stem cells are naïve.
  7. 　人工多能性幹細胞が、ヒト人工多能性幹細胞である請求項１から６のいずれかに記載の人工多能性幹細胞の製造方法。 The method for producing induced pluripotent stem cells according to any one of claims 1 to 6, wherein the induced pluripotent stem cells are human induced pluripotent stem cells.
  8. 　Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種を含み、
    　前記Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物が、Ｊａｒｉｄ２タンパク質のＮ末端の１番目から５５１番目のアミノ酸をコードする遺伝子乃至その遺伝子産物であることを特徴とする人工多能性幹細胞製造用組成物。     Including at least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product,
    A composition for producing an induced pluripotent stem cell, wherein the Jarid2 mutant gene or a gene product thereof is a gene encoding the N-terminal amino acids 1 to 551 of the Jarid2 protein or a gene product thereof.
  9. 　Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物を含む請求項８に記載の人工多能性幹細胞製造用組成物。 At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product is a Jarid2 mutant gene or its gene product The composition for artificial pluripotent stem cell production according to claim 8, comprising:
  10. 　Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、更に、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む請求項９に記載の人工多能性幹細胞製造用組成物。 At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, or a Sall4a gene or its gene product is further selected from the Prdm14 gene or its gene product The composition for producing induced pluripotent stem cells according to claim 9, comprising the Esrrb gene or the gene product thereof, and the Sall4a gene or the gene product thereof.
  11. 　Ｊａｒｉｄ２変異体遺伝子乃至その遺伝子産物、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物からなる群から選択される少なくとも１種が、Ｐｒｄｍ１４遺伝子乃至その遺伝子産物、Ｅｓｒｒｂ遺伝子乃至その遺伝子産物、及びＳａｌｌ４ａ遺伝子乃至その遺伝子産物を含む請求項８に記載の人工多能性幹細胞製造用組成物。 At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, or a Sall4a gene or its gene product is selected from the Prdm14 gene or its gene product, Esrrb The composition for producing induced pluripotent stem cells according to claim 8, comprising a gene or a gene product thereof and a Sall4a gene or a gene product thereof.
  12. 　更に、Ｏｃｔ３／４遺伝子乃至その遺伝子産物、Ｓｏｘ２遺伝子乃至その遺伝子産物、Ｋｌｆ４遺伝子乃至その遺伝子産物、及びｃ－Ｍｙｃ遺伝子乃至その遺伝子産物を含む請求項８から１１のいずれかに記載の人工多能性幹細胞製造用組成物。 The artificial pluripotent according to any one of claims 8 to 11, further comprising an Oct3 / 4 gene or its gene product, a Sox2 gene or its gene product, a Klf4 gene or its gene product, and a c-Myc gene or its gene product. A composition for producing sex stem cells.
  13. 　ｃ－Ｍｙｃ遺伝子と、Ｊａｒｉｄ２変異体遺伝子とが共発現される請求項１２に記載の人工多能性幹細胞製造用組成物。 The composition for producing induced pluripotent stem cells according to claim 12, wherein the c-Myc gene and the Jarid2 mutant gene are co-expressed.
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