WO2021015228A1 - Method for producing specific differentiated cells - Google Patents

Method for producing specific differentiated cells Download PDF

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WO2021015228A1
WO2021015228A1 PCT/JP2020/028402 JP2020028402W WO2021015228A1 WO 2021015228 A1 WO2021015228 A1 WO 2021015228A1 JP 2020028402 W JP2020028402 W JP 2020028402W WO 2021015228 A1 WO2021015228 A1 WO 2021015228A1
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inhibitor
cells
medium
pluripotent stem
culturing
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PCT/JP2020/028402
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French (fr)
Japanese (ja)
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奈穂 山▲崎▼
太一 村口
信一 渡邊
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富士フイルム株式会社
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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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • pluripotent stem cells are selected from the group consisting of histone deacetylase inhibitors, MAPK / ERK kinase inhibitors, STAT3 activators, protein kinase C inhibitors, Wnt signal inhibitors and GSK3 ⁇ inhibitors.
  • the present invention relates to a method for producing a specific differentiated cell, which comprises a step (b) of obtaining a specific differentiated cell by culturing the pluripotent stem cell obtained in (a) in a medium containing a cytokine.
  • iPS cells human induced pluripotent stem cells
  • ES cells embryonic stem cells
  • iPS cells have no ethical problems than ES cells, and are expected to be put into practical use as a cell source with stable quality.
  • in vitro tests have come to be conducted using human primary cultured hepatocytes in drug discovery screening and pharmacokinetic tests.
  • lot-to-lot differences and proliferation ability which is a barrier to research.
  • hepatocytes can also be derived from iPS cells, but the problem is that the differentiation efficiency varies depending on the method and cell line.
  • Patent Document 1 describes (1) a step of differentiating induced pluripotent stem cells into endometrial-like cells, and (2) a step of differentiating endometrial-like cells obtained in step (1) into hepatocellular-like cells.
  • a method for inducing differentiation of induced pluripotent stem cells into hepatocytes which comprises the step of performing at least a part of the culture in the presence of a histone deacetylase inhibitor and / or under oxidative stress loading conditions.
  • HDAC histone deacetylase
  • Valproic acid is a typical drug used for epilepsy, and its main action is ⁇ -amino acid (GABA) transaminase inhibitory action. Furthermore, since valproic acid exhibits an HDAC inhibitory effect, it is expected to be applied to cancer treatment.
  • GABA ⁇ -amino acid
  • Patent Document 1 it is reported that an HDAC inhibitor has an effect of promoting the induction of differentiation of hepatocyte-like cells from iPS cell-derived hepatoblast-like cells, and improves the properties of undifferentiated iPS cells themselves. It's not a thing.
  • the invention of the method for inducing iPS cells has made it possible to obtain stem cells having pluripotency equivalent to that of ES cells, and is expected to be applied to cell therapy and drug discovery support tools.
  • iPS cells have different properties depending on the cell line and clone, and there is a large difference in differentiation potential, and the marker as an iPS cell is expressed.
  • the present invention is a problem to be solved to provide a method for producing a differentiated cell capable of efficiently obtaining a specific differentiated cell of interest.
  • the present inventor efficiently differentiates pluripotent stem cells into target differentiated cells, particularly hepatocytes, by culturing them in the presence of a specific compound. I found that I could do it.
  • the present invention has been completed based on the above findings.
  • Pluripotent stem cells are selected from the group consisting of histone deacetylase inhibitors, MAPK / ERK kinase inhibitors, STAT3 activators, protein kinase C inhibitors, Wnt signal inhibitors and GSK3 ⁇ inhibitors.
  • Step (b) of obtaining specific differentiated cells by culturing the pluripotent stem cells obtained in a) in a medium containing cytokines.
  • a method for producing a specific differentiated cell including. (2) The above step (a) A first culture step in which pluripotent stem cells are cultured in a medium containing a histone deacetylase inhibitor, a MAPK / ERK kinase inhibitor, and a STAT3 activator, and after the first culture step, histone deacetylase.
  • a second culture step of culturing in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, a GSK3 ⁇ inhibitor, and a STAT3 activator without an inhibitor is included (1).
  • the manufacturing method described in. (3) The method according to (1) or (2), wherein the cytokine is at least one selected from the group consisting of activin A, BMP4, bFGF, HGF, and Oncostatin M.
  • the specific differentiated cell is a cell of endoderm lineage.
  • the specific differentiated cell is a hepatocyte.
  • the cells were cultured in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, and a STAT3 activator without containing a histone deacetylase inhibitor.
  • the second culture step is carried out from 3 in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, and a STAT3 activator without containing a histone deacetylase inhibitor.
  • the method according to any one of (2) to (7) which is a step of culturing for 8 days and then culturing in a medium obtained by adding a GSK3 ⁇ inhibitor to the above medium for 3 to 10 days.
  • a specific differentiated cell of interest can be efficiently produced.
  • FIG. 1 shows the degree of methylation of untreated and treated iPS cell lines.
  • FIG. 2 shows the gene expression levels related to the undifferentiated state of the untreated and treated iPS cell lines.
  • FIG. 3A was obtained by measuring the expression of ASGPR1 when untreated and treated human iPS cell lines (strains A to G) were induced to differentiate into hepatocytes by flow cytometry using an Anti-ASGPR1 antibody. Shows the ASGPR1 positive rate.
  • FIG. 3B shows ASGPR1-positive cells whose expression of ASGPR1 when untreated and treated human iPS cell lines (A to G strains) were induced to differentiate into hepatocytes was measured by flow cytometry using Anti-ASGPR1 antibody.
  • FIG. 4 shows the results of measuring the amount of albumin secreted in the culture supernatant when the untreated and treated human iPS cell lines (strains A to G) were induced to differentiate into hepatocytes by the ELISA method.
  • FIG. 5 shows the expression level of an endoderm marker gene when iPS cells are differentiated into endoderm after untreated, valproic acid-treated, and compound-mixed treatment conditions.
  • ALK5 TGF-beta type I receptor
  • bFGF basic Fibroblast Growth Factor
  • BMP4 Basic Fibroblast Growth Factor 34
  • Bone Former 34 Differentiation antigen group 34
  • cTnT cardiac Troponin T
  • DMEM Dulvecco's Modified Eagle Medium
  • DMEM / F12 Dulbecco's Modified Eagle Medium / Nutrition Mixture
  • F-12 Ham DNMT3B DNA (cytosine-5) -methyltransferase beta): DNA (cytosine-5-) methyltransferase 3 beta
  • Dnmt3L DNA Methyltransphase 3 Like
  • DNA methyltransferase 3-like ECAT ES cell assisted transcripts
  • ES cell-related transcription factor ERAs ES cell ess
  • Ras expressed in Erk Extracellular signal-regulated Kinase
  • ESG Embryonic stem cell-specific gene
  • Fbx15 F-Box Protein 15
  • FGF Fibroblast growth factor
  • FOXA2 forkhead box protein A2
  • Fthl17 Ferritin heavy chain polypeptide-like 17
  • GAPDH glycosyl transferase
  • Gdf3 Glyceraldehyde three-phosphate dehydrogenase
  • Grb2 Growth factor receptor binding protein 2
  • GSK Glycogen Synthase Kinase: Glycogen synthase kinase HBM (Hepatocyte Basel Medium): Stem cell basal medium HDAC (Histone Deacetylase): Histone deacetylase IGF (Insulin-like Growth Factor) IGF (Insulin-like Growth Factor) growh factor-binding protein 4): Insulin-like growth factor binding protein 4 IL (interleukin): Interleucine-rich KDR (kinase insert domain-connecting receptor): Kinase insertion domain-containing receptor Klf (Kruppel-like receptor): Kruppell-like factor LGR5 (Leucine-rich repeat-receptor) G protein-coupled receptor containing leucine-rich repeat 5
  • LIF Leukemia inhibitory factor
  • Leukemia inhibitory factor MAP mitogen-activated protein
  • Nr5a1 nuclear receptor subfamily
  • Nr5a2 nuclear receptor subfamily 5, group A, member 2
  • Oct octamer-binding transcription factor
  • PAX6 Paired box 6
  • PCR polymerase chain reaction
  • PDGFRA polymerase chain reaction
  • PDGFRA platelet-developed growth factor receptor alpha
  • Pillot-derived growth factor receptor ⁇ PKC Protein kinase C
  • POU5F1 POU domain, class 5, transcription factor 1
  • Prdm14 PR / SET domain family 14
  • qPCR Quantitative polymerase chain reaction
  • Rex1 Reduced-expression 1
  • Low expression protein-1 ROCK Rastero-associated coiled-coil forming kinase
  • Rho-conjugated coiled-coil-forming kinase RT-PCR Reverse transcription polymerase chain reaction
  • Sall4 Sal-like
  • SCF Stem cell factor
  • Sox SRY (sex determination region Y) -box: SRY (Y chromosome sex-determining gene) Box Stat3 (Signal Transducer and Activator of Tranciption 3): Signaling and transcriptional activator 3
  • T Brachyury: Brachyury Tcl1 encoded by the T gene (T-cell leukemia / lymphoma 1A): T-cell leukemia / lymphoma 1A Tert (Telomerase Reverse Transcriptase): Telomerase Reverse Transcript TGF (Transforming growth factor): Transforming Growth Factor UTF1 (Undifferentiated Embryonic Cell Transcription Factor) Undifferentiated Cell Transcription Factor VEGF (Vascular Endothelial Growth Factor): Vascular Endothelial Growth Factor
  • the method for producing a specific differentiated cell of the present invention uses pluripotent stem cells as a histone deacetylase inhibitor, a MAPK / ERK kinase inhibitor, a STAT3 activator, a protein kinase C inhibitor, a Wnt signal inhibitor and GSK3 ⁇ .
  • pluripotent stem cells By culturing in a medium containing any of the inhibitors selected from the group, pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of DNA was reduced as compared with the pluripotent stem cells before culturing.
  • the step (a) of obtaining, and the step (b) of obtaining specific differentiated cells by culturing the pluripotent stem cells obtained in step (a) in a medium containing cytokines are included.
  • cells that have undergone the same operation under the same culture conditions are regarded as cells having the same properties even if they are different as individuals.
  • the degree of DNA methylation is reduced by culturing pluripotent stem cells in the presence of a specific compound, and the pluripotent stem cells having a reduced degree of DNA methylation are used.
  • the pluripotent stem cells having a reduced degree of DNA methylation are used.
  • valproic acid improves the efficiency of inducing differentiation of human iPS cells into hepatocytes.
  • the step of differentiating hepatoblast-like cells into hepatocyte-like cells is valpro. It is done in the presence of acid.
  • the HDAC inhibitor for example, valproic acid
  • the efficiency of differentiation into endoderm which is in the process of differentiation into hepatocytes, does not increase, while when valproic acid is combined with other compounds, it does not increase. It has been confirmed that the differentiation efficiency increases.
  • the pluripotent stem cell of the present invention may be an undifferentiated cell obtained by introducing a reprogramming factor into a somatic cell.
  • the somatic cell is not particularly limited, and any somatic cell can be used.
  • adult-derived somatic cells that is, mature somatic cells
  • somatic cells include (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, and (3) fibroblasts (skin cells, etc.).
  • Epithelial cells Epithelial cells, hepatocytes, lymphocytes (T cells, B cells), endothelial cells, muscle cells, hair cells, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lungs
  • Examples thereof include differentiated cells such as cells, renal cells, and skin cells.
  • the living body from which the somatic cells are derived is not particularly limited, and examples thereof include humans and non-human animals (for example, monkeys, sheep, cows, horses, dogs, cats, rabbits, rats, mice). Preferably, it is human.
  • the pluripotent stem cells of the present invention are preferably human iPS cells.
  • the reprogramming factor introduced into somatic cells is not particularly limited, but for example, Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, Klf2, L-Myc, N-Myc, Klf5, Lin28, Tert, Fbx15, ERAs, ECAT15-1, ECAT15-2, Tcl1, ⁇ -catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, Fthl17, All4, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5 Cadherin can be mentioned.
  • two or more genes can be selected from these gene groups and introduced in any combination.
  • combinations having at least Oct3 / 4, Sox2, Klf4 and c-Myc, combinations having at least Oct3/4, Sox2, Klf4 and L-Myc, or combinations having at least Oct3 / 4, Sox2, Nanog and Lin28 preferable.
  • the species of the gene to be introduced is the same as the species of the cell to be introduced.
  • the gene introduced into a human-derived cell is preferably a human gene.
  • genes to be introduced into human-derived somatic cells include a combination having at least human OCT3 / 4, human SOX2, human KLF4 and human MYC, a combination having at least OCT3 / 4, SOX2, KLF4 and L-MYC, or A combination having at least human OCT3 / 4, human SOX2, human NANOG and human LIN28 is preferred.
  • the reprogramming factor gene can be introduced into somatic cells using a gene expression vector.
  • the gene expression vector is not particularly limited, and examples thereof include a viral vector, a plasmid vector, an artificial chromosome vector, and a transposon vector.
  • the viral vector include a retrovirus vector, an adenovirus vector, a Sendai virus vector, a lentiviral vector, and an adeno-associated virus vector.
  • Undifferentiated cells obtained by introducing a reprogramming factor into somatic cells may be produced by themselves by introducing a reprogramming factor into somatic cells, but cells provided or sold by research institutes or companies are obtained. You may.
  • -M1, CiRA188Ai-M1 or iRA188Ai-W1 can be obtained and used.
  • NIH National Institutes of Health
  • California Institute of Regenerative Medicine New York Stem Cell Foundation, European Bank Stem Cell, etc. can also be obtained from Stem Cell Foundation, European Bank, etc.
  • the pluripotent stem cell of the present invention may be an undifferentiated cell obtained by introducing a reprogramming factor into a somatic cell, or an undifferentiated cell in an "undifferentiated cell obtained by introducing a reprogramming factor into a somatic cell".
  • the cell means an undifferentiated cell, and is preferably a cell having an ability to differentiate into any one or more of endometrial follicle, meso-embryonic follicle and ectodermal lobe.
  • the above-mentioned "undifferentiated cells obtained by introducing a reprogramming factor into somatic cells” is prepared on a plate coated with feeder cells or a plate coated with a scaffold such as Matrigel (registered trademark) in an appropriate medium. It can be maintained and cultured.
  • the feeder cell is not particularly limited, and examples thereof include mouse embryonic fibroblast (MEF cell) and mouse embryonic fibroblast (STO cell).
  • a commercially available medium such as mTeSR® 1 (Stemcell Technologies) or StemFlex® can be used.
  • DMEM Dulvecco Modified Eagle medium
  • F12 F12
  • Knockout TM D-MEM Invitrogen
  • KSR Knockout TM Medium Replacement (Invitrogen)), bovine fetal serum (FBS), non-essential amino acids (NEAA), L-glutamine, 2-mercaptoethanol, antibiotics (eg, streptomycin, penicillin, puromycin, mitomycin),
  • FBS bovine fetal serum
  • NEAA non-essential amino acids
  • 2-mercaptoethanol antibiotics (eg, streptomycin, penicillin, puromycin, mitomycin)
  • a medium added to any of the above basal media by arbitrarily combining additional components such as bFGF (synonymous with FGF2) can also be mentioned as a medium for maintenance culture.
  • the medium for maintenance culture preferably does not contain ascorbic acid.
  • the culture conditions for the maintenance culture are preferably 37 ° C., 5% CO 2 , 10% O 2 and the like, but are not particularly limited.
  • the "specific differentiated cell” of the present invention means any specific differentiated cell belonging to any of the endoderm lineage, the mesodermal lineage and the ectoderm lineage, and preferably any specific differentiated cell belonging to the endoderm lineage.
  • Differentiated cells such as hepatocytes.
  • pluripotent stem cells are derived from histone deacetylase inhibitors, MAPK / ERK kinase inhibitors, STAT3 activators, protein kinase C inhibitors, Wnt signal inhibitors and GSK3 ⁇ inhibitors.
  • the proportion of 5-methylcytosine in the total cytosine of DNA was 10% or more, 15% or more, 20% or more, 25, as compared with the pluripotent stem cells before culturing. % Or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more , 90% or more, or 95% or more reduced cells.
  • the ratio of 5-methylcytosine to total cytosine of DNA is determined by converting genomic DNA extracted from pluripotent stem cells into EZ-DNA methylation TM Kit (ZYMO RESEACH) according to the procedure of Example 2 described later.
  • the methylation degree ( ⁇ value) may be calculated by converting to Bisulfite (bisulfite) and performing a methylation array analysis with Infinium (registered trademark) methylation EPIC BeadsChip Kit (illumina), but this method is limited.
  • genomic DNA extracted from the cells is treated with Bisulfite, non-methylcytosine is converted to uracil, and then the specific region is amplified by sequence analysis or PCR, and then methylation analysis by sequence analysis or restriction enzyme treatment. May be done.
  • the medium used in the step (a) is a basal medium, Neurobasal (registered trademark) (Thermo Fisher Scientific), B27 (registered trademark) (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific). And any combination of additives such as GlutaMAX® (Thermo Fisher Scientific) or L-Glutamin (Thermo Fisher Scientific) (preferably all of the above additives) in the basal medium.
  • any one selected from the group consisting of histone deacetylase inhibitor, MAPK / ERK kinase inhibitor, STAT3 activator, protein kinase C inhibitor, Wnt signal inhibitor and GSK3 ⁇ inhibitor is added. It may be a medium.
  • step (a) can include the first culture step and the second culture step described later.
  • the first culturing step in the present invention is a step of culturing pluripotent stem cells in a medium containing a histone deacetylase inhibitor, a MAPK / ERK kinase inhibitor, and a STAT3 activator.
  • the medium containing histone deacetylase inhibitor, MAPK / ERK kinase inhibitor, and STAT3 activator was added to the basal medium as Neurobasal (registered trademark) (Thermo Fisher Scientific), B27 (registered).
  • Thermo Fisher Scientific (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), 1-thioglycerol, and GlutaMAX® (Registered Trademarks) (Thermo Fisher Scientific) or L-Glutamin (ThermiScientific) or L-Glutamin (Thermic) (Preferably all of the above additives) were added to the above basal medium, and histone deacetylase inhibitors, MAPK / ERK kinase inhibitors, and STAT3 activators were added.
  • It may be a medium.
  • valproic acid or a salt thereof such as sodium valproate
  • butyric acid or a salt thereof such as sodium butyrate
  • tricostatin A apicidin and the like
  • concentration of the histone deacetylase inhibitor in the medium can be appropriately set according to the type of the histone deacetylase inhibitor and the like.
  • valproic acid it is preferably 1.0 ⁇ mol / L to 5.0 mmol / L, more preferably 0.2 mmol / L to 5.0 mmol / L, and even more preferably 0.5 mmol / L. It is ⁇ 2.0 mmol / L.
  • the MAPK / ERK kinase inhibitor (also referred to as MEK inhibitor) is not particularly limited, but for example, PD0325901 (N-[(2R) -2,3-dihydroxypropoxy] -3,4-difluoro-2-[().
  • the concentration of the MAPK / ERK kinase inhibitor in MAPK / ERK kinase inhibitor can be appropriately set according to the type of the MAPK / ERK kinase inhibitor and the like.
  • PD0325901 it is preferably 0.3 ⁇ mol / L to 5.0 ⁇ mol /. It is L, more preferably 0.6 ⁇ mol / L to 3.0 ⁇ mol / L, because the DNA is more demethylated by culturing in a medium containing PD0325901 at a concentration of 0.6 ⁇ mol / L or more.
  • the STAT3 activator is not particularly limited, but may include a leukemia inhibitory factor (LIF).
  • LIF leukemia inhibitory factor
  • human leukemia inhibitory factors are preferred.
  • the concentration of the STAT3 activator in the medium can be appropriately set according to the type of the STAT3 activator and the like. For example, in the case of LIF, it is preferably 0.1 ng / mL to 200 ng / mL, and more preferably 0.2 ng / mL to 100 ng / mL.
  • the culture conditions in the first culture step are obvious to those skilled in the art, and examples thereof include 37 ° C., 5% CO 2, and 5% O 2 conditions. In particular, it is preferable to culture under the condition of low oxygen (5% O 2 ).
  • the culture period in the first culture step is not particularly limited, but for example, it can be cultured for 1 to 5 days, preferably 2 to 4 days.
  • a MAPK / ERK kinase inhibitor In the second culture step in the present invention, after the first culture step, a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, a GSK3 ⁇ inhibitor, without containing a histone deacetylase inhibitor, And a step of culturing in a medium containing a STAT3 activator.
  • the medium containing the MAPK / ERK kinase inhibitor, protein kinase C inhibitor, Wnt signal inhibitor, GSK3 ⁇ inhibitor, and STAT3 activator without the histone deacetylase inhibitor Neurobasal (registered trademark) (Thermo Fisher Scientific), B27 (registered trademark) (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), 1-thioglycerol, 1-thioglycerol, and 1-thioglycerol in the basal medium.
  • Additives such as Scientific) or L-Glutamin (Thermo Fisher Scientific) are optionally combined (preferably all of the above additives) and added to the basal medium described above, and further, MAPK / ERK kinase.
  • the medium may be supplemented with an inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, a GSK3 ⁇ inhibitor, and a STAT3 activator.
  • the concentration of the MAPK / ERK kinase inhibitor in the medium can be appropriately set according to the type of the MAPK / ERK kinase inhibitor and the like.
  • PD0325901 it is preferably 0.3 ⁇ mol / L to 5.0 ⁇ mol / L, and more preferably 0.6 ⁇ mol / L to 3.0 ⁇ mol / L. This is because DNA is more demethylated by culturing in a medium containing PD0325901 at a concentration of 0.6 ⁇ mol / L or more.
  • the protein kinase C inhibitor (also referred to as PKC inhibitor) is not particularly limited, but is, for example, Go6983 (3- [1- [3- (dimethylamino) propyl] -5-methoxy-1H-indole-3-yl).
  • Go6983 is preferable.
  • the concentration of the PKC inhibitor in the medium can be appropriately set according to the type of the PKC inhibitor and the like. For example, in the case of Go6983, it is preferably 50 nmol / L to 100 ⁇ mol / L, and more preferably 100 nmol / L to 10 ⁇ mol / L.
  • the Wnt signal inhibitor is not particularly limited, but is limited to XAV939 (proteinase inhibitor) (CAS registration number: 284028-89-3), IWP-1, IWP-2, IWP-3, IWP-4, IWR-1, Examples thereof include low molecular weight compounds such as 53AH (above porcupine inhibitor) and KY02111 and their derivatives, and proteins such as IGFBP4, DKK1 and Wnt-C59. Of these, XAV939 is preferable.
  • the concentration of the Wnt signal inhibitor in the medium can be appropriately set according to the type of the Wnt signal inhibitor and the like. For example, in the case of XAV939, it is preferably 50 nmol / L to 100 ⁇ mol / L, and more preferably 100 nmol / L to 10 ⁇ mol / L.
  • the GSK3- ⁇ inhibitor is not particularly limited, but CHIR99021 (CAS Registry Number: 252927-06-9) is preferable.
  • the concentration of the GSK3- ⁇ inhibitor in the medium can be appropriately set according to the type of the GSK3- ⁇ inhibitor and the like.
  • CHIR99021 it is preferably 0.01 ⁇ mol / L to 1.0 ⁇ mol / L, and more preferably 0.1 ⁇ mol / L to 0.5 ⁇ mol / L.
  • the concentration of the STAT3 activator in the medium can be appropriately set according to the type of the STAT3 activator and the like.
  • LIF it is preferably 0.1 ng / mL to 100 ng / mL, and more preferably 0.2 ng / mL to 20 ng / mL.
  • the culture conditions in the second culture step are self-evident to those skilled in the art, and examples thereof include 37 ° C., 5% CO 2 , and 5% O 2 conditions. In particular, it is preferable to culture under the condition of low oxygen (5% O 2 ).
  • the medium preferably does not contain ascorbic acid.
  • the second culture step can be further divided into two culture steps.
  • the two culture steps can each use different media with or without the addition of a GSK3 ⁇ inhibitor. That is, in the second culture step, after culturing in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, and a STAT3 activator without containing a histone deacetylase inhibitor.
  • the step may be a step of further culturing in a medium in which a GSK3 ⁇ inhibitor is added to this medium.
  • the culture period in the second culture step is not particularly limited, but includes a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, and a STAT3 activator without containing a histone deacetylase inhibitor.
  • a medium for, for example, 3 to 8 days, preferably 4 to 7 days the medium can be cultured in a medium containing a GSK3 ⁇ inhibitor, for example, for 3 to 10 days, preferably 4 to 9 days. ..
  • step (a) including the first and second culture steps on the pluripotent stem cells By performing step (a) including the first and second culture steps on the pluripotent stem cells, "the ratio of 5-methylcytosine in the total cytosine of the DNA as compared with the pluripotent stem cells before culture". It is possible to obtain “pluripotent stem cells with reduced”. In one embodiment of the present invention, "pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of DNA is reduced as compared with the pluripotent stem cells before culture” is specified as compared with the cells before culture. It can be said that it is a pluripotent stem cell having a high ability to differentiate into a differentiated cell.
  • the undifferentiated state was It is maintained and has an enhanced ability to differentiate into specific differentiated cells as compared to undifferentiated cells obtained by introducing a reprogramming factor into somatic cells.
  • Completion of step (a) can be evaluated by measuring the degree of methylation and reducing the degree of DNA methylation.
  • the pluripotent stem cells obtained in step (a) have a low incidence of DNA mutations.
  • the low incidence of DNA mutations can be evaluated, for example, by measuring the copy number of the long arm region of chromosome 20 by a conventional method (for example, real-time PCR).
  • the copy number of the long arm region of chromosome 20 in the pluripotent stem cell obtained in the step (a) is preferably 1.5 to 4.5, more preferably 1.5 to 3.5, and even more preferably. Is 1.5 to 2.5.
  • the undifferentiated state of the pluripotent stem cell obtained in the step (a) is not particularly limited, but can be evaluated by measuring the expression of the gene that defines the undifferentiated state.
  • the method for measuring the expression of the gene that defines undifferentiated state is not particularly limited, but for example, it can be measured by quantitative RT-PCR.
  • RT-PCR is a method of synthesizing cDNA using the mRNA to be measured as a template and amplifying it by PCR using this cDNA as a template.
  • quantitative RT-PCR for example, PCR is performed using a primer in which a quencher fluorescent dye and a reporter fluorescent dye are bound to quantify the amount of amplification product in each cycle, and the detected fluorescence intensity increases sharply.
  • a method of measuring the amount of template DNA in a sample can be mentioned.
  • Quantitative RT-PCR techniques are well known in the art and can also be performed using commercially available kits.
  • the expression level or the number of copies of a gene can be measured as a relative value to the expression level or the number of copies of a control housekeeping gene (for example, GAPDH gene).
  • the mRNA of a gene can also be measured by subjecting the amplification product obtained by amplifying the mRNA by ordinary RT-PCR or the like to gel electrophoresis, staining, and then measuring the band intensity.
  • DNA chips can be used to detect or quantify gene mRNA or cDNA.
  • the gene that defines undifferentiated state is not particularly limited, and examples thereof include NANOG, POU5F1, LIN28, SOX2, and DNMT3B.
  • the expression level of the gene defining undifferentiation in the pluripotent stem cells obtained in the step (a) of the present invention is the expression of the same gene in the control group (for example, the pluripotent stem cells not subjected to the step (a)). It is defined that undifferentiated state is maintained when the amount is equal to or higher than the amount, and the equivalent is 80 to 110% of the expression level of the same gene in the control group.
  • the expression level of NANOG in the pluripotent stem cells obtained in the step (a) of the present invention is equal to or higher than the expression level of NANOG in the control group.
  • the expression level of POU5F1 in the pluripotent stem cells obtained in the step (a) of the present invention is equal to or higher than the expression level of POU5F1 in the control group.
  • the expression level of LIN28 in the pluripotent stem cells obtained in the step (a) of the present invention is equal to or higher than the expression level of LIN28 in the control group.
  • the expression level of SOX2 in the pluripotent stem cells obtained in the step (a) of the present invention is equal to or higher than the expression level of SOX2 in the control group.
  • the expression level of DNMT3B in the pluripotent stem cells obtained in the step (a) of the present invention is equal to or higher than the expression level of DNMT3B in the control group.
  • the number of expressed copies of NANOG in the pluripotent stem cell obtained in step (a) is 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more as a ratio to the number of expressed copies of GAPDH. , Or may be 0.05 or more.
  • the number of expressed copies of POU5F1 in the pluripotent stem cells obtained in step (a) is 0.06 or more, 0.07 or more, 0.08, 0.09 or more as a ratio to the number of expressed copies of GAPDH. It may be 0.1 or more, or 0.12 or more.
  • the number of expressed copies of LIN28 in the pluripotent stem cell obtained in step (a) is 0.003 or more, 0.004 or more, 0.005 or more, 0.006 or more as a ratio to the number of expressed copies of GAPDH. , 0.007 or more, or 0.008 or more.
  • the number of expressed copies of SOX2 in the pluripotent stem cells obtained in step (a) is preferably 5.0 ⁇ 10-5 or more, more preferably 6.0 ⁇ 10 ⁇ as a ratio to the number of expressed copies of GAPDH. It is 5 or more, more preferably 7.0 ⁇ 10-5 or more, and particularly preferably 8.0 ⁇ 10-5 or more.
  • pluripotent stem cells obtained in step (a) to differentiate into any one or more of endoderm, mesoderm and ectoderm is possessed by pluripotent stem cells before culture, endoderm, mesoderm and It is improved over the ability to differentiate into any one or more of the ectoderm. More preferably, the ability of the pluripotent stem cell obtained in step (a) to differentiate into endoderm and ectoderm is higher than the ability of the pluripotent stem cell before culture to differentiate into endoderm and ectoderm, respectively.
  • the ability of pluripotent stem cells obtained in step (a) to differentiate into endoderm and mesoderm is greater than the ability of precultured pluripotent stem cells to differentiate into endoderm and mesoderm, respectively. It is improving.
  • the ability of the pluripotent stem cell obtained in step (a) to differentiate into an endoderm is higher than the ability of the pluripotent stem cell before culturing to differentiate into an endoderm.
  • the pluripotent stem cell obtained in step (a) is a cell capable of differentiating into all endoderm, mesoderm and ectoderm.
  • the specific differentiated cell in the present invention is preferably a cell of endoderm lineage, and more preferably a hepatocyte.
  • the ability of a cell to differentiate into an endoderm, the ability of a cell to differentiate into a mesodermal, and the ability of a cell to differentiate into an ectoderm can be used to differentiate a cell into an endoderm, a mesodermal or an ectoderm. It can be evaluated by measuring the expression of each germ layer-specific gene in the cells differentiated into the above three germ layer lineages.
  • the method for measuring the expression of each germ layer-specific gene is not particularly limited, but for example, the measurement can be performed by a quantitative RT-PCR method.
  • the endoderm-specific gene is not particularly limited, and examples thereof include SOX17 and FOXA2.
  • the mesoderm-specific gene is not particularly limited, and examples thereof include T and PDGFRA.
  • the ectoderm-specific gene is not particularly limited, and examples thereof include PAX6 and MAP2.
  • the relative expression level of SOX17 in the cells obtained in step (a) differentiated into endoderm is preferably twice as much as the expression level of SOX17 in the cells obtained by differentiating the pluripotent stem cells into endoderm. More preferably, it may be 3 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 25 times or more, 30 times or more, 35 times or more or 40 times or more. ..
  • the relative expression level of FOXA2 in the cells obtained by differentiating the pluripotent stem cells into the endoderm is preferably doubled with respect to the expression level of FOXA2 in the cells obtained by differentiating the pluripotent stem cells into the endoderm.
  • the above more preferably 3 times or more, 5 times or more, 8 times or more, 9 times or more, 10 times or more, or 12 times or more can be mentioned.
  • the relative expression level of T in the cells obtained in the step (a) differentiated into mesoderm is preferably 2 or more with respect to the expression level of T in the cells obtained by differentiating the pluripotent stem cells into mesoderm. It is more preferable that the number is 3 or more, 5 or more, 8 or more, 9 or more, or 10 or more.
  • the relative expression level of PDGFRA in the cells obtained by differentiating the pluripotent stem cells into mesoderm with respect to the expression level of PDGFRA in the cells obtained by differentiating the pluripotent stem cells into mesoderm is preferably 1. The case where it is 1 or more, more preferably 1.2 or more, 1.3 or more, or 1.4 or more can be mentioned.
  • the relative expression level of PAX6 in the ectoderm-differentiated cells of the pluripotent stem cells obtained in step (a) is preferably 1.1 or more with respect to the expression level of PAX6 in the cells differentiated into pluripotent stem cells. Yes, more preferably 1.2 or more, 1.3 or more, 1.4 or more, or 1.5 or more.
  • the relative expression level of MAP2 in the cells obtained by differentiating the pluripotent stem cells into the ectoderm with respect to the expression level of MAP2 in the cells obtained by differentiating the pluripotent stem cells into the ectoderm is preferably 1. The case where it is 1 or more, more preferably 1.2 or more, or 1.3 or more can be mentioned.
  • the step (a) of the present invention may include a step of culturing in a medium for maintaining and culturing undifferentiated cells containing bFGF after the second culturing step.
  • This step is referred to as a third culture step in the present specification.
  • the third culture step is not an essential step in the method for producing differentiated cells of the present invention. Because "pluripotent stem cells in which the ratio of 5-methylcytosine in total cytosine of DNA is reduced as compared with pluripotent stem cells before culturing" can be obtained by carrying out the above-mentioned first and second culturing steps. Is.
  • the "pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of DNA was reduced as compared with the pluripotent stem cells before culturing" obtained by carrying out the above first and second culturing steps were obtained before culturing. Due to the slower growth rate compared to pluripotent stem cells, it may take time and cost to obtain the same yield.
  • a medium for maintaining and culturing undifferentiated cells a commercially available medium such as mTeSR (registered trademark) 1 (Stemcell Technologies) or StemFlex (registered trademark) can be used.
  • mTeSR Registered trademark
  • StemFlex registered trademark
  • DMEM Dulvecco Modified Eagle medium
  • DMEM / F12 1: 1
  • Knockout TM D-MEM Invitrogen
  • the medium for maintenance culture preferably does not contain ascorbic acid.
  • the culture conditions for the maintenance culture are preferably 37 ° C., 5% CO 2 , 10% O 2 and the like, but are not particularly limited.
  • the culture period in the medium for maintaining and culturing undifferentiated cells is not particularly limited, but can be cultured, for example, for 1 to 3 weeks, preferably 1 to 2 weeks. During the culture period of 1 to 3 weeks or 1 to 2 weeks, the proliferative ability can be enhanced while maintaining the ability to differentiate into specific differentiated cells. Subculture can be performed during this culture period. This is to maintain the cell density in a state suitable for maintaining undifferentiated state by performing subculture.
  • the step (b) of the present invention includes a step (b) of obtaining specific differentiated cells by culturing the pluripotent stem cells obtained in the step (a) in a medium containing a cytokine.
  • the type of differentiated cells obtained in step (b) is not particularly limited. If desired, differentiation can be induced into endoderm lineage cells, mesoderm lineage cells, or ectoderm lineage cells, and preferably into endoderm lineage cells, such as hepatocytes.
  • the culture conditions for inducing differentiation into various cells
  • conditions generally adopted in the culture of animal cells can be adopted. That is, it can be cultured in an environment of, for example, 37 ° C. and 5% CO 2 .
  • basal medium Iskov-modified Dulbecco medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, Gibco, etc.), Dulbecco-modified Eagle's medium (D-MEM) (Nacalai Tesque, Inc., Sigma). , Gibco, etc.), Grasgo basic medium (Gibco, etc.), RPMI1640 medium, and the like can be used.
  • IMDM Iskov-modified Dulbecco medium
  • HamF12 Ham F12 medium
  • D-MEM Dulbecco-modified Eagle's medium
  • Gibco, etc. Gibco, etc.
  • Grasgo basic medium Gibco, etc.
  • RPMI1640 medium and the like
  • basal media Two or more kinds of basal media may be used in combination.
  • components that can be added to the medium include bovine serum albumin (BSA), antibiotics, 2-mercaptoethanol, PVA, non-essential amino acids (NEAA), insulin, transferrin, and selenium.
  • BSA bovine serum albumin
  • antibiotics antibiotics
  • 2-mercaptoethanol 2-mercaptoethanol
  • PVA non-essential amino acids
  • insulin transferrin
  • selenium selenium
  • the medium of step (b) can contain cytokines in the basal medium.
  • the type of cytokine is not particularly limited, and examples thereof include activin A, BMP4, bFGF, HGF, Oncostatin M, VEGF, IL-11, IL-6, IGF-1, SCF, TPO, and TGF- ⁇ .
  • activin A, BMP4, bFGF, HGF, and Oncostatin M can be included.
  • the culture period of step (b) may be 1 to 4 weeks, preferably 1 to 3 weeks.
  • cells are two-dimensionally cultured using a culture dish or the like. However, three-dimensional culture may be carried out using a gel-like culture substrate, a three-dimensional culture plate, or the like.
  • the method of inducing differentiation is not particularly limited.
  • a commercially available StemDiff (registered trademark) Trillineage Differentiation Kit (Stemcell Technologies) can be used to induce differentiation into endoderm, mesoderm and ectoderm, respectively.
  • the induction of differentiation into hepatocytes can be performed by culturing the cells under the conditions described in Example 4 described later. Specifically, cells are seeded in mTeSR® 1 (Stemcell Technologies) or StemFlex® and cultured in a medium containing B27® and Activin A on the 1st to 5th days. , 6th to 10th days were cultured in a medium containing B27®, BMP4, and bFGF, and 11th to 15th days were cultured in a medium containing B27® and HGF. From the 16th day to the 20th day, differentiation can be induced into hepatocytes by culturing in HBM (registered trademark) containing Oncostatin M.
  • HBM registered trademark
  • the induction of differentiation into hepatocytes can be confirmed by analyzing the expression of ASGPR1, which is a marker of mature hepatocytes, with a flow cytometer. Induction of differentiation into hepatocytes can also be confirmed by measuring the amount of albumin secreted in the culture supernatant.
  • ASGPR1 Assay for the expression of ASGPR1, specifically, according to the procedure of Example 4 described later, the cells 20 days after the induction of differentiation were subjected to BD Pharmingen TM PE Mouse Anti-ASGPR1 antibody (Becton Dickinson Japan, Inc.). The cells may be stained and the positive rate of ASGPR1 may be analyzed by flow cytometry Attune TM NxT (Thermo Fisher Scientific). The positive rate of ASGPR1 analyzed by the above method is preferably 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, and more preferably 10% or more, 11% or more, 12% or more.
  • the positive rate of ASGPR1 is preferably increased 1.1 to 100 times, and 1.2 to 100 times, as compared with the case where the step (a) is not performed. Is more preferable, and it is most preferable that the increase is 2 to 50 times.
  • the culture supernatant 20 days after the induction of differentiation was collected, and the amount of albumin secreted in the culture supernatant was measured by ELISA Starter Accessory. It may be quantified by an EnSpire TM plate reader (PerkinElmer) using a Kit, Enzyme Substrate, TMB, Human Albumin ELISA Quantation Set (Bethyl Laboratories, Inc).
  • the amount of albumin secreted analyzed by the above method is 30 ng / mL or more, 40 ng / mL or more, 50 ng / mL or more, 60 ng / mL or more, 70 ng / mL or more, 80 ng / mL or more, 90 ng / mL or more, 100 ng / mL or more.
  • 110 ng / mL or more 120 ng / mL or more, 130 ng / mL or more, 140 ng / mL or more, 150 ng / mL or more, 160 ng / mL or more, 170 ng / mL or more, 180 ng / mL or more, 190 ng / mL or more, 200 ng / mL or more , 250 ng / mL or more, 300 ng / mL or more, 400 ng / mL or more, or 500 ng / mL or more.
  • the amount of albumin secreted is preferably increased 1.1 to 100 times, and 1.2 to 100 times, as compared with the case where the step (a) is not performed. Is more preferable, and the increase is more preferably 2 to 50 times, and particularly preferably 2 to 10 times.
  • the induction of differentiation into blood cells can be performed by culturing the cells under the conditions described in Example 2 of the international patent application PCT / JP2019 / 003336. Specifically, on the first day, the cells were cultured in a medium containing BMP4 and Y27634 (ROCK inhibitor), bFGF and BMP4 were added on the second day, and the cells formed spheroid-like colonies on the third day. After confirming that, the cells were cultured in a medium containing SB431542 (TGF- ⁇ receptor inhibitor), CHIR99021 (GSK3 inhibitor), bFGF and BMP4 (3rd and 4th days), 5th to 6th days.
  • Eyes are cultured in a medium containing VEGF and bFGF, and on days 7 to 10, they are cultured in a medium containing VEGF, bFGF, IL-6, IGF-1, IL-11 and SCF to give blood cells. Differentiation can be induced. The induction of differentiation into blood cells can be confirmed by analyzing the expression of blood cell markers CD34 and KDR with a flow cytometer.
  • Induction of differentiation into cardiomyocytes can be performed, for example, by culturing the cells under the conditions described in Example 5 of the international patent application PCT / JP2019 / 003336. Specifically, cells can be induced to differentiate into cardiomyocytes using a PSC Cardiomyote Difference Kit (Thermo Fisher Scientific) according to a procedure manual. Induction of differentiation into cardiomyocytes can be confirmed by measuring the expression of Cardiac Troponin T (cTnT), which is a cardiomyocyte marker, by flow cytometry.
  • cTnT Cardiac Troponin T
  • Example 5 of the international patent application PCT / JP2019 / 033366 the cells 14 days after the induction of differentiation were stained with Alexa Fluor647 Mouse Anti-Cardiac Troponin T antibody (BD Harmingen) and flowed.
  • the positive rate of cTnT may be analyzed by cytometry Attune NxT (Thermo Fisher Scientific).
  • the positive rate of cTnT analyzed by the above method is preferably 5% or more, more preferably 10% or more, further preferably 20% or more, still more preferably 30% or more, and particularly preferably. It is 40% or more, and most preferably 45% or more.
  • the positive rate of cTnT is preferably increased 1.1 to 100 times, and 1.2 to 100 times, as compared with the case where the step (a) is not performed. Is more preferable, and it is most preferable that the increase is 2 to 50 times.
  • Induction of differentiation into neural stem cells can be performed, for example, by culturing the cells under the conditions described in Example 6 of the international patent application PCT / JP2019 / 003336. Specifically, the cells can be induced to differentiate into neural stem cells using a PSC Natural Indication Medium (Thermo Fisher Scientific) according to a procedure manual. Induction of differentiation into neural stem cells can be confirmed, for example, by immunostaining the SOX1 protein, which is a marker of neural stem cells. When the SOX1 protein is immunostained to quantify the brightness, the brightness can be increased by 1.1 to 10 times by performing the step (a) of the present invention as compared with the case where the step (a) is not performed. It is preferable, and it is more preferable that the increase is 1.5 to 5 times.
  • the pluripotent stem cells obtained in step (a) can be differentiated into endoderm cells by culturing under endoderm cell differentiation conditions.
  • the endoblast lineage cells are not particularly limited, but for example, digestive system cells (hepatocytes, hepatic sinus endothelial cells, cupper cells, hepatic stellate cells, pit cells, bile duct cells, mesenteric cells, pancreatic endocrine cells, glands). Tuft cells, conduit cells, resorbing cells, cup cells, panate cells, intestinal endocrine cells, etc.), cells of tissues such as lung and thyroid.
  • the pluripotent stem cells obtained in step (a) can be differentiated into mesoderm cells by culturing under conditions other than the above for mesoderm cell differentiation conditions.
  • the mesophyll lineage cells are not particularly limited, but are blood cell / lymphocyte lineage cells (hemogenic stem cells, erythrocytes, platelets, macrophages, granulocytes, helper T cells, killer T cells, B lymphocytes, etc.), vasculature cells (vascular lineage cells, etc.) (Vascular endothelial cells, etc.), myocardial cells (for example, atrial muscle cells, ventricular muscle cells, etc.), osteoblasts, bone cells, cartilage cells, tendon cells, fat cells, skeletal muscle cells, smooth muscle cells, and the like.
  • blood cell / lymphocyte lineage cells hemogenic stem cells, erythrocytes, platelets, macrophages, granulocytes, helper T cells, killer T cells, B lymphocytes, etc.
  • the pluripotent stem cells obtained in step (a) can be differentiated into ectoderm cells by culturing under ectoderm cell differentiation conditions other than the above.
  • the ectoderm cells include, but are not limited to, nervous system cells, sensory organ cells (lens, retina, inner ear, etc.), skin epidermal cells, hair follicles, and the like.
  • the cells induced to differentiate using the pluripotent stem cells obtained in step (a) can be used for screening drug candidate compounds for the treatment of various diseases. For example, by adding a drug candidate compound alone or in combination with other drugs to cells in which differentiation has been induced, by detecting changes in cell morphology or function, increase / decrease in various factors, gene expression profiling, etc. , Can be evaluated.
  • the cells are preferably cells having the same phenotype as the disease to be treated, and more preferably, differentiation induction from cells produced by the method of the present invention using somatic cells derived from a patient suffering from the disease. It is a cell.
  • pluripotent stem cells that is, performing the above step (a) on undifferentiated cells obtained by introducing a reprogramming factor into somatic cells
  • pluripotent stem cells that is, performing the above step (a) on undifferentiated cells obtained by introducing a reprogramming factor into somatic cells
  • the compound treatment according to the present invention is applied to pluripotent hepatocytes (that is, the above step (a) is performed on undifferentiated cells obtained by introducing a reprogramming factor into somatic cells. ),
  • the treated cells had an increased efficiency of inducing differentiation into hepatocytes in all seven pluripotent stem cell lines analyzed, as compared with the untreated cells. From the above, it has become possible to reduce the labor and cost for selecting excellent strains for differentiation.
  • the efficiency of inducing differentiation of iPS cells into liver cells can be measured by the positive rate of the hepatocyte marker ASGPR1.
  • a tissue can be prepared from the differentiated cells produced by the production method of the present invention and used in the field of regenerative medicine.
  • the prepared iPS cell-derived liver cells can be applied to the treatment of various liver diseases.
  • it is expected to be used as a material for regeneration / reconstruction of impaired (including dysfunctional) liver tissue, and is expected to contribute to regenerative medicine.
  • iPS cell-derived hepatocytes prepared by the method for inducing differentiation of the present invention.
  • iPS cell-derived hepatocytes can be used to test the metabolism of the test substance. That is, the present invention provides a method for evaluating the metabolism of a test substance.
  • Organic compounds or inorganic compounds of various molecular sizes can be used as the test substance.
  • Existing or candidate ingredients such as pharmaceuticals and nutritional foods are also preferred test substances.
  • a plant extract, a cell extract, a culture supernatant, or the like may be used as a test substance. Moreover, you may use these two or more types in combination.
  • Example 1 The following experiments were conducted to improve and improve the ability of human iPS cells to differentiate into specific cells.
  • Method 1 For human iPS cell lines, strains A to G were sold by FUJIFILM Cellular Dynamics International (FCDI).
  • FCDI FUJIFILM Cellular Dynamics International
  • ⁇ Cell culture and compound treatment> Human iPS cells are placed on a 6-well plate coated with Matrigel® (Corning) in StemFlex® (Thermo Fisher Scientific) medium at 37 ° C., 5% CO 2 , 10% O 2 It was maintained and cultured under the conditions.
  • Day 1 Human iPS cells in culture were exfoliated by treatment with TrypLE TM Select (Invitrogen) at 37 ° C. for 5 minutes to form single cells.
  • the human iPS cells were seeded with mouse embryo-derived fibroblasts (MEF, Lonza) at 0.5x10 6 cells / well ( 6 well plate) or 1x10 5 cells / well seeded in Matrigel-coated wells, as shown in Table 1. It was cultured in medium 1. After that, the cells were cultured under 37 ° C., 5% CO 2 , and 5% O 2 conditions until the 14th day. Days 2-3: Half the amount was replaced with fresh medium 1.
  • Days 4 to 8 The medium was replaced with the medium 2 in Table 1, and the medium was replaced with the same medium every other day until the 8th day.
  • 9th to 14th days Treated with TripLE TM Select at 37 ° C. for 5 minutes to detach cells, and MEF was seeded or Matrigel was seeded in medium 3 to which CHIR99021 (0.3 ⁇ mol / L, Wako) was added to medium 2. (Registered trademark) Subculture was carried out about twice on coated plates over a week. Subsequently, the cells were maintained and cultured in medium 3 under the conditions of 37 ° C., 5% CO 2 , and 5% O 2 .
  • the cells cultured in the above medium 3 were subcultured on a plate coated with Matrigel, and subcultured in StemFlex or mTeSR1 medium under the conditions of 37 ° C., 5% CO 2 , and 10% O 2 for about 2 weeks. It was.
  • human iPS cells cultured in the media 1 to 3 of Table 1 are referred to as “treated” iPS cells, and are StemFlex on a 6-well plate coated with Matrigel (registered trademark) (Corning). (Registered Trademark) (Thermo Fisher Scientific) Cells that have been maintained and cultured in medium but not cultured in media 1 to 3 in Table 1 are referred to as "untreated” iPS cells.
  • Example 2 ⁇ Changes in DNA methylation>
  • the cells were reacted with TripLE TM Select (Gibco) at 37 ° C. for 5 minutes, detached from the plate, and antibody-Feeder Cells-PE, mouse monoclonal ( After staining with Myltenyi) antibody, the MEF negative fraction, which is an iPS fraction, was fractionated with a cell sorter (FACSAriaIII), and DNA was extracted with PureLink (registered trademark) Genome DNA Mini Kit (Thermo Fisher Scientific). ..
  • This DNA was Bisulfite-converted with EZ-DNA Methylation TM Kit (ZYMO RESEACH), and methylation array analysis was performed with Infinium (registered trademark) Methylation EPIC BeadsChip Kit (illumina).
  • Infinium registered trademark
  • Methylation EPIC BeadsChip Kit Infinium (registered trademark) Methylation EPIC BeadsChip Kit (illumina).
  • FIG. 1 shows a violin plot of the degree of methylation, and the width of the graph shows the distribution of regions at each degree of methylation.
  • Example 3 ⁇ Confirmation of undifferentiated state>
  • the cells were reacted with TrypLE TM Select (Gibco) at 37 ° C. for 5 minutes, detached from the plate, and RNAy®.
  • RNA was extracted from cells with a plus Mini Kit (Qiagen).
  • RNA as a template for cDNA synthesized with High Capacity RNA-to- cDNA Kit (Thermo Fisher Scientific) as a template, TaqMan (registered trademark) Gene Expression assembly (Thermo Primer Scientific) by PCR (Thermo Primer Scientific).
  • code name (hereinafter referred to as code name): Hs02387400_g1), POU5F1 (code name: Hs04260367_gH), and LIN28 (code name: Hs00702808_s1) was analyzed.
  • the expression level was corrected with GAPDH (code name: Hs0276624_g1), which is an internal standard gene.
  • Example 4 ⁇ Differentiation into hepatocytes> To examine the ability of A to G strains to differentiate into hepatocytes, untreated or treated iPS cells were reseeded on Matrigel-coated 24-well plates, and the medium in Table 2 was placed in 0.5 mL / well. Maintenance culture was carried out for 5 days each to differentiate into hepatocytes.
  • the media used were mTeSR (registered trademark) 1 (Stemcell Technologies), RPMI1640 medium, and HBM (registered trademark).
  • the compounds used were B27 (registered trademark) (Thermo Fisher Scientific), Activin A (R & D System), bFGF (Fujifilm Wako Pure Chemical Industries), BMP4 (R & D System), HGF (Peprotech). It is M (Peprotech).
  • ⁇ Evaluation method> The efficiency of inducing differentiation into hepatocytes was evaluated by flow cytometry of the expression of ASGPR1, which is a mature hepatocyte marker, in cells 20 days after induction. Twenty days after the induction of differentiation, the cells were treated with TrypLE TM Select (Thermo Fisher Scientific) at 37 ° C. for 5 minutes to detach the cells. Dead cells were stained with ghost Dye TM Violet 510 (TONBO Bioscience). After washing, the cells were stained with BD Harmingen TM PE Mouse Anti-ASGPR1 antibody (Nippon Becton Dickinson Co., Ltd.), and the positive rate of ASGPR1 was analyzed by flow cytometry Attune TM NxT (Thermo Fisher Scientific).
  • the culture supernatant on the 20th day after the induction was collected, and the amount of albumin secreted in the culture supernatant was measured using ELISA Starter Accessory Kit, Enzyme Substrate, TMB, Human Albumin ELISA Quantation Set (Bethyl Laboratories). Quantified by EnSpire TM plate reader (PerkinElmer).
  • the untreated and treated iPS cells were induced to differentiate into hepatocytes, respectively, and the ASGPR1 positive rates in the living cells were compared by flow cytometry.
  • the positive rate of ASGPR1 indicates the efficiency of inducing differentiation of iPS cells into hepatocytes.
  • the ASGPR1 positive rate (%) was 2.90 times that of the A-share when the treated iPS cells were induced to differentiate into hepatocytes, as compared with the case where the untreated iPS cells were induced to differentiate into hepatocytes.
  • the amount of albumin secreted was significantly increased when the treated iPS cells were induced to differentiate, as compared with the case where the untreated iPS cells were induced to differentiate into hepatocytes in all 7 strains (Fig. 4). ).
  • Example 5 ⁇ Comparison of HDAC inhibitor monotherapy and the present invention in endoderm differentiation of iPS cells> The following experiments were conducted to investigate the effect of HDAC inhibitor monotherapy on endoderm differentiation of iPS cells.
  • Sodium valproate was used as the HDAC inhibitor.
  • the iPS cell the D strain was used.
  • Cells in which iPS cells are cultured in StemFlex® on MEF-seeded plates are referred to as "untreated / undifferentiated”.
  • untreated / undifferentiated cells are (1) as they are (referred to as “untreated / endoderm differentiation"), and (2) a medium in which 1 mM of sodium valproate is added to N2B27 medium (referred to as “VPA / endoderm differentiation”).
  • VPA / endoderm differentiation a medium in which 1 mM of sodium valproate is added to N2B27 medium
  • Mix / endoderm differentiation was differentiated into endoderm using the Endoderm medium of the StemDiff Trilineage Differentiation Kit (Stemcell Technologies). These cells were reacted with TripLE Select (Gibco) at 37 ° C.
  • RNeasy registered trademark
  • Mini Kit Qiagen
  • RNA was extracted from the cells with RNeasy (registered trademark) plus Mini Kit (Qiagen).
  • RNA was extracted from the cells with RNeasy (registered trademark) plus Mini Kit (Qiagen).
  • RNeasy registered trademark
  • Mini Kit Qiagen
  • RNA RNA as a template for cDNA synthesized with High Capacity RNA-to- cDNA Kit (Thermo Fisher Scientific) as a template
  • TaqMan Gene Expression assembly Thermo Fisher Scientific
  • Thermo Fisher Scientific Thermo Fisher Scientific
  • SO2 Thermo Fisher Scientific
  • the gene expression of the code name: Hs05036278_s1 was analyzed.
  • the expression level was corrected with GAPDH (code name: Hs0276624_g1), which is an internal standard gene.

Abstract

The present invention addresses the problem of providing a method for producing differentiated cells whereby target specific differentiated cells can be efficiently obtained. According to the present invention, provided is a method for producing specific differentiated cells, said method comprising: step (a) for culturing pluripotent stem cells in a medium, which contains any component selected from the group consisting of a histone deacetylase inhibitor, a MAPK/ERK kinase inhibitor, a STAT3 activator, a protein kinase C inhibitor, a Wnt signal inhibitor and a GSK3β inhibitor, to thereby give pluripotent stem cells in which the ratio of 5-methylcytosine to the total cytosines in DNA is lowered compared to the pluripotent stem cells before culturing; and step (b) for culturing the pluripotent stem cells obtained in step (a) in a cytokine-containing medium to give the specific differentiated cells.

Description

特定の分化細胞の製造方法Method for producing specific differentiated cells
　本発明は、多能性幹細胞を、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、ＳＴＡＴ３活性化剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤およびＧＳＫ３β阻害剤からなる群から選択される何れかを含む培地で培養することにより、培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した多能性幹細胞を得る工程（ａ）、および工程（ａ）で得られる多能性幹細胞を、サイトカインを含む培地で培養することにより、特定の分化細胞を得る工程（ｂ）を含む、特定の分化細胞の製造方法に関する。 In the present invention, pluripotent stem cells are selected from the group consisting of histone deacetylase inhibitors, MAPK / ERK kinase inhibitors, STAT3 activators, protein kinase C inhibitors, Wnt signal inhibitors and GSK3β inhibitors. A step (a) and a step of obtaining pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of DNA is reduced as compared with the pluripotent stem cells before culturing by culturing in a medium containing any of the above. The present invention relates to a method for producing a specific differentiated cell, which comprises a step (b) of obtaining a specific differentiated cell by culturing the pluripotent stem cell obtained in (a) in a medium containing a cytokine.
　多能性幹細胞としては、ヒト人工多能性幹細胞（ｉｎｄｕｃｅｄ　ｐｌｕｒｉｐｏｔｅｎｔ　ｓｔｅｍ　ｃｅｌｌ；ｉＰＳ細胞とも言う）が、２００７年に山中らによって樹立され、胚性幹細胞（ｅｍｂｒｙｏｎｉｃ　ｓｔｅｍ　ｃｅｌｌ：ＥＳ細胞とも言う）と同様の無限の自己複製能と多分化能を有する。ｉＰＳ細胞はＥＳ細胞よりも倫理的問題がなく、品質の安定した細胞供給源として実用化が期待されている。例えば、創薬のスクリーニングや薬物動態試験において、ヒト初代培養肝細胞を用いてｉｎ　ｖｉｔｒｏ試験が行われるようになった。しかし、ロット間差や増殖能に問題があり研究の障壁になっている。肝臓細胞もｉＰＳ細胞から誘導可能であることが知られているが、分化効率は方法や細胞株によってばらつきがあることが問題であった。 As pluripotent stem cells, human induced pluripotent stem cells (also called iPS cells) were established by Yamanaka et al. In 2007, and are similar to embryonic stem cells (embryonic stem cells: ES cells). Has infinite self-renewal ability and pluripotency. iPS cells have no ethical problems than ES cells, and are expected to be put into practical use as a cell source with stable quality. For example, in vitro tests have come to be conducted using human primary cultured hepatocytes in drug discovery screening and pharmacokinetic tests. However, there are problems with lot-to-lot differences and proliferation ability, which is a barrier to research. It is known that hepatocytes can also be derived from iPS cells, but the problem is that the differentiation efficiency varies depending on the method and cell line.
　特許文献１は、（１）人工多能性幹細胞を内胚葉様細胞へと分化させる工程、および（２）工程（１）で得られた内胚葉様細胞を肝細胞様細胞へと分化させる工程であって、ヒストン脱アセチル化酵素阻害剤の存在下及び／又は酸化ストレス負荷条件下で少なくとも一部の培養を実施する工程を含む、人工多能性幹細胞を肝細胞へ分化誘導する方法を開示する。特許文献１には、ヒストン脱アセチル化酵素（ＨＤＡＣ）阻害剤、特にバルプロ酸がヒトｉＰＳ細胞の肝細胞への効率的な分化誘導に効果的であることが記載されている。バルプロ酸はてんかんに用いる代表的薬物であり、その主な作用としてはγ－アミノ酸（ＧＡＢＡ）トランスアミナーゼ阻害作用がある。さらに、バルプロ酸はＨＤＡＣ阻害作用を示すことから、癌治療への応用が期待されている。特許文献１では、ＨＤＡＣ阻害剤はｉＰＳ細胞由来の肝芽細胞様細胞から肝細胞様細胞の分化誘導を促進する効果があるものとして報告されており、未分化のｉＰＳ細胞そのものの性質を向上させるものではない。 Patent Document 1 describes (1) a step of differentiating induced pluripotent stem cells into endometrial-like cells, and (2) a step of differentiating endometrial-like cells obtained in step (1) into hepatocellular-like cells. Disclosed is a method for inducing differentiation of induced pluripotent stem cells into hepatocytes, which comprises the step of performing at least a part of the culture in the presence of a histone deacetylase inhibitor and / or under oxidative stress loading conditions. To do. Patent Document 1 describes that histone deacetylase (HDAC) inhibitors, particularly valproic acid, are effective in inducing efficient differentiation of human iPS cells into hepatocytes. Valproic acid is a typical drug used for epilepsy, and its main action is γ-amino acid (GABA) transaminase inhibitory action. Furthermore, since valproic acid exhibits an HDAC inhibitory effect, it is expected to be applied to cancer treatment. In Patent Document 1, it is reported that an HDAC inhibitor has an effect of promoting the induction of differentiation of hepatocyte-like cells from iPS cell-derived hepatoblast-like cells, and improves the properties of undifferentiated iPS cells themselves. It's not a thing.
国際公開ＷＯ２０１３／１８３５７１号International release WO 2013/183571
　ｉＰＳ細胞の誘導方法の発明により、ＥＳ細胞と同等の多分化能を有する幹細胞を得ることが可能となり、細胞治療や創薬支援ツールの応用が期待されている。しかし、本発明者らのこれまでの検討により、ｉＰＳ細胞は、細胞株やクローンによって性質が異なり、分化能に大きな差があること、およびｉＰＳ細胞としてのマーカーが発現している場合であっても、三胚葉の何れにも分化できない細胞が存在することが見出されている。そのため、ｉＰＳ細胞を、目的とする分化細胞への分化誘導に利用するためには分化優良株の選別に多くの時間とコストがかかるという実情がある。 The invention of the method for inducing iPS cells has made it possible to obtain stem cells having pluripotency equivalent to that of ES cells, and is expected to be applied to cell therapy and drug discovery support tools. However, according to the studies by the present inventors, iPS cells have different properties depending on the cell line and clone, and there is a large difference in differentiation potential, and the marker as an iPS cell is expressed. However, it has been found that there are cells that cannot differentiate in any of the three germ layers. Therefore, in order to use iPS cells for inducing differentiation into target differentiated cells, it takes a lot of time and cost to select excellent strains for differentiation.
　本発明は、目的とする特定の分化細胞を効率よく得ることができる分化細胞の製造方法を提供することを解決すべき課題とする。 The present invention is a problem to be solved to provide a method for producing a differentiated cell capable of efficiently obtaining a specific differentiated cell of interest.
　本発明者は、上記課題を解決すべく鋭意検討した結果、多能性幹細胞を、特定の化合物の存在下で培養することにより、目的とする分化細胞、特に肝臓細胞へと効率よく分化させることができることを見出した。本発明は、上記の知見に基づいて完成したものである。 As a result of diligent studies to solve the above problems, the present inventor efficiently differentiates pluripotent stem cells into target differentiated cells, particularly hepatocytes, by culturing them in the presence of a specific compound. I found that I could do it. The present invention has been completed based on the above findings.
　即ち、本発明によれば、以下の発明が提供される。
（１）多能性幹細胞を、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、ＳＴＡＴ３活性化剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤およびＧＳＫ３β阻害剤からなる群から選択される何れかを含む培地で培養することにより、培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した多能性幹細胞を得る工程（ａ）、および
工程（ａ）で得られる多能性幹細胞を、サイトカインを含む培地で培養することにより、特定の分化細胞を得る工程（ｂ）
を含む、特定の分化細胞の製造方法。
（２）上記工程（ａ）が、
多能性幹細胞を、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、およびＳＴＡＴ３活性化剤を含む培地で培養する第一培養工程、および
第一培養工程の後に、ヒストン脱アセチル化酵素阻害剤を含まずに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、ＧＳＫ３β阻害剤、およびＳＴＡＴ３活性化剤を含む培地で培養する第二培養工程
を含む、（１）に記載の製造方法。
（３）上記サイトカインが、アクチビンＡ、ＢＭＰ４、ｂＦＧＦ、ＨＧＦ、およびＯｎｃｏｓｔａｔｉｎ　Ｍからなる群から選択される少なくとも１つである、（１）または（２）に記載の方法。
（４）上記特定の分化細胞が内胚葉系譜の細胞である、（１）から（３）の何れか一に記載の方法。
（５）上記特定の分化細胞が肝細胞である、（１）から（４）の何れか一に記載の方法。
（６）第一培養工程の培養期間が１から５日間である、（２）から（５）の何れか一に記載の方法。
（７）第二培養工程は、ヒストン脱アセチル化酵素阻害剤を含まずに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、およびＳＴＡＴ３活性化剤を含む培地で培養した後、上記培地にＧＳＫ３β阻害剤を加えた培地で培養を行う工程である、（２）から（６）の何れか一に記載の方法。
（８）第二培養工程は、ヒストン脱アセチル化酵素阻害剤を含まずに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、およびＳＴＡＴ３活性化剤を含む培地で３から８日間培養した後、上記培地にＧＳＫ３β阻害剤を加えた培地で３から１０日間培養を行う工程である、（２）から（７）の何れか一に記載の方法。
（９）上記工程（ｂ）の培養期間が１から４週間である、（１）から（８）の何れか一に記載の方法。
（１０）ヒストン脱アセチル化酵素阻害剤が、バルプロ酸またはその塩である、（１）から（９）の何れか一に記載の方法。
（１１）ＭＡＰＫ／ＥＲＫキナーゼ阻害剤がＰＤ０３２５９０１である、（１）から（１０）の何れか一に記載の方法。
（１２）ＳＴＡＴ３活性化剤が白血病阻止因子である、（１）から（１１）の何れか一に記載の方法。
（１３）プロテインキナーゼＣ阻害剤がＧｏ６９８３である、（１）から（１２）の何れか一に記載の方法。
（１４）Ｗｎｔシグナル阻害剤がＸＡＶ９３９である、（１）から（１３）の何れか一に記載の方法。
（１５）ＧＳＫ３β阻害剤がＣＨＩＲ９９０２１である、（１）から（１４）の何れか一に記載の方法。
（１６）上記第一培養工程の培地がヒストン脱アセチル化酵素阻害剤を濃度１．０μｍｏｌ／Ｌ～５．０ｍｍｏｌ／Ｌの濃度で含む、（２）から（１５）の何れか一に記載の方法。
（１７）上記第一培養工程および第二培養工程の培地がＭＡＰＫ／ＥＲＫキナーゼ阻害剤を濃度０．３μｍｏｌ／Ｌ～５．０μｍｏｌ／Ｌの範囲で含む、（２）から（１６）の何れか一に記載の方法。
（１８）多能性幹細胞がヒトｉＰＳ細胞である、（１）から（１７）の何れか一に記載の方法。 That is, according to the present invention, the following invention is provided.
(1) Pluripotent stem cells are selected from the group consisting of histone deacetylase inhibitors, MAPK / ERK kinase inhibitors, STAT3 activators, protein kinase C inhibitors, Wnt signal inhibitors and GSK3β inhibitors. A step (a) and a step (a) of obtaining pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of DNA is reduced as compared with the pluripotent stem cells before culturing by culturing in a medium containing any of the above. Step (b) of obtaining specific differentiated cells by culturing the pluripotent stem cells obtained in a) in a medium containing cytokines.
A method for producing a specific differentiated cell, including.
(2) The above step (a)
A first culture step in which pluripotent stem cells are cultured in a medium containing a histone deacetylase inhibitor, a MAPK / ERK kinase inhibitor, and a STAT3 activator, and after the first culture step, histone deacetylase. A second culture step of culturing in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, a GSK3β inhibitor, and a STAT3 activator without an inhibitor is included (1). The manufacturing method described in.
(3) The method according to (1) or (2), wherein the cytokine is at least one selected from the group consisting of activin A, BMP4, bFGF, HGF, and Oncostatin M.
(4) The method according to any one of (1) to (3), wherein the specific differentiated cell is a cell of endoderm lineage.
(5) The method according to any one of (1) to (4), wherein the specific differentiated cell is a hepatocyte.
(6) The method according to any one of (2) to (5), wherein the culture period of the first culture step is 1 to 5 days.
(7) In the second culture step, the cells were cultured in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, and a STAT3 activator without containing a histone deacetylase inhibitor. The method according to any one of (2) to (6), which is a step of culturing in a medium in which a GSK3β inhibitor is added to the above medium.
(8) The second culture step is carried out from 3 in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, and a STAT3 activator without containing a histone deacetylase inhibitor. The method according to any one of (2) to (7), which is a step of culturing for 8 days and then culturing in a medium obtained by adding a GSK3β inhibitor to the above medium for 3 to 10 days.
(9) The method according to any one of (1) to (8), wherein the culture period of the step (b) is 1 to 4 weeks.
(10) The method according to any one of (1) to (9), wherein the histone deacetylase inhibitor is valproic acid or a salt thereof.
(11) The method according to any one of (1) to (10), wherein the MAPK / ERK kinase inhibitor is PD0325901.
(12) The method according to any one of (1) to (11), wherein the STAT3 activator is a leukemia inhibitory factor.
(13) The method according to any one of (1) to (12), wherein the protein kinase C inhibitor is Go6983.
(14) The method according to any one of (1) to (13), wherein the Wnt signal inhibitor is XAV939.
(15) The method according to any one of (1) to (14), wherein the GSK3β inhibitor is CHIR99021.
(16) The medium according to any one of (2) to (15), wherein the medium of the first culture step contains a histone deacetylase inhibitor at a concentration of 1.0 μmol / L to 5.0 mmol / L. Method.
(17) Any of (2) to (16), wherein the medium of the first culture step and the second culture step contains a MAPK / ERK kinase inhibitor in a concentration range of 0.3 μmol / L to 5.0 μmol / L. The method described in 1.
(18) The method according to any one of (1) to (17), wherein the pluripotent stem cell is a human iPS cell.
　本発明によれば、目的とする特定の分化細胞を効率よく製造することができる。 According to the present invention, a specific differentiated cell of interest can be efficiently produced.
図１は、未処理および処理済のｉＰＳ細胞株のメチル化度を示す。FIG. 1 shows the degree of methylation of untreated and treated iPS cell lines. 図２は、未処理および処理済のｉＰＳ細胞株の未分化性に関わる遺伝子発現量を示す。FIG. 2 shows the gene expression levels related to the undifferentiated state of the untreated and treated iPS cell lines. 図３Ａは、未処理および処理済のヒトｉＰＳ細胞株（ＡからＧ株）を肝細胞に分化誘導したときのＡＳＧＰＲ１の発現をＡｎｔｉ－ＡＳＧＰＲ１抗体を用いてフローサイトメトリーにて測定して得たＡＳＧＰＲ１陽性率を示す。FIG. 3A was obtained by measuring the expression of ASGPR1 when untreated and treated human iPS cell lines (strains A to G) were induced to differentiate into hepatocytes by flow cytometry using an Anti-ASGPR1 antibody. Shows the ASGPR1 positive rate. 図３Ｂは、未処理および処理済のヒトｉＰＳ細胞株（ＡからＧ株）を肝細胞に分化誘導したときのＡＳＧＰＲ１の発現をＡｎｔｉ－ＡＳＧＰＲ１抗体を用いてフローサイトメトリーにて測定したＡＳＧＰＲ１陽性細胞数の全７株の平均値を示す。FIG. 3B shows ASGPR1-positive cells whose expression of ASGPR1 when untreated and treated human iPS cell lines (A to G strains) were induced to differentiate into hepatocytes was measured by flow cytometry using Anti-ASGPR1 antibody. The average value of all 7 strains of the number is shown. 図４は、未処理および処理済のヒトｉＰＳ細胞株（ＡからＧ株）を肝細胞に分化誘導したときの培養上清中に分泌されたアルブミン量をＥＬＩＳＡ法にて測定した結果を示す。FIG. 4 shows the results of measuring the amount of albumin secreted in the culture supernatant when the untreated and treated human iPS cell lines (strains A to G) were induced to differentiate into hepatocytes by the ELISA method. 図５は、ｉＰＳ細胞を未処理、バルプロ酸処理、化合物混合処理条件後に内胚葉に分化させたときの内胚葉マーカー遺伝子の発現量を示す。FIG. 5 shows the expression level of an endoderm marker gene when iPS cells are differentiated into endoderm after untreated, valproic acid-treated, and compound-mixed treatment conditions.
　以下、本発明を実施するための形態を、詳細に説明する。
　本明細書における略号は以下の意味を有する。
ＡＬＫ５（ＴＧＦ－ｂｅｔａ　ｔｙｐｅ　Ｉ　ｒｅｃｅｐｔｏｒ）：トランスフォーミング増殖因子ベータ　１型受容体
ｂＦＧＦ（ｂａｓｉｃ　Ｆｉｂｒｏｂｌａｓｔ　Ｇｒｏｗｔｈ　Ｆａｃｔｏｒ）：塩基性線維芽細胞成長因子
ＢＭＰ４（Ｂｏｎｅ　Ｍｏｒｐｈｏｇｅｎｅｔｉｃ　Ｐｒｏｔｅｉｎ　４）：骨形成因子
ＣＤ３４（Ｃｌｕｓｔｅｒ　ｏｆ　ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ　３４）：分化抗原群３４ Hereinafter, embodiments for carrying out the present invention will be described in detail.
The abbreviations used herein have the following meanings.
ALK5 (TGF-beta type I receptor): Transforming Growth Factor Beta Type 1 Receptor bFGF (basic Fibroblast Growth Factor): Basic Fibroblast Growth Factor BMP4 (Bone Morphogenetic Factor 34) Bone Former 34): Differentiation antigen group 34
ｃＴｎＴ（ｃａｒｄｉａｃ　Ｔｒｏｐｏｎｉｎ　Ｔ）：心筋トロポニンＴ
ＤＭＥＭ　（Ｄｕｌｂｅｃｃｏ’ｓ　Ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ　Ｍｅｄｉｕｍ）：ダルベッコ改変イーグル培地
ＤＭＥＭ／Ｆ１２（ＤＭＥＭ Ｈａｍ’ｓ Ｆ－１２）：ダルベッコ改変イーグル培地／栄養混合物Ｆ－１２ハム
ＤＮＭＴ３Ｂ（ＤＮＡ（ｃｙｔｏｓｉｎｅ－５－）－ｍｅｔｈｙｌｔｒａｎｓｆｅｒａｓｅ　３　ｂｅｔａ）：ＤＮＡ（シトシン‐５‐）メチルトランスフェラーゼ３ベータ
Ｄｎｍｔ３Ｌ（ＤＮＡ　Ｍｅｔｈｙｌｔｒａｎｓｆｅｒａｓｅ　３　Ｌｉｋｅ）：ＤＮＡメチルトランスフェラーゼ３様
ＥＣＡＴ（ＥＳ　ｃｅｌｌ　ａｓｓｏｃｉａｔｅｄ　ｔｒａｎｓｃｒｉｐｔｓ）：ＥＳ細胞関連転写因子
ＥＲａｓ（ＥＳ　ｃｅｌｌ　ｅｘｐｒｅｓｓｅｄ　Ｒａｓ）：ＥＳ細胞で発現するＲａｓ
Ｅｒｋ（Ｅｘｔｒａｃｅｌｌｕｌａｒ　Ｓｉｇｎａｌ－ｒｅｇｕｌａｔｅｄ　Ｋｉｎａｓｅ）：細胞外シグナル調節キナーゼ cTnT (cardiac Troponin T): Myocardial troponin T
DMEM (Dulvecco's Modified Eagle Medium): Dulbecco's Modified Eagle Medium DMEM / F12 (DMEM Ham's F-12): Dulbecco's Modified Eagle Medium / Nutrition Mixture F-12 Ham DNMT3B (DNA (cytosine-5) -methyltransferase beta): DNA (cytosine-5-) methyltransferase 3 beta Dnmt3L (DNA Methyltransphase 3 Like): DNA methyltransferase 3-like ECAT (ES cell assisted transcripts): ES cell-related transcription factor ERAs (ES cell ess) Ras expressed in
Erk (Extracellular Signal-regulated Kinase): Extracellular signal-regulated kinase
ＥＳＧ（Ｅｍｂｒｙｏｎａｌ　ｓｔｅｍ　ｃｅｌｌ－ｓｐｅｃｉｆｉｃ　ｇｅｎｅ）：胚性幹細胞特異的遺伝子
Ｆｂｘ１５（Ｆ－Ｂｏｘ　Ｐｒｏｔｅｉｎ　１５）：Ｆボックス　タンパク質１５
ＦＧＦ（Ｆｉｂｒｏｂｌａｓｔ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ）：線維芽細胞増殖因子
ＦＯＸＡ２（ｆｏｒｋｈｅａｄ　ｂｏｘ　ｐｒｏｔｅｉｎ　Ａ２）：フォークヘッドボックスタンパク質Ａ２
Ｆｔｈｌ１７（Ｆｅｒｒｉｔｉｎ　ｈｅａｖｙ　ｐｏｌｙｐｅｐｔｉｄｅ－ｌｉｋｅ　１７）：フェリチン重鎖ポリペプチド様１７
ＧＡＰＤＨ（ｇｌｙｃｅｒａｌｄｅｈｙｄｅ－３－ｐｈｏｓｐｈａｔｅ　ｄｅｈｙｄｒｏｇｅｎａｓｅ）：グリセルアルデヒド－３－リン酸デヒドロゲナーゼ
Ｇｄｆ３（Ｇｒｏｗｔｈ　ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ　ｆａｃｔｏｒ－３）：増殖分化因子３ ESG (Embryonal stem cell-specific gene): Embryonic stem cell-specific gene Fbx15 (F-Box Protein 15): F-box protein 15
FGF (Fibroblast growth factor): Fibroblast growth factor FOXA2 (forkhead box protein A2): Forkhead box protein A2
Fthl17 (Ferritin heavy polypeptide-like 17): Ferritin heavy chain polypeptide-like 17
GAPDH (glyceraldehyde-3-phosphate dehydogenesis): Glyceraldehyde-3-phosphate dehydrogenase Gdf3 (Glyceraldehyde three-phosphate dehydrogenase): Growth differentiation factor 3
Ｇｒｂ２（Ｇｒｏｗｔｈ　ｆａｃｔｏｒ　ｒｅｃｅｐｔｏｒ－ｂｏｕｎｄ　ｐｒｏｔｅｉｎ　２）：増殖因子受容体結合タンパク質２
ＧＳＫ（Ｇｌｙｃｏｇｅｎ　Ｓｙｎｔｈａｓｅ　Ｋｉｎａｓｅ）：グリコーゲン合成酵素キナーゼ
ＨＢＭ（Ｈｅｐａｔｏｃｙｔｅ　Ｂａｓａｌ　Ｍｅｄｉｕｍ）：幹細胞基本培地
ＨＤＡＣ（Ｈｉｓｔｏｎｅ　Ｄｅａｃｅｔｙｌａｓｅ）：ヒストン脱アセチル化酵素
ＩＧＦ（Ｉｎｓｕｌｉｎ－ｌｉｋｅ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ）：インスリン様増殖因子
ＩＧＦＢＰ４（Ｉｎｓｕｌｉｎ－ｌｉｋｅ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ－ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎ　４）：インスリン様増殖因子結合タンパク質４
ＩＬ（ｉｎｔｅｒｌｅｕｋｉｎ）：インターロイキン
ＫＤＲ（ｋｉｎａｓｅ　ｉｎｓｅｒｔ　ｄｏｍａｉｎ－ｃｏｎｔａｉｎｉｎｇ　ｒｅｃｅｐｔｏｒ）：キナーゼ挿入ドメイン含有受容体
Ｋｌｆ（Ｋｒｕｐｐｅｌ－ｌｉｋｅ　ｆａｃｔｏｒ）：クルッペル様因子
ＬＧＲ５（Ｌｅｕｃｉｎｅ－ｒｉｃｈ　ｒｅｐｅａｔ－ｃｏｎｔａｉｎｉｎｇ　Ｇ－ｐｒｏｔｅｉｎ　ｃｏｕｐｌｅｄ　ｒｅｃｅｐｔｏｒ　５）：ロイシンリッチリピートを含むＧタンパク質共役受容体５ Grb2 (Growth factor receptor-bound protein 2): Growth factor receptor binding protein 2
GSK (Glycogen Synthase Kinase): Glycogen synthase kinase HBM (Hepatocyte Basel Medium): Stem cell basal medium HDAC (Histone Deacetylase): Histone deacetylase IGF (Insulin-like Growth Factor) IGF (Insulin-like Growth Factor) growh factor-binding protein 4): Insulin-like growth factor binding protein 4
IL (interleukin): Interleucine-rich KDR (kinase insert domain-connecting receptor): Kinase insertion domain-containing receptor Klf (Kruppel-like receptor): Kruppell-like factor LGR5 (Leucine-rich repeat-receptor) G protein-coupled receptor containing leucine-rich repeat 5
ＬＩＦ（Ｌｅｕｋｅｍｉａ　ｉｎｈｉｂｉｔｏｒｙ　ｆａｃｔｏｒ）：白血病阻止因子
ＭＡＰ（ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄ　ｐｒｏｔｅｉｎ）：***促進因子活性化プロテインキナーゼ
Ｎｒ５ａ１（ｎｕｃｌｅａｒ　ｒｅｃｅｐｔｏｒ　ｓｕｂｆａｍｉｌｙ　５，ｇｒｏｕｐ　Ａ，ｍｅｍｂｅｒ　１）：核内受容体サブファミリー５、グループＡ、メンバー１
Ｎｒ５ａ２（ｎｕｃｌｅａｒ　ｒｅｃｅｐｔｏｒ　ｓｕｂｆａｍｉｌｙ　５，ｇｒｏｕｐ　Ａ，ｍｅｍｂｅｒ　２）：核内受容体サブファミリー５、グループＡ、メンバー２
Ｏｃｔ（ｏｃｔａｍｅｒ－ｂｉｎｄｉｎｇ　ｔｒａｎｓｃｒｉｐｔｉｏｎ　ｆａｃｔｏｒ）：オクタマー結合性転写因子 
ＰＡＸ６（ｐａｉｒｅｄ　ｂｏｘ　６）：ペアードボックス６
ＰＣＲ（ｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ）：ポリメラーゼ連鎖反応
ＰＤＧＦＲＡ（ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ　ｒｅｃｅｐｔｏｒ　ａｌｐｈａ）：血小板由来増殖因子受容体α
ＰＫＣ（Ｐｒｏｔｅｉｎ　ｋｉｎａｓｅ　Ｃ）：プロテインキナーゼＣ LIF (Leukemia inhibitory factor): Leukemia inhibitory factor MAP (mitogen-activated protein): Mitogen-activated protein kinase Nr5a1 (nuclear receptor subfamily) Family 5, group A, group A, meb Member 1
Nr5a2 (nuclear receptor subfamily 5, group A, member 2): Nuclear receptor subfamily 5, group A, member 2
Oct (octamer-binding transcription factor): Octamar-binding transcription factor
PAX6 (paired box 6): Paired box 6
PCR (polymerase chain reaction): Polymerase chain reaction PDGFRA (platelet-developed growth factor receptor alpha): Pillot-derived growth factor receptor α
PKC (Protein kinase C): Protein kinase C
ＰＯＵ５Ｆ１（ＰＯＵ　ｄｏｍａｉｎ，　ｃｌａｓｓ　５，　ｔｒａｎｓｃｒｉｐｔｉｏｎ　ｆａｃｔｏｒ　１）：ＰＯＵドメイン、クラス５、転写因子１
Ｐｒｄｍ１４（ＰＲ／ＳＥＴ　ｄｏｍａｉｎ　ｆａｍｉｌｙ　１４）：ＰＲ／ＳＥＴドメインファミリー１４
ｑＰＣＲ（Ｑｕａｎｔｉｔａｔｉｖｅ　ｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ）：定量ポリメラーゼ連鎖反応
Ｒｅｘ１（Ｒｅｄｕｃｅｄ－ｅｘｐｒｅｓｓｉｏｎ　１）：低発現タンパク質－１
ＲＯＣＫ（Ｒｈｏ－ａｓｓｏｃｉａｔｅｄ　ｃｏｉｌｅｄ－ｃｏｉｌ　ｆｏｒｍｉｎｇ　ｋｉｎａｓｅ）：Ｒｈｏ結合コイルドコイル形成キナーゼ
ＲＴ－ＰＣＲ（Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　ｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ）：逆転写ポリメラーゼ連鎖反応
Ｓａｌｌ４（Ｓａｌ－ｌｉｋｅ　ｐｒｏｔｅｉｎ　４）：Ｓａｌ様タンパク質４
ＳＣＦ（Ｓｔｅｍ　ｃｅｌｌ　ｆａｃｔｏｒ）：幹細胞因子 POU5F1 (POU domain, class 5, transcription factor 1): POU domain, class 5, transcription factor 1
Prdm14 (PR / SET domain family 14): PR / SET domain family 14
qPCR (Quantitative polymerase chain reaction): Quantitative polymerase chain reaction Rex1 (Reduced-expression 1): Low expression protein-1
ROCK (Rho-associated coiled-coil forming kinase): Rho-conjugated coiled-coil-forming kinase RT-PCR (Reverse transcription polymerase chain reaction): Reverse transcription polymerase chain reaction Sall4 (Sal-like)
SCF (Stem cell factor): Stem cell factor
Ｓｏｘ：ＳＲＹ （ｓｅｘ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ　Ｙ）－ｂｏｘ：ＳＲＹ（Ｙ染色体性決定遺伝子）ボックス
Ｓｔａｔ３（Ｓｉｇｎａｌ　Ｔｒａｎｓｄｕｃｅｒ　ａｎｄ　Ａｃｔｉｖａｔｏｒ　ｏｆ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　３）：シグナル伝達兼転写活性化因子３
Ｔ：Ｂｒａｃｈｙｕｒｙ：Ｔ　遺伝子にコードされたブラキウリ
Ｔｃｌ１（Ｔ－ｃｅｌｌ　ｌｅｕｋｅｍｉａ／ｌｙｍｐｈｏｍａ　１Ａ）：Ｔ細胞白血病／リンパ腫１Ａ
Ｔｅｒｔ（Ｔｅｌｏｍｅｒａｓｅ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔａｓｅ）：テロメラーゼ逆転写酵素
ＴＧＦ（Ｔｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ）：トランスフォーミング増殖因子
ＵＴＦ１（Ｕｎｄｉｆｆｅｒｅｎｔｉａｔｅｄ　Ｅｍｂｒｙｏｎｉｃ　Ｃｅｌｌ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｆａｃｔｏｒ　１）：未分化胚性幹細胞転写因子１
ＶＥＧＦ（Ｖａｓｃｕｌａｒ　Ｅｎｄｏｔｈｅｌｉａｌ　Ｇｒｏｗｔｈ　Ｆａｃｔｏｒ）：血管内皮増殖因子 Sox: SRY (sex determination region Y) -box: SRY (Y chromosome sex-determining gene) Box Stat3 (Signal Transducer and Activator of Tranciption 3): Signaling and transcriptional activator 3
T: Brachyury: Brachyury Tcl1 encoded by the T gene (T-cell leukemia / lymphoma 1A): T-cell leukemia / lymphoma 1A
Tert (Telomerase Reverse Transcriptase): Telomerase Reverse Transcript TGF (Transforming growth factor): Transforming Growth Factor UTF1 (Undifferentiated Embryonic Cell Transcription Factor) Undifferentiated Cell Transcription Factor
VEGF (Vascular Endothelial Growth Factor): Vascular Endothelial Growth Factor
　本発明の特定の分化細胞の製造方法は、多能性幹細胞を、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、ＳＴＡＴ３活性化剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤およびＧＳＫ３β阻害剤からなる群から選択される何れかを含む培地で培養することにより、培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した多能性幹細胞を得る工程（ａ）、および工程（ａ）で得られる多能性幹細胞を、サイトカインを含む培地で培養することにより、特定の分化細胞を得る工程（ｂ）を含む。なお、本発明において、同じ培養条件で同一の操作をした細胞は、個体として別であっても同一の性質を持つ細胞とみなす。 The method for producing a specific differentiated cell of the present invention uses pluripotent stem cells as a histone deacetylase inhibitor, a MAPK / ERK kinase inhibitor, a STAT3 activator, a protein kinase C inhibitor, a Wnt signal inhibitor and GSK3β. By culturing in a medium containing any of the inhibitors selected from the group, pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of DNA was reduced as compared with the pluripotent stem cells before culturing. The step (a) of obtaining, and the step (b) of obtaining specific differentiated cells by culturing the pluripotent stem cells obtained in step (a) in a medium containing cytokines are included. In the present invention, cells that have undergone the same operation under the same culture conditions are regarded as cells having the same properties even if they are different as individuals.
　本発明においては、多能性幹細胞を、特定の化合物の存在下で培養することにより、そのＤＮＡのメチル化度が低下することを見出し、ＤＮＡのメチル化度を低下させた多能性幹細胞を特定の分化細胞への分化誘導に用いることにより、効率よく特定の分化細胞を製造することに成功したものである。 In the present invention, it has been found that the degree of DNA methylation is reduced by culturing pluripotent stem cells in the presence of a specific compound, and the pluripotent stem cells having a reduced degree of DNA methylation are used. By using it to induce differentiation into specific differentiated cells, we have succeeded in efficiently producing specific differentiated cells.
　ところで、バルプロ酸がヒトｉＰＳ細胞から肝臓細胞への分化誘導効率を向上させるという報告があり（特許文献１）、この文献では、肝芽細胞様細胞を肝細胞様細胞へと分化させる工程をバルプロ酸の存在下で行っている。しかしながら、本発明者らの検討では、ＨＤＡＣ阻害剤（例えばバルプロ酸）単独では、未分化のｉＰＳ細胞そのものの性質が向上しないことが明らかとなった。後記の実施例では、バルプロ酸単独で処理した場合には、肝臓細胞への分化途中段階である内胚葉への分化効率が上昇しないこと、一方、バルプロ酸を他の化合物と組み合わせた場合には分化効率が上昇することを確認している。 By the way, there is a report that valproic acid improves the efficiency of inducing differentiation of human iPS cells into hepatocytes (Patent Document 1). In this document, the step of differentiating hepatoblast-like cells into hepatocyte-like cells is valpro. It is done in the presence of acid. However, in the study by the present inventors, it was clarified that the properties of undifferentiated iPS cells themselves are not improved by the HDAC inhibitor (for example, valproic acid) alone. In the examples described below, when treated with valproic acid alone, the efficiency of differentiation into endoderm, which is in the process of differentiation into hepatocytes, does not increase, while when valproic acid is combined with other compounds, it does not increase. It has been confirmed that the differentiation efficiency increases.
　本発明の多能性幹細胞は、体細胞に初期化因子を導入して得られる未分化細胞であってもよい。体細胞としては、特に限定されず、任意の体細胞を利用することができる。例えば、胎児期の体細胞のほか、成人由来の体細胞（即ち、成熟した体細胞）を用いてもよい。体細胞としては、例えば、（１）神経幹細胞、造血幹細胞、間葉系幹細胞、歯髄幹細胞等の組織幹細胞（体性幹細胞）、（２）組織前駆細胞、（３）線維芽細胞（皮膚細胞等）、上皮細胞、肝細胞、リンパ球（Ｔ細胞、Ｂ細胞）、内皮細胞、筋肉細胞、毛細胞、胃粘膜細胞、腸細胞、脾細胞、膵細胞（膵外分泌細胞等）、脳細胞、肺細胞、腎細胞、皮膚細胞等の分化した細胞が挙げられる。体細胞の由来となる生体としては、特に限定されないが、例えば、ヒト、非ヒト動物（例えば、サル、ヒツジ、ウシ、ウマ、イヌ、ネコ、ウサギ、ラット、マウス）が挙げられる。好ましくは、ヒトである。本発明の多能性幹細胞は、好ましくはヒトｉＰＳ細胞である。 The pluripotent stem cell of the present invention may be an undifferentiated cell obtained by introducing a reprogramming factor into a somatic cell. The somatic cell is not particularly limited, and any somatic cell can be used. For example, in addition to fetal somatic cells, adult-derived somatic cells (that is, mature somatic cells) may be used. Examples of somatic cells include (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, and (3) fibroblasts (skin cells, etc.). ), Epithelial cells, hepatocytes, lymphocytes (T cells, B cells), endothelial cells, muscle cells, hair cells, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lungs Examples thereof include differentiated cells such as cells, renal cells, and skin cells. The living body from which the somatic cells are derived is not particularly limited, and examples thereof include humans and non-human animals (for example, monkeys, sheep, cows, horses, dogs, cats, rabbits, rats, mice). Preferably, it is human. The pluripotent stem cells of the present invention are preferably human iPS cells.
　体細胞に導入される初期化因子としては特に限定されないが、例えば、Ｏｃｔ３／４、Ｋｌｆ４、ｃ－Ｍｙｃ、Ｓｏｘ２、Ｎａｎｏｇ、Ｋｌｆ２、Ｌ－Ｍｙｃ、Ｎ－Ｍｙｃ、Ｋｌｆ５、Ｌｉｎ２８、Ｔｅｒｔ、Ｆｂｘ１５、ＥＲａｓ、ＥＣＡＴ１５－１、ＥＣＡＴ１５－２、Ｔｃｌ１、β－カテニン、ＥＣＡＴ１、Ｅｓｇ１、Ｄｎｍｔ３Ｌ、ＥＣＡＴ８、Ｇｄｆ３、Ｓｏｘ１５、Ｆｔｈｌ１７、Ｓａｌｌ４、Ｒｅｘ１、ＵＴＦ１、Ｓｔｅｌｌａ、Ｓｔａｔ３、Ｇｒｂ２、Ｐｒｄｍ１４、Ｎｒ５ａ１、Ｎｒ５ａ２、Ｅ－ｃａｄｈｅｒｉｎが挙げられる。ここで、これらの遺伝子群の中から２以上の遺伝子を選択して任意に組み合わせて導入することができる。なかでも、Ｏｃｔ３／４、Ｓｏｘ２、Ｋｌｆ４およびｃ－Ｍｙｃを少なくとも有する組み合わせ、Ｏｃｔ３／４、Ｓｏｘ２、Ｋｌｆ４およびＬ－Ｍｙｃを少なくとも有する組み合わせ、またはＯｃｔ３／４、Ｓｏｘ２、ＮａｎｏｇおよびＬｉｎ２８を少なくとも有する組み合わせが好ましい。 The reprogramming factor introduced into somatic cells is not particularly limited, but for example, Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, Klf2, L-Myc, N-Myc, Klf5, Lin28, Tert, Fbx15, ERAs, ECAT15-1, ECAT15-2, Tcl1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, Fthl17, All4, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5 Cadherin can be mentioned. Here, two or more genes can be selected from these gene groups and introduced in any combination. Among them, combinations having at least Oct3 / 4, Sox2, Klf4 and c-Myc, combinations having at least Oct3/4, Sox2, Klf4 and L-Myc, or combinations having at least Oct3 / 4, Sox2, Nanog and Lin28 preferable.
　また、導入する遺伝子の種は、導入先の細胞の種と同一であることが好ましい。例えば、ヒト由来の細胞へ導入される遺伝子はヒト遺伝子であることが好ましい。例えば、ヒト由来の体細胞へ導入される遺伝子としては、ヒトＯＣＴ３／４、ヒトＳＯＸ２、ヒトＫＬＦ４およびヒトＭＹＣを少なくとも有する組み合わせ、ＯＣＴ３／４、ＳＯＸ２、ＫＬＦ４およびL－ＭＹＣを少なくとも有する組み合わせ、またはヒトＯＣＴ３／４、ヒトＳＯＸ２、ヒトＮＡＮＯＧおよびヒトＬＩＮ２８を少なくとも有する組み合わせが好ましい。 Further, it is preferable that the species of the gene to be introduced is the same as the species of the cell to be introduced. For example, the gene introduced into a human-derived cell is preferably a human gene. For example, genes to be introduced into human-derived somatic cells include a combination having at least human OCT3 / 4, human SOX2, human KLF4 and human MYC, a combination having at least OCT3 / 4, SOX2, KLF4 and L-MYC, or A combination having at least human OCT3 / 4, human SOX2, human NANOG and human LIN28 is preferred.
　初期化因子の遺伝子は、遺伝子発現ベクターを用いて体細胞に導入することができる。遺伝子発現ベクターとしては、特に限定されないが、例えば、ウイルスベクター、プラスミドベクター、人工染色体ベクター、トランスポゾンベクターが挙げられる。ウイルスベクターとしては、レトロウイルスベクター、アデノウイルスベクター、センダイウイルスベクター、レンチウイルスベクター、アデノ随伴ウイルスベクターが挙げられる。 The reprogramming factor gene can be introduced into somatic cells using a gene expression vector. The gene expression vector is not particularly limited, and examples thereof include a viral vector, a plasmid vector, an artificial chromosome vector, and a transposon vector. Examples of the viral vector include a retrovirus vector, an adenovirus vector, a Sendai virus vector, a lentiviral vector, and an adeno-associated virus vector.
　体細胞に初期化因子を導入して得られる未分化細胞は、体細胞に初期化因子を導入することによって自ら作製してもよいが、研究機関や企業から提供または販売されている細胞を入手してもよい。 Undifferentiated cells obtained by introducing a reprogramming factor into somatic cells may be produced by themselves by introducing a reprogramming factor into somatic cells, but cells provided or sold by research institutes or companies are obtained. You may.
　例えば、京都大学ｉＰＳ細胞研究所から提供されている２０１Ｂ７、２５３Ｇ１、２５３Ｇ４、１２０１Ｃ１、１２０５Ｄ１、１２１０Ｂ２、１２３１Ａ３、１３８３Ｄ２、１３８３Ｄ６、ｉＰＳ－ＴＩＧ１２０－３ｆ７、ｉＰＳ－ＴＩＧ１２０－４ｆ１、ｉＰＳ－ＴＩＧ１１４－４ｆ１、ＣｉＲＡ０８６Ａｉ－ｍ１、ＣｉＲＡ１８８Ａｉ－Ｍ１、またはｉＲＡ１８８Ａｉ－Ｗ１を入手して、使用することができる。 For example, 201B7, 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1231A3, 1383D2, 1383D6, iPS-TIG120-3f7, iPS-TIG120-4f1, iPS-TIG114-4f1, CiRA086Ai provided by the Center for iPS Cell Research and Application, Kyoto University. -M1, CiRA188Ai-M1 or iRA188Ai-W1 can be obtained and used.
　また、ＮＩＨ（Ｎａｔｉｏｎａｌ　Ｉｎｓｔｉｔｕｔｅｓ　ｏｆ　Ｈｅａｌｔｈ）やＣａｌｉｆｏｒｎｉａ　Ｉｎｓｔｉｔｕｔｅ　ｏｆ　Ｒｅｇｅｎｅｒａｔｉｖｅ　Ｍｅｄｉｃｉｎｅ、Ｎｅｗ　Ｙｏｒｋ　Ｓｔｅｍ　Ｃｅｌｌ　Ｆｏｕｎｄａｔｉｏｎ、Ｅｕｒｏｐｅａｎ　Ｂａｎｋ　ｆｏｒ　ｉｎｄｕｃｅｄ　Ｐｌｕｒｉｐｏｔｅｎｔ　Ｓｔｅｍ　Ｃｅｌｌｓ等が作成しているｉＰＳ細胞バンクから入手することもできる。 In addition, NIH (National Institutes of Health), California Institute of Regenerative Medicine, New York Stem Cell Foundation, European Bank Stem Cell, etc. can also be obtained from Stem Cell Foundation, European Bank, etc.
　本発明の多能性幹細胞は、体細胞に初期化因子を導入して得られる未分化細胞であってもよく、「体細胞に初期化因子を導入して得られる未分化細胞」における未分化細胞とは、分化していない細胞を意味し、好ましくは内胚葉、中胚葉および外胚葉の何れか一つ以上に分化する能力を有する細胞である。 The pluripotent stem cell of the present invention may be an undifferentiated cell obtained by introducing a reprogramming factor into a somatic cell, or an undifferentiated cell in an "undifferentiated cell obtained by introducing a reprogramming factor into a somatic cell". The cell means an undifferentiated cell, and is preferably a cell having an ability to differentiate into any one or more of endometrial follicle, meso-embryonic follicle and ectodermal lobe.
　上記「体細胞に初期化因子を導入して得られる未分化細胞」は、フィーダー細胞をコートしたプレートまたはマトリゲル（Ｍａｔｒｉｇｅｌ）（登録商標）などの足場をコートしたプレート上で、適当な培地にて維持培養することができる。フィーダー細胞としては、特に限定されないが、マウス胚性線維芽細胞（ＭＥＦ細胞）、マウス胎児繊維芽細胞（ＳＴＯ細胞）が挙げられる。 The above-mentioned "undifferentiated cells obtained by introducing a reprogramming factor into somatic cells" is prepared on a plate coated with feeder cells or a plate coated with a scaffold such as Matrigel (registered trademark) in an appropriate medium. It can be maintained and cultured. The feeder cell is not particularly limited, and examples thereof include mouse embryonic fibroblast (MEF cell) and mouse embryonic fibroblast (STO cell).
　未分化細胞の維持培養の際の培地としては、ｍＴｅＳＲ（登録商標）１（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）またはＳｔｅｍＦｌｅｘ（登録商標）などの市販の培地を使用することができる。あるいはまた、例えば、基礎培地として、ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ　Ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ　ｍｅｄｉｕｍ）、ＤＭＥＭとＦ１２の混合培地（ＤＭＥＭ／Ｆ１２＝１：１）、ＫｎｏｃｋｏｕｔＴＭ　Ｄ－ＭＥＭ（Ｉｎｖｉｔｒｏｇｅｎ社）などが挙げられ、代替血清（ＫＳＲ；ＫｎｏｃｋｏｕｔＴＭ　Ｓｅｒｕｍ　Ｒｅｐｌａｃｅｍｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ社））、ウシ胎児血清（ＦＢＳ）、非必須アミノ酸（ＮＥＡＡ）、Ｌ－グルタミン、２－メルカプトエタノール、抗生物質（例えば、ストレプトマイシン、ペニシリン、ピューロマイシン、マイトマイシン）、ｂＦＧＦ（ＦＧＦ２と同義である）等の添加成分を任意に組み合わせて、上記いずれかの基礎培地に添加してした培地も、維持培養の際の培地として挙げることができる。維持培養の際の培地は、アスコルビン酸を含まないことが好ましい。維持培養の培養条件は、３７℃、５％ＣＯ２、１０％Ｏ２条件下などが好ましいが、特に限定されない。 As the medium for the maintenance culture of undifferentiated cells, a commercially available medium such as mTeSR® 1 (Stemcell Technologies) or StemFlex® can be used. Alternatively, for example, DMEM (Dulvecco Modified Eagle medium), a mixed medium of DMEM and F12 (DMEM / F12 = 1: 1), Knockout TM D-MEM (Invitrogen), and the like can be mentioned as the basal medium, and alternative sera (DMEM). KSR; Knockout TM Medium Replacement (Invitrogen)), bovine fetal serum (FBS), non-essential amino acids (NEAA), L-glutamine, 2-mercaptoethanol, antibiotics (eg, streptomycin, penicillin, puromycin, mitomycin), A medium added to any of the above basal media by arbitrarily combining additional components such as bFGF (synonymous with FGF2) can also be mentioned as a medium for maintenance culture. The medium for maintenance culture preferably does not contain ascorbic acid. The culture conditions for the maintenance culture are preferably 37 ° C., 5% CO 2 , 10% O 2 and the like, but are not particularly limited.
　本発明の「特定の分化細胞」とは、内胚葉系譜、中胚葉系譜および外胚葉系譜の何れかに属する、任意の特定の分化した細胞を意味し、好ましくは内胚葉系譜に属する任意の特定の分化した細胞であり、例えば肝細胞である。 The "specific differentiated cell" of the present invention means any specific differentiated cell belonging to any of the endoderm lineage, the mesodermal lineage and the ectoderm lineage, and preferably any specific differentiated cell belonging to the endoderm lineage. Differentiated cells, such as hepatocytes.
［工程（ａ）］
　本発明の工程（ａ）は、多能性幹細胞を、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、ＳＴＡＴ３活性化剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤およびＧＳＫ３β阻害剤からなる群から選択される何れかを含む培地で培養することにより、培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した工程（ａ）を得る工程である。 [Step (a)]
In step (a) of the present invention, pluripotent stem cells are derived from histone deacetylase inhibitors, MAPK / ERK kinase inhibitors, STAT3 activators, protein kinase C inhibitors, Wnt signal inhibitors and GSK3β inhibitors. In the step (a) of obtaining the step (a) in which the proportion of 5-methylcytosine in the total cytosine of DNA was reduced as compared with the pluripotent stem cells before culturing by culturing in a medium containing any of the groups selected from is there.
　工程（ａ）で得られる多能性幹細胞は、培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が、１０％以上、１５％以上、２０％以上、２５％以上、３０％以上、３５％以上、４０％以上、４５％以上、５０％以上、５５％以上、６０％以上、６５％以上、７０％以上、７５％以上、８０％以上、８５％以上、９０％以上、または９５％以上低下した細胞であってもよい。ＤＮＡの全シトシンにおける５メチルシトシンの割合は、具体的には、後記の実施例２の手順に準じて、多能性幹細胞から抽出したゲノムＤＮＡを、ＥＺ－ＤＮＡ　ＭｅｔｈｙｌａｔｉｏｎＴＭ　Ｋｉｔ（ＺＹＭＯ　ＲＥＳＥＡＣＨ）にてＢｉｓｕｌｆｉｔｅ（亜硫酸水素塩）変換し、Ｉｎｆｉｎｉｕｍ（登録商標）　Ｍｅｔｈｙｌａｔｉｏｎ　ＥＰＩＣ　ＢｅａｄｓＣｈｉｐ　Ｋｉｔ（ｉｌｌｕｍｉｎａ）にてメチル化アレイ解析を実施し、メチル化度（β値）を算出すればよいが、この方法に限定される必要はなく、細胞から抽出したゲノムＤＮＡをＢｉｓｕｌｆｉｔｅで処理し、非メチルシトシンをウラシルに変換した後に、シーケンス解析やＰＣＲによる特異的領域を増幅した後にシーケンス解析または制限酵素処理によるメチル化解析を行ってもよい。 In the pluripotent stem cells obtained in step (a), the proportion of 5-methylcytosine in the total cytosine of DNA was 10% or more, 15% or more, 20% or more, 25, as compared with the pluripotent stem cells before culturing. % Or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more , 90% or more, or 95% or more reduced cells. Specifically, the ratio of 5-methylcytosine to total cytosine of DNA is determined by converting genomic DNA extracted from pluripotent stem cells into EZ-DNA methylation TM Kit (ZYMO RESEACH) according to the procedure of Example 2 described later. The methylation degree (β value) may be calculated by converting to Bisulfite (bisulfite) and performing a methylation array analysis with Infinium (registered trademark) methylation EPIC BeadsChip Kit (illumina), but this method is limited. It is not necessary to be performed, and the genomic DNA extracted from the cells is treated with Bisulfite, non-methylcytosine is converted to uracil, and then the specific region is amplified by sequence analysis or PCR, and then methylation analysis by sequence analysis or restriction enzyme treatment. May be done.
　工程（ａ）で用いる培地は、基礎培地に、Ｎｅｕｒｏｂａｓａｌ（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、Ｂ２７（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、Ｎ２（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、１－チオグリセロール、およびＧｌｕｔａＭＡＸ（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）またはＬ－Ｇｌｕｔａｍｉｎ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）などの添加成分を任意に組み合わせて（好ましくは上記の添加成分の全てを組み合わせて）、上記の基礎培地に添加し、さらに、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、ＳＴＡＴ３活性化剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤およびＧＳＫ３β阻害剤からなる群から選択される何れかを添加した培地であってもよい。上記基礎培地としては、ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ　Ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ　ｍｅｄｉｕｍ）、ＤＭＥＭとＦ１２の混合培地（ＤＭＥＭ／Ｆ１２＝１：１）、ＫｎｏｃｋｏｕｔＴＭ　Ｄ－ＭＥＭ（Ｉｎｖｉｔｒｏｇｅｎ社）などを挙げることができる。 The medium used in the step (a) is a basal medium, Neurobasal (registered trademark) (Thermo Fisher Scientific), B27 (registered trademark) (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific). And any combination of additives such as GlutaMAX® (Thermo Fisher Scientific) or L-Glutamin (Thermo Fisher Scientific) (preferably all of the above additives) in the basal medium. In addition, any one selected from the group consisting of histone deacetylase inhibitor, MAPK / ERK kinase inhibitor, STAT3 activator, protein kinase C inhibitor, Wnt signal inhibitor and GSK3β inhibitor is added. It may be a medium. Examples of the basal medium include DMEM (Dulvecco Modified Eagle medium), a mixed medium of DMEM and F12 (DMEM / F12 = 1: 1), Knockout TM D-MEM (Invitrogen), and the like.
　ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、ＳＴＡＴ３活性化剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤およびＧＳＫ３β阻害剤の種類や濃度並びに培養条件は、後記の［第一培養工程について］および［第二培養工程について］にて具体的に記載する。
　上記工程（ａ）は、後記の第一培養工程および第二培養工程を含むことができる。 The types and concentrations of histone deacetylase inhibitors, MAPK / ERK kinase inhibitors, STAT3 activators, protein kinase C inhibitors, Wnt signal inhibitors and GSK3β inhibitors and the culture conditions are described in [First culture step] below. ] And [About the second culture step] will be described in detail.
The above step (a) can include the first culture step and the second culture step described later.
［第一培養工程について］
　本発明における第一培養工程は、多能性幹細胞を、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、およびＳＴＡＴ３活性化剤を含む培地で培養する工程である。 [About the first culture process]
The first culturing step in the present invention is a step of culturing pluripotent stem cells in a medium containing a histone deacetylase inhibitor, a MAPK / ERK kinase inhibitor, and a STAT3 activator.
　第一培養工程において、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、およびＳＴＡＴ３活性化剤を含む培地は、基礎培地に、Ｎｅｕｒｏｂａｓａｌ（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、Ｂ２７（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、Ｎ２（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、１－チオグリセロール、およびＧｌｕｔａＭＡＸ（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）またはＬ－Ｇｌｕｔａｍｉｎ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）などの添加成分を任意に組み合わせて（好ましくは上記の添加成分の全てを組み合わせて）、上記の基礎培地に添加し、さらに、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、およびＳＴＡＴ３活性化剤を添加した培地であってもよい。上記基礎培地としては、ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ　Ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ　ｍｅｄｉｕｍ）、ＤＭＥＭとＦ１２の混合培地（ＤＭＥＭ／Ｆ１２＝１：１）、ＫｎｏｃｋｏｕｔＴＭ　Ｄ－ＭＥＭ（Ｉｎｖｉｔｒｏｇｅｎ社）などを挙げることができる。 In the first culture step, the medium containing histone deacetylase inhibitor, MAPK / ERK kinase inhibitor, and STAT3 activator was added to the basal medium as Neurobasal (registered trademark) (Thermo Fisher Scientific), B27 (registered). Trademarks) (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), 1-thioglycerol, and GlutaMAX® (Registered Trademarks) (Thermo Fisher Scientific) or L-Glutamin (ThermiScientific) or L-Glutamin (Thermic) (Preferably all of the above additives) were added to the above basal medium, and histone deacetylase inhibitors, MAPK / ERK kinase inhibitors, and STAT3 activators were added. It may be a medium. Examples of the basal medium include DMEM (Dulvecco Modified Eagle medium), a mixed medium of DMEM and F12 (DMEM / F12 = 1: 1), Knockout TM D-MEM (Invitrogen), and the like.
　ヒストン脱アセチル化酵素阻害剤としては、バルプロ酸またはその塩（バルプロ酸ナトリウムなど）、酪酸またはその塩（酪酸ナトリウムなど）、トリコスタチンＡ、およびアピシジンなどを使用することができるが、特に限定されない。培地におけるヒストン脱アセチル化酵素阻害剤の濃度は、ヒストン脱アセチル化酵素阻害剤の種類などに応じて適宜設定することができる。例えば、バルプロ酸の場合には、好ましくは１．０μｍｏｌ／Ｌ～５．０ｍｍｏｌ／Ｌであり、より好ましくは０．２ｍｍｏｌ／Ｌ～５．０ｍｍｏｌ／Ｌであり、さらに好ましくは０．５ｍｍｏｌ／Ｌ～２．０ｍｍｏｌ／Ｌである。 As the histone deacetylase inhibitor, valproic acid or a salt thereof (such as sodium valproate), butyric acid or a salt thereof (such as sodium butyrate), tricostatin A, apicidin and the like can be used, but is not particularly limited. .. The concentration of the histone deacetylase inhibitor in the medium can be appropriately set according to the type of the histone deacetylase inhibitor and the like. For example, in the case of valproic acid, it is preferably 1.0 μmol / L to 5.0 mmol / L, more preferably 0.2 mmol / L to 5.0 mmol / L, and even more preferably 0.5 mmol / L. It is ~ 2.0 mmol / L.
　ＭＡＰＫ／ＥＲＫキナーゼ阻害剤（ＭＥＫ阻害剤ともいう）としては、特に限定されないが、例えば、ＰＤ０３２５９０１（Ｎ－［（２Ｒ）－２，３－ジヒドロキシプロポキシ］－３，４－ジフルオロ－２－［（２－フルオロ－４－ヨードフェニル）アミノ］－ベンズアミド；ＣＡＳ登録番号：３９１２１０－１０－９）、Ｕ０１２６（１，４－ジアミノ－２，３－ジシアノ－１，４－ビス［２－アミノフェニルチオ］ブタジエン；ＣＡＳ登録番号：１０９５１１－５８－２）、ＰＤ９８０５９（２－（２－アミノ－３－メトキシフェニル）－４Ｈ－１－ベンゾピラン－４－オン；ＣＡＳ登録番号：１６７８６９－２１－８）、ＰＤ１８４３５２（２－(２－クロロ－４－ヨードフェニルアミノ)－Ｎ－シクロプロピルメトキシ－３，４－ジフルオロベンズアミド；ＣＡＳ登録番号：２１２６３１－７９－３が挙げられる。なかでも、ＰＤ０３２５９０１が好ましい。培地におけるＭＡＰＫ／ＥＲＫキナーゼ阻害剤の濃度は、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤の種類などに応じて適宜設定することができる。例えば、ＰＤ０３２５９０１の場合には、好ましくは０．３μｍｏｌ／Ｌ～５．０μｍｏｌ／Ｌであり、より好ましくは０．６μｍｏｌ／Ｌ～３．０μｍｏｌ／Ｌである。これはＰＤ０３２５９０１を濃度０．６μｍｏｌ／Ｌ以上含む培地で培養することにより、ＤＮＡがより脱メチル化されるためである。本発明の一実施態様では、ＤＮＡ変異を最小限に抑えるためにはＰＤ０３２５９０１を０．８μｍｏｌ／Ｌ～２．０μｍｏｌ／Ｌで添加することが望ましい。 The MAPK / ERK kinase inhibitor (also referred to as MEK inhibitor) is not particularly limited, but for example, PD0325901 (N-[(2R) -2,3-dihydroxypropoxy] -3,4-difluoro-2-[(). 2-Fluoro-4-iodophenyl) amino] -benzamide; CAS registration number: 391210-10-9), U0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio] ] Butadiene; CAS registration number: 109511-58-2), PD98059 (2- (2-amino-3-methoxyphenyl) -4H-1-benzopyran-4-one; CAS registration number: 167869-21-8), PD184352 (2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluorobenzamide; CAS registration number: 212631-79-3; among them, PD0325901 is preferable. The concentration of the MAPK / ERK kinase inhibitor in MAPK / ERK kinase inhibitor can be appropriately set according to the type of the MAPK / ERK kinase inhibitor and the like. For example, in the case of PD0325901, it is preferably 0.3 μmol / L to 5.0 μmol /. It is L, more preferably 0.6 μmol / L to 3.0 μmol / L, because the DNA is more demethylated by culturing in a medium containing PD0325901 at a concentration of 0.6 μmol / L or more. There is. In one embodiment of the invention, it is desirable to add PD0325901 at 0.8 μmol / L to 2.0 μmol / L in order to minimize DNA mutations.
　ＳＴＡＴ３活性化剤としては、特に限定されないが、白血病阻止因子（ＬＩＦ）を挙げることができる。特に、ヒト白血病阻止因子が好ましい。培地におけるＳＴＡＴ３活性化剤の濃度は、ＳＴＡＴ３活性化剤の種類などに応じて適宜設定することができる。例えば、ＬＩＦの場合には、好ましくは０．１ｎｇ／ｍＬ～２００ｎｇ／ｍＬであり、より好ましくは０．２ｎｇ／ｍＬ～１００ｎｇ／ｍＬである。 The STAT3 activator is not particularly limited, but may include a leukemia inhibitory factor (LIF). In particular, human leukemia inhibitory factors are preferred. The concentration of the STAT3 activator in the medium can be appropriately set according to the type of the STAT3 activator and the like. For example, in the case of LIF, it is preferably 0.1 ng / mL to 200 ng / mL, and more preferably 0.2 ng / mL to 100 ng / mL.
　第一培養工程における培養条件は、当業者には自明であり、一例としては、３７℃、５％ＣＯ２、５％Ｏ２条件下を挙げることができる。特に、低酸素（５％Ｏ２）の条件下で培養することが好ましい。 The culture conditions in the first culture step are obvious to those skilled in the art, and examples thereof include 37 ° C., 5% CO 2, and 5% O 2 conditions. In particular, it is preferable to culture under the condition of low oxygen (5% O 2 ).
　第一培養工程における培養期間は特に限定されないが、例えば、１日～５日間、好ましくは２日～４日間培養することができる。 The culture period in the first culture step is not particularly limited, but for example, it can be cultured for 1 to 5 days, preferably 2 to 4 days.
［第二培養工程について］
　本発明における第二培養工程は、第一培養工程の後に、ヒストン脱アセチル化酵素阻害剤を含まずに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、ＧＳＫ３β阻害剤、およびＳＴＡＴ３活性化剤を含む培地で培養する工程である。 [About the second culture process]
In the second culture step in the present invention, after the first culture step, a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, a GSK3β inhibitor, without containing a histone deacetylase inhibitor, And a step of culturing in a medium containing a STAT3 activator.
　第二培養工程において、ヒストン脱アセチル化酵素阻害剤を含まずに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、ＧＳＫ３β阻害剤、およびＳＴＡＴ３活性化剤を含む培地は、基礎培地に、Ｎｅｕｒｏｂａｓａｌ（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、Ｂ２７（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、Ｎ２（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、１－チオグリセロール、およびＧｌｕｔａＭＡＸ（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）またはＬ－Ｇｌｕｔａｍｉｎ　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）などの添加成分を任意に組み合わせて（好ましくは上記の添加成分の全てを組み合わせて）、上記の基礎培地に添加し、さらに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、ＧＳＫ３β阻害剤、およびＳＴＡＴ３活性化剤を添加した培地であってもよい。上記基礎培地としては、ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ　Ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ　ｍｅｄｉｕｍ）、ＤＭＥＭとＦ１２の混合培地（ＤＭＥＭ／Ｆ１２＝１：１）、ＫｎｏｃｋｏｕｔＴＭ　Ｄ－ＭＥＭ（Ｉｎｖｉｔｒｏｇｅｎ社）などを挙げることができる。 In the second culture step, the medium containing the MAPK / ERK kinase inhibitor, protein kinase C inhibitor, Wnt signal inhibitor, GSK3β inhibitor, and STAT3 activator without the histone deacetylase inhibitor Neurobasal (registered trademark) (Thermo Fisher Scientific), B27 (registered trademark) (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), 1-thioglycerol, 1-thioglycerol, and 1-thioglycerol in the basal medium. Additives such as Scientific) or L-Glutamin (Thermo Fisher Scientific) are optionally combined (preferably all of the above additives) and added to the basal medium described above, and further, MAPK / ERK kinase. The medium may be supplemented with an inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, a GSK3β inhibitor, and a STAT3 activator. Examples of the basal medium include DMEM (Dulvecco Modified Eagle medium), a mixed medium of DMEM and F12 (DMEM / F12 = 1: 1), Knockout TM D-MEM (Invitrogen), and the like.
　ＭＡＰＫ／ＥＲＫキナーゼ阻害剤の具体例は上記の通りである。培地におけるＭＡＰＫ／ＥＲＫキナーゼ阻害剤の濃度は、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤の種類などに応じて適宜設定することができる。例えば、ＰＤ０３２５９０１の場合には、好ましくは０．３μｍｏｌ／Ｌ～５．０μｍｏｌ／Ｌであり、より好ましくは０．６μｍｏｌ／Ｌ～３．０μｍｏｌ／Ｌである。これはＰＤ０３２５９０１を濃度０．６μｍｏｌ／Ｌ以上含む培地で培養することにより、ＤＮＡがより脱メチル化されるためである。本発明の一実施態様では、ＤＮＡ変異を最小限に抑えるためにはＰＤ０３２５９０１を０．８μｍｏｌ／Ｌ～２．０μｍｏｌ／Ｌで添加することが望ましい。 Specific examples of the MAPK / ERK kinase inhibitor are as described above. The concentration of the MAPK / ERK kinase inhibitor in the medium can be appropriately set according to the type of the MAPK / ERK kinase inhibitor and the like. For example, in the case of PD0325901, it is preferably 0.3 μmol / L to 5.0 μmol / L, and more preferably 0.6 μmol / L to 3.0 μmol / L. This is because DNA is more demethylated by culturing in a medium containing PD0325901 at a concentration of 0.6 μmol / L or more. In one embodiment of the invention, it is desirable to add PD0325901 at 0.8 μmol / L to 2.0 μmol / L in order to minimize DNA mutations.
　プロテインキナーゼＣ阻害剤（ＰＫＣ阻害剤ともいう）としては、特に限定されないが、例えば、Ｇｏ６９８３（３－［１－［３－（ジメチルアミノ）プロピル］－５－メトキシ－１Ｈ－インドール－３－イル］－４－（１Ｈ－インドール－３－イル）－１Ｈ－ピロール－２，５－ジオン；ＣＡＳ登録番号：１３３０５３－１９－７）、ＧＦ１０９２０３Ｘ（３－（１－（３－ジメチルアミノ）プロピル）－１Ｈ－インドール－３－イル）－４－（１Ｈ－インドール－３－イル）－１Ｈ－ピロール－２，５－ジオン；ＣＡＳ登録番号：１３３０５２－９０－１）が挙げられる。なかでも、Ｇｏ６９８３が好ましい。培地におけるＰＫＣ阻害剤の濃度は、ＰＫＣ阻害剤の種類などに応じて適宜設定することができる。例えば、Ｇｏ６９８３の場合には、好ましくは５０ｎｍｏｌ／Ｌ～１００μｍｏｌ／Ｌであり、より好ましくは１００ｎｍｏｌ／Ｌ～１０μｍｏｌ／Ｌである。 The protein kinase C inhibitor (also referred to as PKC inhibitor) is not particularly limited, but is, for example, Go6983 (3- [1- [3- (dimethylamino) propyl] -5-methoxy-1H-indole-3-yl). ] -4- (1H-Indole-3-yl) -1H-Pyrrole-2,5-dione; CAS registration number: 133053-19-7), GF109203X (3- (1- (3-dimethylamino) propyl)) -1H-indole-3-yl) -4- (1H-indole-3-yl) -1H-pyrrole-2,5-dione; CAS registration number: 133052-90-1). Of these, Go6983 is preferable. The concentration of the PKC inhibitor in the medium can be appropriately set according to the type of the PKC inhibitor and the like. For example, in the case of Go6983, it is preferably 50 nmol / L to 100 μmol / L, and more preferably 100 nmol / L to 10 μmol / L.
　Ｗｎｔシグナル阻害剤としては、特に限定されないが、ＸＡＶ９３９（ｔａｎｋｙｒａｓｅ阻害剤）（ＣＡＳ登録番号：２８４０２８－８９－３）、ＩＷＰ－１、ＩＷＰ－２、ＩＷＰ－３、ＩＷＰ－４、ＩＷＲ－１、５３ＡＨ（以上ｐｏｒｃｕｐｉｎｅ阻害剤）、ＫＹ０２１１１などの低分子化合物およびそれらの誘導体や、ＩＧＦＢＰ４、ＤＫＫ１、Ｗｎｔ－Ｃ５９などのタンパク質が挙げられる。なかでも、ＸＡＶ９３９が好ましい。培地におけるＷｎｔシグナル阻害剤の濃度は、Ｗｎｔシグナル阻害剤の種類などに応じて適宜設定することができる。例えば、ＸＡＶ９３９の場合には、好ましくは５０ｎｍｏｌ／Ｌ～１００μｍｏｌ／Ｌであり、より好ましくは１００ｎｍｏｌ／Ｌ～１０μｍｏｌ／Ｌである。 The Wnt signal inhibitor is not particularly limited, but is limited to XAV939 (proteinase inhibitor) (CAS registration number: 284028-89-3), IWP-1, IWP-2, IWP-3, IWP-4, IWR-1, Examples thereof include low molecular weight compounds such as 53AH (above porcupine inhibitor) and KY02111 and their derivatives, and proteins such as IGFBP4, DKK1 and Wnt-C59. Of these, XAV939 is preferable. The concentration of the Wnt signal inhibitor in the medium can be appropriately set according to the type of the Wnt signal inhibitor and the like. For example, in the case of XAV939, it is preferably 50 nmol / L to 100 μmol / L, and more preferably 100 nmol / L to 10 μmol / L.
　ＧＳＫ３－β阻害剤としては、特に限定されないが、ＣＨＩＲ９９０２１（ＣＡＳ登録番号：２５２９２７－０６－９）が好ましい。培地におけるＧＳＫ３－β阻害剤の濃度は、ＧＳＫ３－β阻害剤の種類などに応じて適宜設定することができる。例えば、ＣＨＩＲ９９０２１の場合には、好ましくは０．０１μｍｏｌ／Ｌ～１．０μｍｏｌ／Ｌであり、より好ましくは０．１μｍｏｌ／Ｌ～０．５μｍｏｌ／Ｌである。 The GSK3-β inhibitor is not particularly limited, but CHIR99021 (CAS Registry Number: 252927-06-9) is preferable. The concentration of the GSK3-β inhibitor in the medium can be appropriately set according to the type of the GSK3-β inhibitor and the like. For example, in the case of CHIR99021, it is preferably 0.01 μmol / L to 1.0 μmol / L, and more preferably 0.1 μmol / L to 0.5 μmol / L.
　ＳＴＡＴ３活性化剤の具体例は上記の通りである。培地におけるＳＴＡＴ３活性化剤の濃度は、ＳＴＡＴ３活性化剤の種類などに応じて適宜設定することができる。例えば、ＬＩＦの場合には、好ましくは０．１ｎｇ／ｍＬ～１００ｎｇ／ｍＬであり、より好ましくは０．２ｎｇ／ｍＬ～２０ｎｇ／ｍＬである。 Specific examples of the STAT3 activator are as described above. The concentration of the STAT3 activator in the medium can be appropriately set according to the type of the STAT3 activator and the like. For example, in the case of LIF, it is preferably 0.1 ng / mL to 100 ng / mL, and more preferably 0.2 ng / mL to 20 ng / mL.
　第二培養工程における培養条件は、当業者には自明であり、一例としては、３７℃、５％ＣＯ２、５％Ｏ２条件下を挙げることができる。特に、低酸素（５％Ｏ２）の条件下で培養することが好ましい。なお、第一および第二培養工程において、培地はアスコルビン酸を含まないことが好ましい。 The culture conditions in the second culture step are self-evident to those skilled in the art, and examples thereof include 37 ° C., 5% CO 2 , and 5% O 2 conditions. In particular, it is preferable to culture under the condition of low oxygen (5% O 2 ). In the first and second culture steps, the medium preferably does not contain ascorbic acid.
　本発明の一実施態様において、第二培養工程は、さらに２つの培養工程に分けて実施することができる。２つの培養工程は、それぞれ、ＧＳＫ３β阻害剤の添加の有無について異なる培地を使用することができる。すなわち、第二培養工程は、ヒストン脱アセチル化酵素阻害剤を含まずに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、およびＳＴＡＴ３活性化剤を含む培地で培養した後、この培地にＧＳＫ３β阻害剤を加えた培地でさらに培養を行う工程であってもよい。 In one embodiment of the present invention, the second culture step can be further divided into two culture steps. The two culture steps can each use different media with or without the addition of a GSK3β inhibitor. That is, in the second culture step, after culturing in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, and a STAT3 activator without containing a histone deacetylase inhibitor. , The step may be a step of further culturing in a medium in which a GSK3β inhibitor is added to this medium.
　第二培養工程における培養期間は特に限定されないが、ヒストン脱アセチル化酵素阻害剤を含まずに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、およびＳＴＡＴ３活性化剤を含む培地で、例えば、３から８日間、好ましくは４から７日間培養した後、この培地にＧＳＫ３β阻害剤を加えた培地で、例えば、３から１０日間、好ましくは４から９日間培養することができる。 The culture period in the second culture step is not particularly limited, but includes a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, and a STAT3 activator without containing a histone deacetylase inhibitor. After culturing in a medium for, for example, 3 to 8 days, preferably 4 to 7 days, the medium can be cultured in a medium containing a GSK3β inhibitor, for example, for 3 to 10 days, preferably 4 to 9 days. ..
　多能性幹細胞に対して、第一および第二培養工程を含む工程（ａ）を実施することにより、「培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した多能性幹細胞」を得ることができる。本発明の一実施態様において、「培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した多能性幹細胞」は、培養前の細胞と比較して特定の分化細胞に分化する能力が高い多能性幹細胞であるということができる。工程（ａ）を実施することにより得られた、「培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した多能性幹細胞」では、未分化状態は維持されており、且つ体細胞に初期化因子を導入して得られる未分化細胞と比較して特定の分化細胞に分化する能力が亢進されている。 By performing step (a) including the first and second culture steps on the pluripotent stem cells, "the ratio of 5-methylcytosine in the total cytosine of the DNA as compared with the pluripotent stem cells before culture". It is possible to obtain "pluripotent stem cells with reduced". In one embodiment of the present invention, "pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of DNA is reduced as compared with the pluripotent stem cells before culture" is specified as compared with the cells before culture. It can be said that it is a pluripotent stem cell having a high ability to differentiate into a differentiated cell. In the "pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of the DNA was reduced as compared with the pluripotent stem cells before culture" obtained by carrying out the step (a), the undifferentiated state was It is maintained and has an enhanced ability to differentiate into specific differentiated cells as compared to undifferentiated cells obtained by introducing a reprogramming factor into somatic cells.
　工程（ａ）が完了したことは、メチル化度を測定し、ＤＮＡのメチル化度が低下したことで評価することができる。 Completion of step (a) can be evaluated by measuring the degree of methylation and reducing the degree of DNA methylation.
　また、工程（ａ）で得られる多能性幹細胞は、ＤＮＡ変異の発生率が低いことが好ましい。ＤＮＡ変異の発生率が低いことは、例えば、２０番染色体長腕領域のコピー数を、常法（例えば、リアルタイムＰＣＲなど）により測定することにより評価することができる。工程（ａ）で得られる多能性幹細胞における２０番染色体長腕領域のコピー数は、好ましくは１．５～４．５であり、より好ましくは１．５～３．５であり、さらに好ましくは１．５～２．５である。 Further, it is preferable that the pluripotent stem cells obtained in step (a) have a low incidence of DNA mutations. The low incidence of DNA mutations can be evaluated, for example, by measuring the copy number of the long arm region of chromosome 20 by a conventional method (for example, real-time PCR). The copy number of the long arm region of chromosome 20 in the pluripotent stem cell obtained in the step (a) is preferably 1.5 to 4.5, more preferably 1.5 to 3.5, and even more preferably. Is 1.5 to 2.5.
　工程（ａ）で得られる多能性幹細胞の未分化性は、特に限定されないが、未分化性を定義する遺伝子の発現を測定することにより評価することができる。未分化性を定義する遺伝子の発現の測定方法は特に限定されないが、例えば、定量的ＲＴ－ＰＣＲにより測定を行うことができる。ＲＴ－ＰＣＲは、測定対象となるｍＲＮＡを鋳型としてｃＤＮＡを合成し、このｃＤＮＡを鋳型としてＰＣＲにより増幅する方法である。定量的ＲＴ－ＰＣＲとしては、例えば、クエンチャー蛍光色素とレポーター蛍光色素が結合されたプライマーを用いてＰＣＲを行って各サイクル毎に増幅産物量を定量し、検出される蛍光強度が急激に増大するサイクル数から、試料中の鋳型ＤＮＡ量を測定する方法（リアルタイムＰＣＲ）等を挙げることができる。定量的ＲＴ－ＰＣＲの手法は本技術分野において周知であり、市販のキットを使用して実施することもできる。定量的ＲＴ－ＰＣＲによれば、遺伝子の発現量またはコピー数を、対照となるハウスキーピング遺伝子（例えば、ＧＡＰＤＨ遺伝子）の発現量またはコピー数に対する相対値として測定することができる。なお、遺伝子のｍＲＮＡの測定は、通常のＲＴ－ＰＣＲなどによりｍＲＮＡの増幅を行うことにより得た増幅産物をゲル電気泳動にかけ、染色後、バンド強度を測定することによっても行うことができる。あるいは、ＤＮＡチップを用いて遺伝子のｍＲＮＡまたはｃＤＮＡを検出または定量することもできる。 The undifferentiated state of the pluripotent stem cell obtained in the step (a) is not particularly limited, but can be evaluated by measuring the expression of the gene that defines the undifferentiated state. The method for measuring the expression of the gene that defines undifferentiated state is not particularly limited, but for example, it can be measured by quantitative RT-PCR. RT-PCR is a method of synthesizing cDNA using the mRNA to be measured as a template and amplifying it by PCR using this cDNA as a template. As quantitative RT-PCR, for example, PCR is performed using a primer in which a quencher fluorescent dye and a reporter fluorescent dye are bound to quantify the amount of amplification product in each cycle, and the detected fluorescence intensity increases sharply. From the number of cycles to be performed, a method of measuring the amount of template DNA in a sample (real-time PCR) and the like can be mentioned. Quantitative RT-PCR techniques are well known in the art and can also be performed using commercially available kits. According to quantitative RT-PCR, the expression level or the number of copies of a gene can be measured as a relative value to the expression level or the number of copies of a control housekeeping gene (for example, GAPDH gene). The mRNA of a gene can also be measured by subjecting the amplification product obtained by amplifying the mRNA by ordinary RT-PCR or the like to gel electrophoresis, staining, and then measuring the band intensity. Alternatively, DNA chips can be used to detect or quantify gene mRNA or cDNA.
　未分化性を定義する遺伝子としては、特に限定されないが、ＮＡＮＯＧ、ＰＯＵ５Ｆ１、ＬＩＮ２８、ＳＯＸ２、ＤＮＭＴ３Ｂなどを挙げることができる。本発明の工程（ａ）で得られる多能性幹細胞における未分化性を定義する遺伝子の発現量が、対照群（例えば、工程（ａ）に供していない多能性幹細胞）における同じ遺伝子の発現量と同等であるか、またはそれ以上である場合に未分化性が維持されていると定義し、同等とは対照群における同じ遺伝子の発現量の８０～１１０％とする。 The gene that defines undifferentiated state is not particularly limited, and examples thereof include NANOG, POU5F1, LIN28, SOX2, and DNMT3B. The expression level of the gene defining undifferentiation in the pluripotent stem cells obtained in the step (a) of the present invention is the expression of the same gene in the control group (for example, the pluripotent stem cells not subjected to the step (a)). It is defined that undifferentiated state is maintained when the amount is equal to or higher than the amount, and the equivalent is 80 to 110% of the expression level of the same gene in the control group.
　好ましくは、本発明の工程（ａ）で得られる多能性幹細胞におけるＮＡＮＯＧの発現量は、対照群におけるＮＡＮＯＧの発現量と同等であるか、またはそれ以上である。
　好ましくは、本発明の工程（ａ）で得られる多能性幹細胞におけるＰＯＵ５Ｆ１の発現量は、対照群におけるＰＯＵ５Ｆ１の発現量と同等であるか、またはそれ以上である。
　好ましくは、本発明の工程（ａ）で得られる多能性幹細胞におけるＬＩＮ２８の発現量は、対照群におけるＬＩＮ２８の発現量と同等であるか、またはそれ以上である。
　好ましくは、本発明の工程（ａ）で得られる多能性幹細胞におけるＳＯＸ２の発現量は、対照群におけるＳＯＸ２の発現量と同等であるか、またはそれ以上である。
　好ましくは、本発明の工程（ａ）で得られる多能性幹細胞におけるＤＮＭＴ３Ｂの発現量は、対照群におけるＤＮＭＴ３Ｂの発現量と同等であるか、またはそれ以上である。 Preferably, the expression level of NANOG in the pluripotent stem cells obtained in the step (a) of the present invention is equal to or higher than the expression level of NANOG in the control group.
Preferably, the expression level of POU5F1 in the pluripotent stem cells obtained in the step (a) of the present invention is equal to or higher than the expression level of POU5F1 in the control group.
Preferably, the expression level of LIN28 in the pluripotent stem cells obtained in the step (a) of the present invention is equal to or higher than the expression level of LIN28 in the control group.
Preferably, the expression level of SOX2 in the pluripotent stem cells obtained in the step (a) of the present invention is equal to or higher than the expression level of SOX2 in the control group.
Preferably, the expression level of DNMT3B in the pluripotent stem cells obtained in the step (a) of the present invention is equal to or higher than the expression level of DNMT3B in the control group.
　好ましくは、工程（ａ）で得られる多能性幹細胞におけるＮＡＮＯＧの発現コピー数は、ＧＡＰＤＨの発現コピー数に対する比率として、０．０１以上、０．０２以上、０．０３以上、０．０４以上、または０．０５以上であってもよい。 Preferably, the number of expressed copies of NANOG in the pluripotent stem cell obtained in step (a) is 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more as a ratio to the number of expressed copies of GAPDH. , Or may be 0.05 or more.
　好ましくは、工程（ａ）で得られる多能性幹細胞におけるＰＯＵ５Ｆ１の発現コピー数は、ＧＡＰＤＨの発現コピー数に対する比率として、０．０６以上、０．０７以上、０．０８、０．０９以上、０．１以上、または０．１２以上であってもよい。 Preferably, the number of expressed copies of POU5F1 in the pluripotent stem cells obtained in step (a) is 0.06 or more, 0.07 or more, 0.08, 0.09 or more as a ratio to the number of expressed copies of GAPDH. It may be 0.1 or more, or 0.12 or more.
　好ましくは、工程（ａ）で得られる多能性幹細胞におけるＬＩＮ２８の発現コピー数は、ＧＡＰＤＨの発現コピー数に対する比率として、０．００３以上、０．００４以上、０．００５以上、０．００６以上、０．００７以上、または０．００８以上であってもよい。 Preferably, the number of expressed copies of LIN28 in the pluripotent stem cell obtained in step (a) is 0.003 or more, 0.004 or more, 0.005 or more, 0.006 or more as a ratio to the number of expressed copies of GAPDH. , 0.007 or more, or 0.008 or more.
　工程（ａ）で得られる多能性幹細胞におけるＳＯＸ２の発現コピー数は、ＧＡＰＤＨの発現コピー数に対する比率として、好ましくは５．０×１０－５以上であり、より好ましくは６．０×１０－５以上であり、さらに好ましくは７．０×１０－５以上であり、特に好ましくは８．０×１０－５以上である。 The number of expressed copies of SOX2 in the pluripotent stem cells obtained in step (a) is preferably 5.0 × 10-5 or more, more preferably 6.0 × 10 − as a ratio to the number of expressed copies of GAPDH. It is 5 or more, more preferably 7.0 × 10-5 or more, and particularly preferably 8.0 × 10-5 or more.
　工程（ａ）で得られる多能性幹細胞が有する、内胚葉、中胚葉および外胚葉の何れか一つ以上に分化する能力は、培養前の多能性幹細胞が有する、内胚葉、中胚葉および外胚葉の何れか一つ以上に分化する能力よりも向上している。
　より好ましくは、工程（ａ）で得られる多能性幹細胞が有する内胚葉および外胚葉に分化する能力は、培養前の多能性幹細胞が有する内胚葉および外胚葉に分化する能力よりもそれぞれ向上しているか、または工程（ａ）で得られる多能性幹細胞が有する内胚葉および中胚葉に分化する能力は、培養前の多能性幹細胞が有する内胚葉および中胚葉に分化する能力よりもそれぞれ向上している。
　特により好ましくは、工程（ａ）で得られる多能性幹細胞が有する内胚葉に分化する能力は、培養前の多能性幹細胞が有する内胚葉に分化する能力よりも向上している。 The ability of pluripotent stem cells obtained in step (a) to differentiate into any one or more of endoderm, mesoderm and ectoderm is possessed by pluripotent stem cells before culture, endoderm, mesoderm and It is improved over the ability to differentiate into any one or more of the ectoderm.
More preferably, the ability of the pluripotent stem cell obtained in step (a) to differentiate into endoderm and ectoderm is higher than the ability of the pluripotent stem cell before culture to differentiate into endoderm and ectoderm, respectively. The ability of pluripotent stem cells obtained in step (a) to differentiate into endoderm and mesoderm is greater than the ability of precultured pluripotent stem cells to differentiate into endoderm and mesoderm, respectively. It is improving.
Particularly preferably, the ability of the pluripotent stem cell obtained in step (a) to differentiate into an endoderm is higher than the ability of the pluripotent stem cell before culturing to differentiate into an endoderm.
　さらに好ましくは、工程（ａ）で得られる多能性幹細胞は、内胚葉、中胚葉および外胚葉の全てに分化することができる細胞である。 More preferably, the pluripotent stem cell obtained in step (a) is a cell capable of differentiating into all endoderm, mesoderm and ectoderm.
　本発明における特定の分化細胞は、好ましくは、内胚葉系譜の細胞であり、より好ましくは、肝細胞である。 The specific differentiated cell in the present invention is preferably a cell of endoderm lineage, and more preferably a hepatocyte.
　本発明において、細胞が有する内胚葉に分化する能力、細胞が有する中胚葉に分化する能力、および細胞が有する外胚葉に分化する能力は、細胞を内胚葉、中胚葉または外胚葉
に分化させ、上記の三胚葉系譜に分化させた細胞における各胚葉特異的遺伝子の発現を測定することにより評価することができる。 In the present invention, the ability of a cell to differentiate into an endoderm, the ability of a cell to differentiate into a mesodermal, and the ability of a cell to differentiate into an ectoderm can be used to differentiate a cell into an endoderm, a mesodermal or an ectoderm. It can be evaluated by measuring the expression of each germ layer-specific gene in the cells differentiated into the above three germ layer lineages.
　各胚葉特異的遺伝子の発現の測定方法は特に限定されないが、例えば、定量的ＲＴ－ＰＣＲ法により測定を行うことができる。
　内胚葉特異的遺伝子としては、特に限定されないが、ＳＯＸ１７およびＦＯＸＡ２などを挙げることができる。
　中胚葉特異的遺伝子としては、特に限定されないが、ＴおよびＰＤＧＦＲＡなどを挙げることができる。
　外胚葉特異的遺伝子としては、特に限定されないが、ＰＡＸ６およびＭＡＰ２などを挙げることができる。 The method for measuring the expression of each germ layer-specific gene is not particularly limited, but for example, the measurement can be performed by a quantitative RT-PCR method.
The endoderm-specific gene is not particularly limited, and examples thereof include SOX17 and FOXA2.
The mesoderm-specific gene is not particularly limited, and examples thereof include T and PDGFRA.
The ectoderm-specific gene is not particularly limited, and examples thereof include PAX6 and MAP2.
　工程（ａ）で得られる多能性幹細胞が有する、内胚葉に分化する能力が、培養前の多能性幹細胞が有する内胚葉に分化する能力よりも向上している場合としては、培養前の多能性幹細胞を内胚葉に分化させた細胞におけるＳＯＸ１７の発現量に対する、工程（ａ）で得られる多能性幹細胞を内胚葉に分化させた細胞におけるＳＯＸ１７の相対発現量が、好ましくは２倍以上であり、より好ましくは３倍以上、５倍以上、１０倍以上、１５倍以上、２０倍以上、２５倍以上、３０倍以上、３５倍以上または４０倍以上である場合を挙げることができる。 When the ability of the pluripotent stem cells obtained in step (a) to differentiate into endoderm is higher than the ability of the pluripotent stem cells before culturing to differentiate into endoderm, the ability to differentiate into endoderm is improved before culturing. The relative expression level of SOX17 in the cells obtained in step (a) differentiated into endoderm is preferably twice as much as the expression level of SOX17 in the cells obtained by differentiating the pluripotent stem cells into endoderm. More preferably, it may be 3 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 25 times or more, 30 times or more, 35 times or more or 40 times or more. ..
　工程（ａ）で得られる多能性幹細胞が有する、内胚葉に分化する能力が、培養前の多能性幹細胞が有する内胚葉に分化する能力よりも向上している場合としては、培養前の多能性幹細胞を内胚葉に分化させた細胞におけるＦＯＸＡ２の発現量に対する、工程（ａ）で得られる多能性幹細胞を内胚葉に分化させた細胞におけるＦＯＸＡ２の相対発現量が、好ましくは２倍以上であり、より好ましくは３倍以上、５倍以上、８倍以上、９倍以上、１０倍以上または１２倍以上である場合を挙げることができる。 When the ability of the pluripotent stem cells obtained in step (a) to differentiate into endoderm is higher than the ability of the pluripotent stem cells before culturing to differentiate into endoderm, the ability to differentiate into endoderm is improved before culturing. The relative expression level of FOXA2 in the cells obtained by differentiating the pluripotent stem cells into the endoderm is preferably doubled with respect to the expression level of FOXA2 in the cells obtained by differentiating the pluripotent stem cells into the endoderm. The above, more preferably 3 times or more, 5 times or more, 8 times or more, 9 times or more, 10 times or more, or 12 times or more can be mentioned.
　工程（ａ）で得られる多能性幹細胞が有する、中胚葉に分化する能力が、培養前の多能性幹細胞が有する中胚葉に分化する能力よりも向上している場合としては、培養前の多能性幹細胞を中胚葉に分化させた細胞におけるＴの発現量に対する、工程（ａ）で得られる多能性幹細胞を内胚葉に分化させた細胞におけるＴの相対発現量が、好ましくは２以上であり、より好ましくは３以上、５以上、８以上、９以上、または１０以上である場合を挙げることができる。 When the ability of the pluripotent stem cells obtained in step (a) to differentiate into mesoderm is higher than the ability of the pluripotent stem cells before culturing to differentiate into mesoderm, the ability to differentiate into mesoderm is improved before culturing. The relative expression level of T in the cells obtained in the step (a) differentiated into mesoderm is preferably 2 or more with respect to the expression level of T in the cells obtained by differentiating the pluripotent stem cells into mesoderm. It is more preferable that the number is 3 or more, 5 or more, 8 or more, 9 or more, or 10 or more.
　工程（ａ）で得られる多能性幹細胞が有する、中胚葉に分化する能力が、培養前の多能性幹細胞が有する中胚葉に分化する能力よりも向上している場合としては、培養前の多能性幹細胞を中胚葉に分化させた細胞におけるＰＤＧＦＲＡの発現量に対する、工程（ａ）で得られる多能性幹細胞を内胚葉に分化させた細胞におけるＰＤＧＦＲＡの相対発現量が、好ましくは１．１以上であり、より好ましくは１．２以上、１．３以上または１．４以上である場合を挙げることができる。 When the ability of the pluripotent stem cells obtained in step (a) to differentiate into mesoderm is higher than the ability of the pluripotent stem cells before culturing to differentiate into mesoderm, the ability to differentiate into mesoderm is improved before culturing. The relative expression level of PDGFRA in the cells obtained by differentiating the pluripotent stem cells into mesoderm with respect to the expression level of PDGFRA in the cells obtained by differentiating the pluripotent stem cells into mesoderm is preferably 1. The case where it is 1 or more, more preferably 1.2 or more, 1.3 or more, or 1.4 or more can be mentioned.
　工程（ａ）で得られる多能性幹細胞が有する、外胚葉に分化する能力が、培養前の多能性幹細胞が有する外胚葉に分化する能力よりも向上している場合としては、培養前の多能性幹細胞に分化させた細胞におけるＰＡＸ６の発現量に対する、工程（ａ）で得られる多能性幹細胞を外胚葉に分化させた細胞におけるＰＡＸ６の相対発現量が、好ましくは１．１以上であり、より好ましくは１．２以上、１．３以上、１．４以上または１．５以上である場合を挙げることができる。 When the ability of the pluripotent stem cells obtained in step (a) to differentiate into ectoderm is higher than the ability of the pluripotent stem cells before culturing to differentiate into ectoderm, the ability to differentiate into ectoderm is improved before culturing. The relative expression level of PAX6 in the ectoderm-differentiated cells of the pluripotent stem cells obtained in step (a) is preferably 1.1 or more with respect to the expression level of PAX6 in the cells differentiated into pluripotent stem cells. Yes, more preferably 1.2 or more, 1.3 or more, 1.4 or more, or 1.5 or more.
　工程（ａ）で得られる多能性幹細胞が有する、外胚葉に分化する能力が、培養前の多能性幹細胞が有する外胚葉に分化する能力よりも向上している場合としては、培養前の多能性幹細胞を外胚葉に分化させた細胞におけるＭＡＰ２の発現量に対する、工程（ａ）で得られる多能性幹細胞を外胚葉に分化させた細胞におけるＭＡＰ２の相対発現量が、好ましくは１．１以上であり、より好ましくは１．２以上、または１．３以上である場合を挙げることができる。 When the ability of the pluripotent stem cells obtained in step (a) to differentiate into ectoderm is higher than the ability of the pluripotent stem cells before culturing to differentiate into ectoderm, the ability to differentiate into ectoderm is improved before culturing. The relative expression level of MAP2 in the cells obtained by differentiating the pluripotent stem cells into the ectoderm with respect to the expression level of MAP2 in the cells obtained by differentiating the pluripotent stem cells into the ectoderm is preferably 1. The case where it is 1 or more, more preferably 1.2 or more, or 1.3 or more can be mentioned.
［第三培養工程について］
　本発明の特定の実施態様において、本発明の工程（ａ）は、上記第二培養工程の後に、ｂＦＧＦを含む未分化細胞を維持培養するための培地で培養する工程を含んでいてもよい。この工程を、本明細書中、第三培養工程という。第三培養工程は、本発明の分化細胞の製造方法の必須の工程ではない。「培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した多能性幹細胞」は、上記第一および第二培養工程を実施することにより、得られるためである。 [About the third culture process]
In a specific embodiment of the present invention, the step (a) of the present invention may include a step of culturing in a medium for maintaining and culturing undifferentiated cells containing bFGF after the second culturing step. This step is referred to as a third culture step in the present specification. The third culture step is not an essential step in the method for producing differentiated cells of the present invention. Because "pluripotent stem cells in which the ratio of 5-methylcytosine in total cytosine of DNA is reduced as compared with pluripotent stem cells before culturing" can be obtained by carrying out the above-mentioned first and second culturing steps. Is.
　上記第一および第二培養工程を実施して得られた「培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した多能性幹細胞」は、培養前の多能性幹細胞に比較して、増殖速度が遅いため、同じ収率を得るために時間とコストがかかる場合がある。上記第一および第二培養工程を実施して得られた「培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した多能性幹細胞」を、第三培養工程において、さらに培養することにより、特定の分化細胞に分化する能力が高い状態を維持しつつ、増殖能を亢進させることができる。 The "pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of DNA was reduced as compared with the pluripotent stem cells before culturing" obtained by carrying out the above first and second culturing steps were obtained before culturing. Due to the slower growth rate compared to pluripotent stem cells, it may take time and cost to obtain the same yield. The third "pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of DNA was reduced as compared with the pluripotent stem cells before culturing" obtained by carrying out the first and second culture steps. By further culturing in the culturing step, it is possible to enhance the proliferative ability while maintaining a state in which the ability to differentiate into specific differentiated cells is high.
　未分化細胞を維持培養するための培地としては、ｍＴｅＳＲ（登録商標）１（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）またはＳｔｅｍＦｌｅｘ（登録商標）などの市販の培地を使用することができる。あるいはまた、例えば、基礎培地として、ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ　Ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ　ｍｅｄｉｕｍ）、ＤＭＥＭとＦ１２の混合培地（ＤＭＥＭ／Ｆ１２＝１：１）、ＫｎｏｃｋｏｕｔＴＭ　Ｄ－ＭＥＭ（Ｉｎｖｉｔｒｏｇｅｎ社）などが挙げられ、代替血清（ＫＳＲ；ＫｎｏｃｋｏｕｔＴＭ　Ｓｅｒｕｍ　Ｒｅｐｌａｃｅｍｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ社））、ウシ胎児血清（ＦＢＳ）、非必須アミノ酸（ＮＥＡＡ）、Ｌ－グルタミン、２－メルカプトエタノール、抗生物質（例えば、ストレプトマイシン、ペニシリン、ピューロマイシン、マイトマイシン）、ｂＦＧＦ等の添加成分を任意に組み合わせて、上記いずれかの基礎培地に添加したものも、未分化細胞を維持培養の際の培地として、挙げることができる。維持培養の際の培地は、アスコルビン酸を含まないことが好ましい。維持培養の培養条件は、３７℃、５％ＣＯ２、１０％Ｏ２条件下などが好ましいが、特に限定されない。 As a medium for maintaining and culturing undifferentiated cells, a commercially available medium such as mTeSR (registered trademark) 1 (Stemcell Technologies) or StemFlex (registered trademark) can be used. Alternatively, for example, as the basal medium, DMEM (Dulvecco Modified Eagle medium), a mixed medium of DMEM and F12 (DMEM / F12 = 1: 1), Knockout TM D-MEM (Invitrogen), and the like can be mentioned as alternative sera (DMEM). KSR; Knockout TM Medium Replacement (Invitrogen)), fetal bovine serum (FBS), non-essential amino acids (NEAA), L-glutamine, 2-mercaptoethanol, antibiotics (eg, streptomycin, penicillin, puromycin, mitomycin), An arbitrary combination of additive components such as bFGF and addition to any of the above basal media can also be mentioned as a medium for maintenance culture of undifferentiated cells. The medium for maintenance culture preferably does not contain ascorbic acid. The culture conditions for the maintenance culture are preferably 37 ° C., 5% CO 2 , 10% O 2 and the like, but are not particularly limited.
　未分化細胞を維持培養するための培地での培養期間は、特に限定されないが、例えば、１から３週間、好ましくは１から２週間培養することができる。１から３週間あるいは１から２週間の培養期間であれば、特定の分化細胞に分化する能力を維持しつつ、増殖能を亢進させることができる。この培養期間に、継代を行うことができる。継代培養を行うことにより、細胞密度を未分化性の維持に適した状態に保つためである。 The culture period in the medium for maintaining and culturing undifferentiated cells is not particularly limited, but can be cultured, for example, for 1 to 3 weeks, preferably 1 to 2 weeks. During the culture period of 1 to 3 weeks or 1 to 2 weeks, the proliferative ability can be enhanced while maintaining the ability to differentiate into specific differentiated cells. Subculture can be performed during this culture period. This is to maintain the cell density in a state suitable for maintaining undifferentiated state by performing subculture.
［工程（ｂ）］
　本発明の工程（ｂ）は、工程（ａ）で得られる多能性幹細胞を、サイトカインを含む培地で培養することにより、特定の分化細胞を得る工程（ｂ）を含む。工程（ｂ）により得られる分化細胞の種類は、特に限定されない。所望により、内胚葉系譜の細胞、中胚葉系譜の細胞、または外胚葉系譜の細胞に分化誘導することができ、好ましくは内胚葉系譜の細胞、例えば肝細胞に分化誘導することができる。 [Step (b)]
The step (b) of the present invention includes a step (b) of obtaining specific differentiated cells by culturing the pluripotent stem cells obtained in the step (a) in a medium containing a cytokine. The type of differentiated cells obtained in step (b) is not particularly limited. If desired, differentiation can be induced into endoderm lineage cells, mesoderm lineage cells, or ectoderm lineage cells, and preferably into endoderm lineage cells, such as hepatocytes.
　各種細胞への分化誘導の培養条件（培養温度など）は、動物細胞の培養において一般に採用されている条件を採用することができる。すなわち、例えば３７℃、５％ＣＯ２の環境下で培養することができる。また、基礎培地として、イスコフ改変ダルベッコ培地（ＩＭＤＭ）（ＧＩＢＣＯ社等）、ハムＦ１２培地（ＨａｍＦ１２）（ＳＩＧＭＡ社、Ｇｉｂｃｏ社等）、ダルベッコ改変イーグル培地（Ｄ－ＭＥＭ）（ナカライテスク株式会社、シグマ社、Ｇｉｂｃｏ社等）、グラスゴー基本培地（Ｇｉｂｃｏ社等）、ＲＰＭＩ１６４０培地等を用いることができる。二種以上の基礎培地を併用することにしてもよい。培地に添加可能な成分の例としてウシ血清アルブミン（ＢＳＡ）、抗生物質、２－メルカプトエタノール、ＰＶＡ、非必須アミノ酸（ＮＥＡＡ）、インスリン、トランスフェリン、セレニウムを挙げることができる。 As the culture conditions (culture temperature, etc.) for inducing differentiation into various cells, conditions generally adopted in the culture of animal cells can be adopted. That is, it can be cultured in an environment of, for example, 37 ° C. and 5% CO 2 . In addition, as basal medium, Iskov-modified Dulbecco medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, Gibco, etc.), Dulbecco-modified Eagle's medium (D-MEM) (Nacalai Tesque, Inc., Sigma). , Gibco, etc.), Grasgo basic medium (Gibco, etc.), RPMI1640 medium, and the like can be used. Two or more kinds of basal media may be used in combination. Examples of components that can be added to the medium include bovine serum albumin (BSA), antibiotics, 2-mercaptoethanol, PVA, non-essential amino acids (NEAA), insulin, transferrin, and selenium.
　工程（ｂ）の培地は、上記基礎培地に、サイトカインを含むことができる。サイトカインの種類は、特に限定されないがアクチビンＡ、ＢＭＰ４、ｂＦＧＦ、ＨＧＦ、Ｏｎｃｏｓｔａｔｉｎ　Ｍ、ＶＥＧＦ、ＩＬ－１１、ＩＬ－６、ＩＧＦ－１、ＳＣＦ、ＴＰＯ、およびＴＧＦ－β等を挙げることができる。肝細胞の分化誘導をする場合には、例えばアクチビンＡ、ＢＭＰ４、ｂＦＧＦ、ＨＧＦ、およびＯｎｃｏｓｔａｔｉｎ　Ｍからなる群から選択される少なくとも１つを含むことができる。工程（ｂ）の培養期間は、１から４週間であってもよく、好ましくは１から３週間である。典型的には培養皿などを用いて二次元的に細胞を培養する。但し、ゲル状の培養基材あるいは３次元培養プレートなどを用いた３次元培養を実施することにしてもよい。 The medium of step (b) can contain cytokines in the basal medium. The type of cytokine is not particularly limited, and examples thereof include activin A, BMP4, bFGF, HGF, Oncostatin M, VEGF, IL-11, IL-6, IGF-1, SCF, TPO, and TGF-β. When inducing differentiation of hepatocytes, for example, at least one selected from the group consisting of activin A, BMP4, bFGF, HGF, and Oncostatin M can be included. The culture period of step (b) may be 1 to 4 weeks, preferably 1 to 3 weeks. Typically, cells are two-dimensionally cultured using a culture dish or the like. However, three-dimensional culture may be carried out using a gel-like culture substrate, a three-dimensional culture plate, or the like.
　分化誘導する方法は、特に限定されない。例えば、市販のＳｔｅｍＤｉｆｆ(登録商標)Ｔｒｉｌｉｎｅａｇｅ　Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ　Ｋｉｔ（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）を用いて、内胚葉、中胚葉および外胚葉のそれぞれに分化誘導することができる。 The method of inducing differentiation is not particularly limited. For example, a commercially available StemDiff (registered trademark) Trillineage Differentiation Kit (Stemcell Technologies) can be used to induce differentiation into endoderm, mesoderm and ectoderm, respectively.
　また、肝細胞への分化誘導は、後記する実施例４に記載の条件で細胞を培養することにより行うことができる。具体的には、細胞をｍＴｅＳＲ（登録商標）１（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）またはＳｔｅｍＦｌｅｘ（登録商標）に播種し、１日目～５日目は、Ｂ２７（登録商標）およびＡｃｔｉｖｉｎ　Ａを含む培地で培養し、６日目から１０日目は、Ｂ２７（登録商標）、ＢＭＰ４、およびｂＦＧＦを含む培地で培養し、１１日目から１５日目は、Ｂ２７（登録商標）およびＨＧＦを含む培地で培養し、１６日目から２０日目は、Ｏｎｃｏｓｔａｔｉｎ　Ｍを含むＨＢＭ（登録商標）で培養することにより、肝細胞に分化誘導することができる。なお、肝細胞への分化誘導は、成熟化肝細胞のマーカーであるＡＳＧＰＲ１の発現をフローサイトメーターにて解析することにより確認することができる。また、肝細胞への分化誘導は培養上清中に分泌されたアルブミンの量を測定することによっても確認することができる。 Further, the induction of differentiation into hepatocytes can be performed by culturing the cells under the conditions described in Example 4 described later. Specifically, cells are seeded in mTeSR® 1 (Stemcell Technologies) or StemFlex® and cultured in a medium containing B27® and Activin A on the 1st to 5th days. , 6th to 10th days were cultured in a medium containing B27®, BMP4, and bFGF, and 11th to 15th days were cultured in a medium containing B27® and HGF. From the 16th day to the 20th day, differentiation can be induced into hepatocytes by culturing in HBM (registered trademark) containing Oncostatin M. The induction of differentiation into hepatocytes can be confirmed by analyzing the expression of ASGPR1, which is a marker of mature hepatocytes, with a flow cytometer. Induction of differentiation into hepatocytes can also be confirmed by measuring the amount of albumin secreted in the culture supernatant.
　ＡＳＧＰＲ１の発現について、具体的には、後記の実施例４の手順に準じて、分化誘導後２０日目の細胞について、ＢＤ　ＰｈａｒｍｉｎｇｅｎＴＭ　ＰＥ　Ｍｏｕｓｅ　Ａｎｔｉ－ＡＳＧＰＲ１抗体（日本ベクトン・ディッキンソン株式会社）にて染色し、フローサイトメトリーＡｔｔｕｎｅＴＭ　ＮｘＴ　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）にてＡＳＧＰＲ１の陽性率を解析すればよい。上記方法で解析したＡＳＧＰＲ１の陽性率は、好ましくは５％以上、６％以上、７％以上、８％以上、９％以上であり、より好ましくは１０％以上、１１％以上、１２％以上、１３％以上、１４％以上であり、さらに好ましくは１５％以上、１６％以上、１７％以上、１８％以上、１９％以上であり、特に好ましくは２０％以上、２１％以上、２２％以上、２３％以上、２４％以上、または２５％以上である。本発明の工程（ａ）を行うことにより、工程（ａ）を行わない場合と比べて、ＡＳＧＰＲ１の陽性率が１．１～１００倍上昇することが好ましく、１．２～１００倍上昇することがさらに好ましく、２～５０倍上昇することが最も好ましい。 Regarding the expression of ASGPR1, specifically, according to the procedure of Example 4 described later, the cells 20 days after the induction of differentiation were subjected to BD Pharmingen TM PE Mouse Anti-ASGPR1 antibody (Becton Dickinson Japan, Inc.). The cells may be stained and the positive rate of ASGPR1 may be analyzed by flow cytometry Attune TM NxT (Thermo Fisher Scientific). The positive rate of ASGPR1 analyzed by the above method is preferably 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, and more preferably 10% or more, 11% or more, 12% or more. 13% or more, 14% or more, more preferably 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, particularly preferably 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, or 25% or more. By performing the step (a) of the present invention, the positive rate of ASGPR1 is preferably increased 1.1 to 100 times, and 1.2 to 100 times, as compared with the case where the step (a) is not performed. Is more preferable, and it is most preferable that the increase is 2 to 50 times.
　アルブミンの分泌量について、具体的には、後記の実施例４の手順に準じて、分化誘導後２０日目の培養上清を回収し、培養上清中に分泌されたアルブミン量をＥＬＩＳＡ　Ｓｔａｒｔｅｒ　Ａｃｃｅｓｓｏｒｙ　Ｋｉｔ、Ｅｎｚｙｍｅ　Ｓｕｂｓｔｒａｔｅ、ＴＭＢ、Ｈｕｍａｎ　Ａｌｂｕｍｉｎ　ＥＬＩＳＡ　Ｑｕａｎｔｉｔａｔｉｏｎ　Ｓｅｔ（Ｂｅｔｈｙｌ　Ｌａｂｏｒａｔｏｒｉｅｓ，　Ｉｎｃ）を用いてＥｎＳｐｉｒｅTMプレートリーダー（ＰｅｒｋｉｎＥｌｍｅｒ）により定量すればよい。上記方法で解析したアルブミンの分泌量は、３０ｎｇ／ｍＬ以上、４０ｎｇ／ｍＬ以上、５０ｎｇ／ｍＬ以上、６０ｎｇ／ｍＬ以上、７０ｎｇ／ｍＬ以上、８０ｎｇ／ｍＬ以上、９０ｎｇ／ｍＬ以上、１００ｎｇ／ｍＬ以上、１１０ｎｇ／ｍＬ以上、１２０ｎｇ／ｍＬ以上、１３０ｎｇ／ｍＬ以上、１４０ｎｇ／ｍＬ以上、１５０ｎｇ／ｍＬ以上、１６０ｎｇ／ｍＬ以上、１７０ｎｇ／ｍＬ以上、１８０ｎｇ／ｍＬ以上、１９０ｎｇ／ｍＬ以上、２００ｎｇ／ｍＬ以上、２５０ｎｇ／ｍＬ以上、３００ｎｇ／ｍＬ以上、４００ｎｇ／ｍＬ以上、または５００ｎｇ／ｍＬ以上であってもよい。本発明の工程（ａ）を行うことにより、工程（ａ）を行わない場合と比べて、アルブミンの分泌量が１．１～１００倍上昇することが好ましく、１．２～１００倍上昇することがさらに好ましく、２～５０倍上昇することがより好ましく、２～１０倍上昇することが特に好ましい。 Regarding the amount of albumin secreted, specifically, according to the procedure of Example 4 described later, the culture supernatant 20 days after the induction of differentiation was collected, and the amount of albumin secreted in the culture supernatant was measured by ELISA Starter Accessory. It may be quantified by an EnSpire TM plate reader (PerkinElmer) using a Kit, Enzyme Substrate, TMB, Human Albumin ELISA Quantation Set (Bethyl Laboratories, Inc). The amount of albumin secreted analyzed by the above method is 30 ng / mL or more, 40 ng / mL or more, 50 ng / mL or more, 60 ng / mL or more, 70 ng / mL or more, 80 ng / mL or more, 90 ng / mL or more, 100 ng / mL or more. , 110 ng / mL or more, 120 ng / mL or more, 130 ng / mL or more, 140 ng / mL or more, 150 ng / mL or more, 160 ng / mL or more, 170 ng / mL or more, 180 ng / mL or more, 190 ng / mL or more, 200 ng / mL or more , 250 ng / mL or more, 300 ng / mL or more, 400 ng / mL or more, or 500 ng / mL or more. By performing the step (a) of the present invention, the amount of albumin secreted is preferably increased 1.1 to 100 times, and 1.2 to 100 times, as compared with the case where the step (a) is not performed. Is more preferable, and the increase is more preferably 2 to 50 times, and particularly preferably 2 to 10 times.
　また、血液細胞への分化誘導は、国際特許出願ＰＣＴ／ＪＰ２０１９／００３３３６の実施例２に記載の条件で細胞を培養することにより行うことができる。具体的には、１日目は、ＢＭＰ４およびＹ２７６３４（ＲＯＣＫ阻害剤）を含む培地で培養し、２日目にｂＦＧＦおよびＢＭＰ４を添加し、３日目に細胞がスフェロイド状のコロニーを形成していることを確認して、ＳＢ４３１５４２（ＴＧＦ-β受容体阻害剤）、ＣＨＩＲ９９０２１（ＧＳＫ３阻害剤）、ｂＦＧＦおよびＢＭＰ４を含む培地で培養し（３日目および４日目）、５日目～６日目は、ＶＥＧＦおよびｂＦＧＦを含む培地で培養し、７日目～１０日目はＶＥＧＦ、ｂＦＧＦ、ＩＬ－６、ＩＧＦ－１、ＩＬ－１１およびＳＣＦを含む培地で培養することにより、血液細胞に分化誘導することができる。なお、血液細胞への分化誘導は、血液細胞のマーカーであるＣＤ３４とＫＤＲの発現をフローサイトメーターにて解析することにより確認することができる。 Further, the induction of differentiation into blood cells can be performed by culturing the cells under the conditions described in Example 2 of the international patent application PCT / JP2019 / 003336. Specifically, on the first day, the cells were cultured in a medium containing BMP4 and Y27634 (ROCK inhibitor), bFGF and BMP4 were added on the second day, and the cells formed spheroid-like colonies on the third day. After confirming that, the cells were cultured in a medium containing SB431542 (TGF-β receptor inhibitor), CHIR99021 (GSK3 inhibitor), bFGF and BMP4 (3rd and 4th days), 5th to 6th days. Eyes are cultured in a medium containing VEGF and bFGF, and on days 7 to 10, they are cultured in a medium containing VEGF, bFGF, IL-6, IGF-1, IL-11 and SCF to give blood cells. Differentiation can be induced. The induction of differentiation into blood cells can be confirmed by analyzing the expression of blood cell markers CD34 and KDR with a flow cytometer.
　心筋細胞への分化誘導は、例えば、国際特許出願ＰＣＴ／ＪＰ２０１９／００３３３６の実施例５に記載の条件で細胞を培養することにより行うことができる。具体的には、細胞を、ＰＳＣ　Ｃａｒｄｉｏｍｙｏｃｙｔｅ　Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ　Ｋｉｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）を用いて手順書に従い、心筋細胞に分化誘導することができる。心筋細胞への分化誘導は、心筋細胞マーカーであるＣａｒｄｉａｃ　Ｔｒｏｐｏｎｉｎ　Ｔ（ｃＴｎＴ）の発現をフローサイトメトリーで測定することにより確認することができる。国際特許出願ＰＣＴ／ＪＰ２０１９／００３３３６の実施例５の手順に準じて、分化誘導後１４日目の細胞について、Ａｌｅｘａ　Ｆｌｕｏｒ６４７　Ｍｏｕｓｅ　Ａｎｔｉ－Ｃａｒｄｉａｃ　Ｔｒｏｐｏｎｉｎ　Ｔ抗体（ＢＤ　Ｐｈａｒｍｉｎｇｅｎ）にて細胞内を染色し、フローサイトメトリーＡｔｔｕｎｅ　ＮｘＴ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）にてｃＴｎＴの陽性率を解析すればよい。上記方法で解析したｃＴｎＴの陽性率は、好ましくは５％以上であり、より好ましくは１０％以上であり、さらに好ましくは２０％以上であり、さらに一層好ましくは３０％以上であり、特に好ましくは４０％以上であり、最も好ましくは４５％以上である。本発明の工程（ａ）を行うことにより、工程（ａ）を行わない場合と比べて、ｃＴｎＴの陽性率が１．１～１００倍上昇することが好ましく、１．２～１００倍上昇することがさらに好ましく、２～５０倍上昇することが最も好ましい。 Induction of differentiation into cardiomyocytes can be performed, for example, by culturing the cells under the conditions described in Example 5 of the international patent application PCT / JP2019 / 003336. Specifically, cells can be induced to differentiate into cardiomyocytes using a PSC Cardiomyote Difference Kit (Thermo Fisher Scientific) according to a procedure manual. Induction of differentiation into cardiomyocytes can be confirmed by measuring the expression of Cardiac Troponin T (cTnT), which is a cardiomyocyte marker, by flow cytometry. According to the procedure of Example 5 of the international patent application PCT / JP2019 / 03336, the cells 14 days after the induction of differentiation were stained with Alexa Fluor647 Mouse Anti-Cardiac Troponin T antibody (BD Harmingen) and flowed. The positive rate of cTnT may be analyzed by cytometry Attune NxT (Thermo Fisher Scientific). The positive rate of cTnT analyzed by the above method is preferably 5% or more, more preferably 10% or more, further preferably 20% or more, still more preferably 30% or more, and particularly preferably. It is 40% or more, and most preferably 45% or more. By performing the step (a) of the present invention, the positive rate of cTnT is preferably increased 1.1 to 100 times, and 1.2 to 100 times, as compared with the case where the step (a) is not performed. Is more preferable, and it is most preferable that the increase is 2 to 50 times.
　神経幹細胞への分化誘導は、例えば、国際特許出願ＰＣＴ／ＪＰ２０１９／００３３３６の実施例６に記載の条件で細胞を培養することにより行うことができる。具体的には、細胞をＰＳＣ　Ｎｅｕｒａｌ　Ｉｎｄｕｃｔｉｏｎ　Ｍｅｄｉｕｍ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｃｎｔｉｆｉｃ）を用いて手順書に従い、神経幹細胞に分化誘導することができる。神経幹細胞への分化誘導は、例えば、神経幹細胞のＭａｒｋｅｒであるＳＯＸ１蛋白質を免疫染色することにより確認することができる。ＳＯＸ１蛋白質を免疫染色して輝度を定量化した場合、本発明の工程（ａ）を行うことにより、工程（ａ）を行わない場合と比べて、輝度が１．１～１０倍上昇することが好ましく、１．５～５倍上昇することがさらに好ましい。 Induction of differentiation into neural stem cells can be performed, for example, by culturing the cells under the conditions described in Example 6 of the international patent application PCT / JP2019 / 003336. Specifically, the cells can be induced to differentiate into neural stem cells using a PSC Natural Indication Medium (Thermo Fisher Scientific) according to a procedure manual. Induction of differentiation into neural stem cells can be confirmed, for example, by immunostaining the SOX1 protein, which is a marker of neural stem cells. When the SOX1 protein is immunostained to quantify the brightness, the brightness can be increased by 1.1 to 10 times by performing the step (a) of the present invention as compared with the case where the step (a) is not performed. It is preferable, and it is more preferable that the increase is 1.5 to 5 times.
　工程（ａ）で得られる多能性幹細胞は、内胚葉系細胞分化条件にて培養することにより、内胚葉系細胞へ分化することができる。内胚葉系細胞としては、特に限定されないが、例えば、消化器系細胞（肝細胞、肝類洞内皮細胞、クッパー細胞、肝星細胞、ピット細胞、胆管細胞、中皮細胞、膵内分泌細胞、腺房細胞、導管細胞、吸収細胞、杯細胞、パネート細胞、腸内分泌細胞等）、肺、甲状腺等の組織の細胞が挙げられる。 The pluripotent stem cells obtained in step (a) can be differentiated into endoderm cells by culturing under endoderm cell differentiation conditions. The endoblast lineage cells are not particularly limited, but for example, digestive system cells (hepatocytes, hepatic sinus endothelial cells, cupper cells, hepatic stellate cells, pit cells, bile duct cells, mesenteric cells, pancreatic endocrine cells, glands). Tuft cells, conduit cells, resorbing cells, cup cells, panate cells, intestinal endocrine cells, etc.), cells of tissues such as lung and thyroid.
　工程（ａ）で得られる多能性幹細胞は、上記以外の中胚葉系細胞分化条件にて培養することにより、中胚葉系細胞へ分化することができる。中胚葉系細胞としては、特に限定されないが、血球・リンパ球系細胞（造血幹細胞、赤血球、血小板、マクロファージ、顆粒球、ヘルパーＴ細胞、キラーＴ細胞、Ｂリンパ球等）、脈管系細胞（血管内皮細胞等）、心筋細胞（例えば心房筋細胞、心室筋細胞等）、骨芽細胞、骨細胞，軟骨細胞，腱細胞，脂肪細胞、骨格筋細胞、平滑筋細胞等が挙げられる。 The pluripotent stem cells obtained in step (a) can be differentiated into mesoderm cells by culturing under conditions other than the above for mesoderm cell differentiation conditions. The mesophyll lineage cells are not particularly limited, but are blood cell / lymphocyte lineage cells (hemogenic stem cells, erythrocytes, platelets, macrophages, granulocytes, helper T cells, killer T cells, B lymphocytes, etc.), vasculature cells (vascular lineage cells, etc.) (Vascular endothelial cells, etc.), myocardial cells (for example, atrial muscle cells, ventricular muscle cells, etc.), osteoblasts, bone cells, cartilage cells, tendon cells, fat cells, skeletal muscle cells, smooth muscle cells, and the like.
　工程（ａ）で得られる多能性幹細胞は、上記以外の外胚葉系細胞分化条件にて培養することにより、外胚葉系細胞へ分化することができる。外胚葉系細胞としては、特に限定されないが、神経系細胞、感覚器細胞（水晶体、網膜、内耳など）、皮膚表皮細胞、毛包などが挙げられる。 The pluripotent stem cells obtained in step (a) can be differentiated into ectoderm cells by culturing under ectoderm cell differentiation conditions other than the above. The ectoderm cells include, but are not limited to, nervous system cells, sensory organ cells (lens, retina, inner ear, etc.), skin epidermal cells, hair follicles, and the like.
　本発明において、工程（ａ）で得られる多能性幹細胞を用いて分化誘導した細胞は、各種疾患の治療用医薬品候補化合物のスクリーニングに用いることができる。例えば、単独でまたは他の薬剤と組み合わせて、医薬品候補化合物を、分化誘導した細胞に添加することによって、細胞の形態または機能的な変化、各種因子の増減、遺伝子発現プロファイリング等を検出することにより、評価を行うことができる。ここで、細胞は、治療対象となる疾患と同様の表現型を有する細胞が好ましく、より好ましくは、疾患に罹患した患者に由来する体細胞を用いて本発明の方法により製造した細胞から分化誘導した細胞である。 In the present invention, the cells induced to differentiate using the pluripotent stem cells obtained in step (a) can be used for screening drug candidate compounds for the treatment of various diseases. For example, by adding a drug candidate compound alone or in combination with other drugs to cells in which differentiation has been induced, by detecting changes in cell morphology or function, increase / decrease in various factors, gene expression profiling, etc. , Can be evaluated. Here, the cells are preferably cells having the same phenotype as the disease to be treated, and more preferably, differentiation induction from cells produced by the method of the present invention using somatic cells derived from a patient suffering from the disease. It is a cell.
　本発明による化合物処理を多能性幹細胞に施すこと（即ち、体細胞に初期化因子を導入して得られる未分化細胞に対して、上記工程（ａ）を実施すること）は、多能性幹細胞としての未分化性を損なわせるものではない。 Applying the compound treatment according to the present invention to pluripotent stem cells (that is, performing the above step (a) on undifferentiated cells obtained by introducing a reprogramming factor into somatic cells) is pluripotent. It does not impair the undifferentiated nature of stem cells.
　後記の実施例において、本発明による化合物処理を多能性幹細胞に施すこと（即ち、体細胞に初期化因子を導入して得られる未分化細胞に対して、上記工程（ａ）を実施すること）で、処理した細胞は未処理細胞と比較して、解析した全７種類の多能性幹細胞株において、肝細胞への分化誘導効率が上昇した。以上より、分化優良株を選別する労力やコスト削減が可能となった。好ましくは、ｉＰＳ細胞からの肝臓細胞への分化誘導効率は、肝細胞マーカーＡＳＧＰＲ１の陽性率により測定することができる。 In the examples described below, the compound treatment according to the present invention is applied to pluripotent hepatocytes (that is, the above step (a) is performed on undifferentiated cells obtained by introducing a reprogramming factor into somatic cells. ), The treated cells had an increased efficiency of inducing differentiation into hepatocytes in all seven pluripotent stem cell lines analyzed, as compared with the untreated cells. From the above, it has become possible to reduce the labor and cost for selecting excellent strains for differentiation. Preferably, the efficiency of inducing differentiation of iPS cells into liver cells can be measured by the positive rate of the hepatocyte marker ASGPR1.
　本発明において、本発明の製造方法により製造された分化細胞から組織を作製して、再生医療の分野で使用することができる。作製したｉＰＳ細胞由来の肝臓細胞は各種肝疾患の治療に適用可能である。特に、障害された（機能不全を含む）肝臓組織の再生・再建用の材料としての利用が想定され、再生医療への貢献が期待できる。 In the present invention, a tissue can be prepared from the differentiated cells produced by the production method of the present invention and used in the field of regenerative medicine. The prepared iPS cell-derived liver cells can be applied to the treatment of various liver diseases. In particular, it is expected to be used as a material for regeneration / reconstruction of impaired (including dysfunctional) liver tissue, and is expected to contribute to regenerative medicine.
　本発明の分化誘導方法で作成したｉＰＳ細胞由来の肝細胞の別の用途として各種のｉｎ　ｖｉｔｒｏアッセイが提供される。例えば、ｉＰＳ細胞由来肝細胞を用いて被験物質の代謝について試験することができる。即ち、本発明は被験物質の代謝を評価する方法を提供する。被験物質には様々な分子サイズの有機化合物または無機化合物を用いることができる。医薬や栄養食品等の既存成分あるいは候補成分も好ましい被験物質である。植物抽出液、細胞抽出液、培養上清などを被験物質として用いてもよい。またこれらの２種類以上を組み合わせて用いてもよい。 Various in vitro assays are provided as another use of iPS cell-derived hepatocytes prepared by the method for inducing differentiation of the present invention. For example, iPS cell-derived hepatocytes can be used to test the metabolism of the test substance. That is, the present invention provides a method for evaluating the metabolism of a test substance. Organic compounds or inorganic compounds of various molecular sizes can be used as the test substance. Existing or candidate ingredients such as pharmaceuticals and nutritional foods are also preferred test substances. A plant extract, a cell extract, a culture supernatant, or the like may be used as a test substance. Moreover, you may use these two or more types in combination.
　以下の実施例により本発明をさらに具体的に説明するが、本発明は実施例によって限定されるものではない。 The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.
（実施例１）
　ヒトｉＰＳ細胞の特定細胞への分化能を改善および向上させるために以下の実験を行った。
［方法］
＜細胞＞
　ヒトｉＰＳ細胞株について、ＡからＧ株は、ＦＵＪＩＦＩＬＭ　Ｃｅｌｌｕｌａｒ　Ｄｙｎａｍｉｃｓ　Ｉｎｔｅｒｎａｔｉｏｎａｌ（ＦＣＤＩ）より分譲を受けた。
＜細胞培養および化合物処理＞
　ヒトｉＰＳ細胞は、Ｍａｔｒｉｇｅｌ（マトリゲル）（登録商標）（Ｃｏｒｎｉｎｇ）をコートした６ウェルプレート上でＳｔｅｍＦｌｅｘ（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）培地にて３７℃、５％ＣＯ２、１０％Ｏ２条件下で維持培養した。 (Example 1)
The following experiments were conducted to improve and improve the ability of human iPS cells to differentiate into specific cells.
[Method]
<Cell>
For human iPS cell lines, strains A to G were sold by FUJIFILM Cellular Dynamics International (FCDI).
<Cell culture and compound treatment>
Human iPS cells are placed on a 6-well plate coated with Matrigel® (Corning) in StemFlex® (Thermo Fisher Scientific) medium at 37 ° C., 5% CO 2 , 10% O 2 It was maintained and cultured under the conditions.
１日目：培養中のヒトｉＰＳ細胞をＴｒｙｐＬＥＴＭ　Ｓｅｌｅｃｔ（Ｉｎｖｉｔｒｏｇｅｎ）で３７℃、５分間の処理により剥離し、シングルセル化した。このヒトｉＰＳ細胞を、マウス胎仔由来線維芽細胞（ＭＥＦ、Ｌｏｎｚａ）を０．５ｘ１０６ｃｅｌｌｓ／ｗｅｌｌ（６ｗｅｌｌ　ｐｌａｔｅ）で播種済またはマトリゲルコート済のウェルに１ｘ１０５ｃｅｌｌｓ／ｗｅｌｌ播種し、表１の培地１で培養した。以降１４日目まで３７℃、５％ＣＯ２、５％Ｏ２条件下で培養した。
２～３日目：新たな培地１に半量交換した。
４～８日目：表１の培地２に培地を交換し、８日目まで１日おきに同培地に交換した。
９日目～１４日目：ＴｒｙｐＬＥＴＭ　Ｓｅｌｅｃｔで３７℃で５分処理して細胞を剥離し、培地２にＣＨＩＲ９９０２１（０．３μｍｏｌ／Ｌ、Ｗａｋｏ）を添加した培地３でＭＥＦを播種済またはマトリゲル（登録商標）コートしたプレートにて約２回継代培養を１週間かけて行った。以降、培地３により、細胞を３７℃、５％ＣＯ２、５％Ｏ２条件下で維持培養した。 Day 1: Human iPS cells in culture were exfoliated by treatment with TrypLE TM Select (Invitrogen) at 37 ° C. for 5 minutes to form single cells. The human iPS cells were seeded with mouse embryo-derived fibroblasts (MEF, Lonza) at 0.5x10 6 cells / well ( 6 well plate) or 1x10 5 cells / well seeded in Matrigel-coated wells, as shown in Table 1. It was cultured in medium 1. After that, the cells were cultured under 37 ° C., 5% CO 2 , and 5% O 2 conditions until the 14th day.
Days 2-3: Half the amount was replaced with fresh medium 1.
Days 4 to 8: The medium was replaced with the medium 2 in Table 1, and the medium was replaced with the same medium every other day until the 8th day.
9th to 14th days: Treated with TripLE TM Select at 37 ° C. for 5 minutes to detach cells, and MEF was seeded or Matrigel was seeded in medium 3 to which CHIR99021 (0.3 μmol / L, Wako) was added to medium 2. (Registered trademark) Subculture was carried out about twice on coated plates over a week. Subsequently, the cells were maintained and cultured in medium 3 under the conditions of 37 ° C., 5% CO 2 , and 5% O 2 .
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　その後、上記培地３で培養した細胞をマトリゲルコートしたプレート上に継代し、ＳｔｅｍＦｌｅｘまたはｍＴｅＳＲ１培地で、３７℃、５％ＣＯ２、１０％Ｏ２条件下で、約２週間継代培養を行った。 Then, the cells cultured in the above medium 3 were subcultured on a plate coated with Matrigel, and subcultured in StemFlex or mTeSR1 medium under the conditions of 37 ° C., 5% CO 2 , and 10% O 2 for about 2 weeks. It was.
　以下の実施例において、表１の培地１～３で培養したヒトｉＰＳ細胞を「処理済」のｉＰＳ細胞といい、Ｍａｔｒｉｇｅｌ（マトリゲル）（登録商標）（Ｃｏｒｎｉｎｇ）をコートした６ウェルプレート上でＳｔｅｍＦｌｅｘ（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）培地にて維持培養したが、表１の培地１～３では培養していない細胞を「未処理」のｉＰＳ細胞という。 In the following examples, human iPS cells cultured in the media 1 to 3 of Table 1 are referred to as "treated" iPS cells, and are StemFlex on a 6-well plate coated with Matrigel (registered trademark) (Corning). (Registered Trademark) (Thermo Fisher Scientific) Cells that have been maintained and cultured in medium but not cultured in media 1 to 3 in Table 1 are referred to as "untreated" iPS cells.
（実施例２）
＜ＤＮＡメチル化度の変化＞
　Ａ株について未処理または処理済のｉＰＳ細胞からＭＥＦを除去するために、細胞をＴｒｙｐＬＥＴＭ　Ｓｅｌｅｃｔ（Ｇｉｂｃｏ）にて３７℃５分間反応させプレートから剥離し、Ａｎｔｉ－Ｆｅｅｄｅｒ　Ｃｅｌｌｓ－ＰＥ，　ｍｏｕｓｅ　ｍｏｎｏｃｌｏｎａｌ　（Ｍｙｌｔｅｎｙｉ）抗体を用いて染色した後、セルソーター（ＦＡＣＳＡｒｉａＩＩＩ）にてｉＰＳ分画であるＭＥＦ陰性分画を分取し、ＰｕｒｅＬｉｎｋ（登録商標）　Ｇｅｎｏｍｅ　ＤＮＡ　Ｍｉｎｉ　Ｋｉｔ　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）にてＤＮＡを抽出した。このＤＮＡを、ＥＺ－ＤＮＡ　ＭｅｔｈｙｌａｔｉｏｎＴＭ　Ｋｉｔ　（ＺＹＭＯ　ＲＥＳＥＡＣＨ）にてＢｉｓｕｌｆｉｔｅ変換し、Ｉｎｆｉｎｉｕｍ（登録商標）　Ｍｅｔｈｙｌａｔｉｏｎ　ＥＰＩＣ　ＢｅａｄｓＣｈｉｐ　Ｋｉｔ（ｉｌｌｕｍｉｎａ）にてメチル化アレイ解析を実施した。解析結果は、メチル化アレイ用解析ソフトＧｅｎｏｍｅＳｔｕｄｉｏ（ｉｌｌｕｍｉｎａ）によりメチル化度（（β値）＝５メチルシトシン／全シトシン）を算出し、アレイ内の内部標準用プローブにて測定された値を用いて正規化した。 (Example 2)
<Changes in DNA methylation>
In order to remove MEF from untreated or treated iPS cells for strain A, the cells were reacted with TripLE TM Select (Gibco) at 37 ° C. for 5 minutes, detached from the plate, and antibody-Feeder Cells-PE, mouse monoclonal ( After staining with Myltenyi) antibody, the MEF negative fraction, which is an iPS fraction, was fractionated with a cell sorter (FACSAriaIII), and DNA was extracted with PureLink (registered trademark) Genome DNA Mini Kit (Thermo Fisher Scientific). .. This DNA was Bisulfite-converted with EZ-DNA Methylation TM Kit (ZYMO RESEACH), and methylation array analysis was performed with Infinium (registered trademark) Methylation EPIC BeadsChip Kit (illumina). For the analysis results, the degree of methylation ((β value) = 5 methylcytosine / total cytosine) was calculated using the methylation array analysis software Genome Studio (illumina), and the values measured by the internal standard probe in the array were used. Normalized.
＜結果＞
　処理済のｉＰＳ細胞のＤＮＡメチル化度は、未処理のｉＰＳ細胞のメチル化度に比較し、４８％低下していた（図１）。図１はメチル化度のバイオリンプロット図を表し、グラフの幅は各メチル化度にある領域の分布を表している。 <Result>
The DNA methylation degree of the treated iPS cells was 48% lower than that of the untreated iPS cells (Fig. 1). FIG. 1 shows a violin plot of the degree of methylation, and the width of the graph shows the distribution of regions at each degree of methylation.
（実施例３）
＜未分化性の確認＞
　ＡからＤ株について未処理または処理済のヒトｉＰＳ細胞の未分化性を評価するために、細胞をＴｒｙｐＬＥＴＭ　Ｓｅｌｅｃｔ（Ｇｉｂｃｏ）にて３７℃５分間反応させプレートから剥離し、ＲＮｅａｓｙ（登録商標）　ｐｌｕｓ　Ｍｉｎｉ　Ｋｉｔ（Ｑｉａｇｅｎ）にて細胞からＲＮＡを抽出した。このＲＮＡを用いてＨｉｇｈ　Ｃａｐａｃｉｔｙ　ＲＮＡ－ｔｏ－ｃＤＮＡ　Ｋｉｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）にて合成したｃＤＮＡを鋳型にしてＴａｑＭａｎ（登録商標）　Ｇｅｎｅ　Ｅｘｐｒｅｓｓｉｏｎ　ａｓｓａｙ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）によりｑＰＣＲで、ＮＡＮＯＧ（Ｐｒｏｂｅ　ｐｒｉｍｅｒ　ｓｅｔのコード名（以下コード名と記載）：Ｈｓ０２３８７４００＿ｇ１）、　ＰＯＵ５Ｆ１（コード名：Ｈｓ０４２６０３６７＿ｇＨ）、およびＬＩＮ２８（コード名：Ｈｓ００７０２８０８＿ｓ１）の遺伝子発現を解析した。発現量は内部標準遺伝子であるＧＡＰＤＨ（コード名：Ｈｓ０２７８６６２４＿ｇ１）で補正した。 (Example 3)
<Confirmation of undifferentiated state>
In order to evaluate the undifferentiated state of untreated or treated human iPS cells for strains A to D, the cells were reacted with TrypLE TM Select (Gibco) at 37 ° C. for 5 minutes, detached from the plate, and RNAy®. RNA was extracted from cells with a plus Mini Kit (Qiagen). Using this RNA as a template for cDNA synthesized with High Capacity RNA-to- cDNA Kit (Thermo Fisher Scientific) as a template, TaqMan (registered trademark) Gene Expression assembly (Thermo Primer Scientific) by PCR (Thermo Primer Scientific). The gene expression of code name (hereinafter referred to as code name): Hs02387400_g1), POU5F1 (code name: Hs04260367_gH), and LIN28 (code name: Hs00702808_s1) was analyzed. The expression level was corrected with GAPDH (code name: Hs0276624_g1), which is an internal standard gene.
＜結果＞
　未処理および処理済のｉＰＳ細胞における遺伝子発現を解析したところ、ＡからＤ株の全４株で未処理のｉＰＳ細胞と比較して処理済のｉＰＳ細胞では未分化性に関わる遺伝子（ＮＡＮＯＧ、ＰＯＵ５Ｆ１、ＬＩＮ２８）の発現が同等以上であった（図２）。この結果は、上記実施例１の表１の培地１～３での培養がｉＰＳ細胞の未分化性を維持あるいは向上させるものであって、未分化性を損なわせる処理ではないこと、即ち処理済のｉＰＳ細胞が別の細胞に分化したわけではないことを示唆する。 <Result>
Analysis of gene expression in untreated and treated iPS cells revealed that all 4 strains A to D were compared with untreated iPS cells, and the treated iPS cells had genes related to undifferentiation (NANOG, POU5F1). , LIN28) was equal to or better than that (Fig. 2). The result is that the culture in the medium 1 to 3 of Table 1 of Example 1 maintains or improves the undifferentiated state of iPS cells, and is not a treatment that impairs the undifferentiated state, that is, has been treated. It is suggested that the iPS cells in the above did not differentiate into other cells.
（実施例４）
＜肝細胞への分化＞
　ＡからＧ株について肝細胞への分化能を検討するために、未処理または処理済のｉＰＳ細胞をマトリゲルコートした２４ウェルプレートに再播種し、表２の培地を０．５ｍＬ／ｗｅｌｌに入れ、各５日間ずつ維持培養を行い、肝細胞に分化させた。なお、使用した培地は、ｍＴｅＳＲ（登録商標）１（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）、ＲＰＭＩ１６４０培地、ＨＢＭ（登録商標）である。また、使用した化合物は、Ｂ２７（登録商標）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、Ａｃｔｉｖｉｎ　Ａ（Ｒ＆Ｄ　Ｓｙｓｔｅｍ社）、ｂＦＧＦ（富士フイルム和光純薬社）、ＢＭＰ４（Ｒ＆Ｄ　Ｓｙｓｔｅｍ社）、ＨＧＦ（Ｐｅｐｒｏｔｅｃｈ）およびＯｎｃｏｓｔａｔｉｎ　Ｍ（Ｐｅｐｒｏｔｅｃｈ）である。
Figure JPOXMLDOC01-appb-T000002
 (Example 4)
<Differentiation into hepatocytes>
To examine the ability of A to G strains to differentiate into hepatocytes, untreated or treated iPS cells were reseeded on Matrigel-coated 24-well plates, and the medium in Table 2 was placed in 0.5 mL / well. Maintenance culture was carried out for 5 days each to differentiate into hepatocytes. The media used were mTeSR (registered trademark) 1 (Stemcell Technologies), RPMI1640 medium, and HBM (registered trademark). The compounds used were B27 (registered trademark) (Thermo Fisher Scientific), Activin A (R & D System), bFGF (Fujifilm Wako Pure Chemical Industries), BMP4 (R & D System), HGF (Peprotech). It is M (Peprotech).
Figure JPOXMLDOC01-appb-T000002
＜評価法＞
　肝細胞への分化誘導効率は誘導２０日後の細胞における成熟化肝細胞マーカーであるＡＳＧＰＲ１の発現をフローサイトメトリーにて評価した。分化誘導後２０日目の細胞をＴｒｙｐＬＥＴＭ　Ｓｅｌｅｃｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｃｎｔｉｆｉｃ）を用いて３７℃で５分処理して細胞を剥離した。Ｇｈｏｓｔ　ＤｙｅＴＭ　Ｖｉｏｌｅｔ　５１０　（ＴＯＮＢＯ　Ｂｉｏｓｃｉｅｎｃｅ）にて死細胞を染色した。洗浄後、ＢＤ　ＰｈａｒｍｉｎｇｅｎＴＭ　ＰＥ　Ｍｏｕｓｅ　Ａｎｔｉ－ＡＳＧＰＲ１抗体（日本ベクトン・ディッキンソン株式会社）にて染色し、フローサイトメトリーＡｔｔｕｎｅＴＭ　ＮｘＴ　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）にてＡＳＧＰＲ１の陽性率を解析した。 <Evaluation method>
The efficiency of inducing differentiation into hepatocytes was evaluated by flow cytometry of the expression of ASGPR1, which is a mature hepatocyte marker, in cells 20 days after induction. Twenty days after the induction of differentiation, the cells were treated with TrypLE TM Select (Thermo Fisher Scientific) at 37 ° C. for 5 minutes to detach the cells. Dead cells were stained with Ghost Dye TM Violet 510 (TONBO Bioscience). After washing, the cells were stained with BD Harmingen TM PE Mouse Anti-ASGPR1 antibody (Nippon Becton Dickinson Co., Ltd.), and the positive rate of ASGPR1 was analyzed by flow cytometry Attune TM NxT (Thermo Fisher Scientific).
　また、誘導後２０日目の培養上清を回収し、培養上清中に分泌されたアルブミン量をＥＬＩＳＡ　Ｓｔａｒｔｅｒ　Ａｃｃｅｓｓｏｒｙ　Ｋｉｔ、Ｅｎｚｙｍｅ　Ｓｕｂｓｔｒａｔｅ、ＴＭＢ、Ｈｕｍａｎ　Ａｌｂｕｍｉｎ　ＥＬＩＳＡ　Ｑｕａｎｔｉｔａｔｉｏｎ　Ｓｅｔ（Ｂｅｔｈｙｌ　Ｌａｂｏｒａｔｏｒｉｅｓ，　Ｉｎｃ）を用いてＥｎＳｐｉｒｅTMプレートリーダー（ＰｅｒｋｉｎＥｌｍｅｒ）により定量した。 In addition, the culture supernatant on the 20th day after the induction was collected, and the amount of albumin secreted in the culture supernatant was measured using ELISA Starter Accessory Kit, Enzyme Substrate, TMB, Human Albumin ELISA Quantation Set (Bethyl Laboratories). Quantified by EnSpire TM plate reader (PerkinElmer).
＜結果＞
　未処理および処理済のｉＰＳ細胞をそれぞれ肝細胞に分化誘導させ、フローサイトメトリーにて生細胞中のＡＳＧＰＲ１陽性率を比較した。ＡＳＧＰＲ１の陽性率はｉＰＳ細胞からの肝細胞への分化誘導効率を示す。結果、ＡＳＧＰＲ１陽性率（％）は、未処理のｉＰＳ細胞を肝細胞に分化誘導したときと比較として、処理済のｉＰＳ細胞を肝細胞に分化誘導したときでは、Ａ株２．９０倍、Ｂ株２．１９倍、Ｃ株２．９７倍、Ｄ株２．０９倍、Ｅ株８．６８倍、Ｆ株２．６９倍、およびＧ株２．２０倍にそれぞれ増加し、全７株平均３．３９倍の増加であった（図３Ａ）。さらに、ＡＳＧＰＲ１陽性細胞数は、未処理のｉＰＳ細胞を肝細胞に分化誘導したときと比較して、処理済のｉＰＳ細胞を肝細胞に分化誘導したときでは、全７株平均で３．４５倍増加した（図３Ｂ、ｐ＝０．０２８）。このように、全７株で処理済のｉＰＳ細胞を分化誘導したときのＡＳＧＰＲ１陽性率が上昇した。すなわち、肝細胞への分化誘導効率が上昇した。
　また、アルブミン分泌量については、全７株において、未処理のｉＰＳ細胞を肝細胞に分化誘導したときと比較として、処理済のｉＰＳ細胞を分化誘導したとき有意に分泌量が上昇した（図４）。 <Result>
The untreated and treated iPS cells were induced to differentiate into hepatocytes, respectively, and the ASGPR1 positive rates in the living cells were compared by flow cytometry. The positive rate of ASGPR1 indicates the efficiency of inducing differentiation of iPS cells into hepatocytes. As a result, the ASGPR1 positive rate (%) was 2.90 times that of the A-share when the treated iPS cells were induced to differentiate into hepatocytes, as compared with the case where the untreated iPS cells were induced to differentiate into hepatocytes. Shares increased 2.19 times, C shares 2.97 times, D shares 2.09 times, E shares 8.68 times, F shares 2.69 times, and G shares 2.20 times, respectively, averaging all 7 shares The increase was 3.39 times (Fig. 3A). Furthermore, the number of ASGPR1-positive cells was 3.45 times higher on average for all 7 strains when the treated iPS cells were induced to differentiate into hepatocytes than when the untreated iPS cells were induced to differentiate into hepatocytes. Increased (Fig. 3B, p = 0.028). In this way, the ASGPR1 positive rate when the treated iPS cells were induced to differentiate in all 7 strains increased. That is, the efficiency of inducing differentiation into hepatocytes increased.
Regarding the amount of albumin secreted, the amount of albumin secreted was significantly increased when the treated iPS cells were induced to differentiate, as compared with the case where the untreated iPS cells were induced to differentiate into hepatocytes in all 7 strains (Fig. 4). ).
（実施例５）
＜ｉＰＳ細胞の内胚葉分化におけるＨＤＡＣ阻害剤単独処理と本発明の比較＞
　ｉＰＳ細胞の内胚葉分化におけるＨＤＡＣ阻害剤単独処理の影響を検討するため、以下の実験を行った。ＨＤＡＣ阻害剤として、バルプロ酸ナトリウムを使用した。ｉＰＳ細胞はＤ株を用いた。
　ｉＰＳ細胞をＭＥＦ播種済のプレート上でＳｔｅｍＦｌｅｘ（登録商標）にて培養した細胞を「未処理・未分化」とする。この「未処理・未分化」細胞を（１）そのまま（「未処理・内胚葉分化」という）、（２）Ｎ２Ｂ２７培地にバルプロ酸ナトリウムを１ｍＭ添加した培地（「ＶＰＡ・内胚葉分化」という）、または（３）上記実施例１の培地（表１の培地１～３）（「Ｍｉｘ・内胚葉分化」という）にて１０日間３７℃、５％ＣＯ２、５％Ｏ２で培養した細胞をＳｔｅｍＤｉｆｆ　Ｔｒｉｌｉｎｅａｇｅ　Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ　Ｋｉｔ　（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）のＥｎｄｏｄｅｒｍ　ｍｅｄｉｕｍを用いて内胚葉に分化させた。これらの細胞をＴｒｙｐＬＥ　Ｓｅｌｅｃｔ（Ｇｉｂｃｏ）にて３７℃５分間反応させプレートから剥離し、ＲＮｅａｓｙ（登録商標）　ｐｌｕｓ　Ｍｉｎｉ　Ｋｉｔ（Ｑｉａｇｅｎ）にて細胞からＲＮＡを抽出した。このＲＮＡを用いてＨｉｇｈ　Ｃａｐａｃｉｔｙ　ＲＮＡ－ｔｏ－ｃＤＮＡ　Ｋｉｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）にて合成したｃＤＮＡを鋳型にしてＴａｑＭａｎ　Ｇｅｎｅ　Ｅｘｐｒｅｓｓｉｏｎ　ａｓｓａｙ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）によりｑＰＣＲで、ＳＯＸ１７（コード名：Ｈｓ００７５１７５２＿ｓ１）、ＦＯＸＡ２（コード名：Ｈｓ０５０３６２７８＿ｓ１）の遺伝子発現を解析した。発現量は内部標準遺伝子であるＧＡＰＤＨ（コード名：Ｈｓ０２７８６６２４＿ｇ１）で補正した。 (Example 5)
<Comparison of HDAC inhibitor monotherapy and the present invention in endoderm differentiation of iPS cells>
The following experiments were conducted to investigate the effect of HDAC inhibitor monotherapy on endoderm differentiation of iPS cells. Sodium valproate was used as the HDAC inhibitor. As the iPS cell, the D strain was used.
Cells in which iPS cells are cultured in StemFlex® on MEF-seeded plates are referred to as "untreated / undifferentiated". These "untreated / undifferentiated" cells are (1) as they are (referred to as "untreated / endoderm differentiation"), and (2) a medium in which 1 mM of sodium valproate is added to N2B27 medium (referred to as "VPA / endoderm differentiation"). Or (3) Cells cultured at 37 ° C., 5% CO 2 , 5% O 2 for 10 days in the medium of Example 1 (mediums 1 to 3 in Table 1) (referred to as "Mix / endoderm differentiation"). Was differentiated into endoderm using the Endoderm medium of the StemDiff Trilineage Differentiation Kit (Stemcell Technologies). These cells were reacted with TripLE Select (Gibco) at 37 ° C. for 5 minutes, detached from the plate, and RNA was extracted from the cells with RNeasy (registered trademark) plus Mini Kit (Qiagen). Using this RNA as a template for cDNA synthesized with High Capacity RNA-to- cDNA Kit (Thermo Fisher Scientific) as a template, TaqMan Gene Expression assembly (Thermo Fisher Scientific) (Thermo Fisher Scientific) (Thermo Fisher Scientific) by qPCR by qPCR, SO2 (Thermo Fisher Scientific) The gene expression of the code name: Hs05036278_s1) was analyzed. The expression level was corrected with GAPDH (code name: Hs0276624_g1), which is an internal standard gene.
＜結果＞
　「未処理・未分化」と比較して、「未処理・内胚葉分化」の細胞は内胚葉マーカーであるＳＯＸ１７とＦＯＸＡ２の発現が上昇しており（図５）、本実施例で行った分化誘導法により細胞が内胚葉分化可能であることが確認できた。そして、内胚葉分化した細胞を比較すると、「未処理・内胚葉分化」の細胞のＳＯＸ１７遺伝子発現に対して、「ＶＰＡ・内胚葉分化」のそれは０．１４倍、「Ｍｉｘ・内胚葉分化」のそれは４０．８倍であった。また、「未処理・内胚葉分化」の細胞のＦＯＸＡ２遺伝子発現に対して、「ＶＰＡ・内胚葉分化」のそれは０．２１倍、「Ｍｉｘ・内胚葉分化」のそれは１２．９倍であった（図５）。「未処理・内胚葉分化」と比較して、「Ｍｉｘ・内胚葉分化」では顕著にこれらの遺伝子発現が上昇したものの、「ＶＰＡ・内胚葉分化」では遺伝子発現がむしろ低下した。即ち、未処理と比較して、上記実施例１の培地（表１の培地１～３）（Ｍｉｘ）で培養したｉＰＳ細胞は内胚葉に効率よく分化したが、バルプロ酸ナトリウムのみの処理（ＶＰＡ）では内胚葉への分化が促進されないことが示された。 <Result>
Compared with "untreated / undifferentiated", the expression of endoderm markers SOX17 and FOXA2 was increased in the "untreated / endoderm differentiated" cells (Fig. 5), and the differentiation performed in this example. It was confirmed that the cells were capable of endoderm differentiation by the induction method. Comparing the endoderm-differentiated cells, the SOX17 gene expression of the "untreated / endoderm-differentiated" cells was 0.14 times that of "VPA / endoderm-differentiated", and "Mix-endoderm-differentiated". That was 40.8 times. In addition, the FOXA2 gene expression in "untreated / endoderm differentiation" cells was 0.21 times that of "VPA / endoderm differentiation" and 12.9 times that of "Mix / endoderm differentiation". (Fig. 5). Compared with "untreated / endoderm differentiation", the expression of these genes was significantly increased in "Mix / endoderm differentiation", but the gene expression was rather decreased in "VPA / endoderm differentiation". That is, as compared with the untreated iPS cells, the iPS cells cultured in the medium of Example 1 (mediums 1 to 3 in Table 1) (Mix) differentiated efficiently into endoderm, but treated with sodium valproate only (VPA). ) Showed that differentiation into endoderm was not promoted.

Claims (18)

  1. 多能性幹細胞を、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、ＳＴＡＴ３活性化剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤およびＧＳＫ３β阻害剤からなる群から選択される何れかを含む培地で培養することにより、培養前の多能性幹細胞と比較して、ＤＮＡの全シトシンにおける５メチルシトシンの割合が低下した多能性幹細胞を得る工程（ａ）、および
    工程（ａ）で得られる多能性幹細胞を、サイトカインを含む培地で培養することにより、特定の分化細胞を得る工程（ｂ）
    を含む、特定の分化細胞の製造方法。     Pluripotent stem cells are selected from the group consisting of histone deacetylase inhibitors, MAPK / ERK kinase inhibitors, STAT3 activators, protein kinase C inhibitors, Wnt signal inhibitors and GSK3β inhibitors. In the steps (a) and (a) of obtaining pluripotent stem cells in which the proportion of 5-methylcytosine in the total cytosine of the DNA is reduced as compared with the pluripotent stem cells before culturing by culturing in the containing medium. Step (b) of obtaining specific differentiated cells by culturing the obtained pluripotent stem cells in a medium containing cytokines.
    A method for producing a specific differentiated cell, including.
  2. 前記工程（ａ）が、
    多能性幹細胞を、ヒストン脱アセチル化酵素阻害剤、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、およびＳＴＡＴ３活性化剤を含む培地で培養する第一培養工程、および
    第一培養工程の後に、ヒストン脱アセチル化酵素阻害剤を含まずに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、ＧＳＫ３β阻害剤、およびＳＴＡＴ３活性化剤を含む培地で培養する第二培養工程
    を含む、請求項１に記載の製造方法。     The step (a)
    A first culture step in which pluripotent stem cells are cultured in a medium containing a histone deacetylase inhibitor, a MAPK / ERK kinase inhibitor, and a STAT3 activator, and after the first culture step, histone deacetylase. 1. A second culture step of culturing in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, a GSK3β inhibitor, and a STAT3 activator without an inhibitor. The manufacturing method described in.
  3. 前記サイトカインが、アクチビンＡ、ＢＭＰ４、ｂＦＧＦ、ＨＧＦ、およびＯｎｃｏｓｔａｔｉｎ　Ｍからなる群から選択される少なくとも１つである、請求項１または２に記載の方法。 The method of claim 1 or 2, wherein the cytokine is at least one selected from the group consisting of activin A, BMP4, bFGF, HGF, and Oncostatin M.
  4. 前記特定の分化細胞が内胚葉系譜の細胞である、請求項１から３の何れか一項に記載の方法。 The method according to any one of claims 1 to 3, wherein the specific differentiated cell is a cell of endoderm lineage.
  5. 前記特定の分化細胞が肝細胞である、請求項１から４の何れか一項に記載の方法。 The method according to any one of claims 1 to 4, wherein the specific differentiated cell is a hepatocyte.
  6. 第一培養工程の培養期間が１から５日間である、請求項２から５の何れか一項に記載の方法。 The method according to any one of claims 2 to 5, wherein the culture period of the first culture step is 1 to 5 days.
  7. 第二培養工程は、ヒストン脱アセチル化酵素阻害剤を含まずに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、およびＳＴＡＴ３活性化剤を含む培地で培養した後、前記培地にＧＳＫ３β阻害剤を加えた培地で培養を行う工程である、請求項２から６の何れか一項に記載の方法。 In the second culture step, after culturing in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, and a STAT3 activator without containing a histone deacetylase inhibitor, the above-mentioned The method according to any one of claims 2 to 6, which is a step of culturing in a medium in which a GSK3β inhibitor is added to the medium.
  8. 第二培養工程は、ヒストン脱アセチル化酵素阻害剤を含まずに、ＭＡＰＫ／ＥＲＫキナーゼ阻害剤、プロテインキナーゼＣ阻害剤、Ｗｎｔシグナル阻害剤、およびＳＴＡＴ３活性化剤を含む培地で３から８日間培養した後、前記培地にＧＳＫ３β阻害剤を加えた培地で３から１０日間培養を行う工程である、請求項２から７の何れか一項に記載の方法。 The second culture step is culturing for 3 to 8 days in a medium containing a MAPK / ERK kinase inhibitor, a protein kinase C inhibitor, a Wnt signal inhibitor, and a STAT3 activator without the histone deacetylase inhibitor. The method according to any one of claims 2 to 7, which is a step of culturing in the medium to which a GSK3β inhibitor is added for 3 to 10 days.
  9. 前記工程（ｂ）の培養期間が１から４週間である、請求項１から８の何れか一項に記載の方法。 The method according to any one of claims 1 to 8, wherein the culture period of the step (b) is 1 to 4 weeks.
  10. ヒストン脱アセチル化酵素阻害剤が、バルプロ酸またはその塩である、請求項１から９の何れか一項に記載の方法。 The method according to any one of claims 1 to 9, wherein the histone deacetylase inhibitor is valproic acid or a salt thereof.
  11. ＭＡＰＫ／ＥＲＫキナーゼ阻害剤がＰＤ０３２５９０１である、請求項１から１０の何れか一項に記載の方法。 The method according to any one of claims 1 to 10, wherein the MAPK / ERK kinase inhibitor is PD0325901.
  12. ＳＴＡＴ３活性化剤が白血病阻止因子である、請求項１から１１の何れか一項に記載の方法。 The method according to any one of claims 1 to 11, wherein the STAT3 activator is a leukemia inhibitory factor.
  13. プロテインキナーゼＣ阻害剤がＧｏ６９８３である、請求項１から１２の何れか一項に記載の方法。 The method according to any one of claims 1 to 12, wherein the protein kinase C inhibitor is Go6983.
  14. Ｗｎｔシグナル阻害剤がＸＡＶ９３９である、請求項１から１３の何れか一項に記載の方法。 The method according to any one of claims 1 to 13, wherein the Wnt signal inhibitor is XAV939.
  15. ＧＳＫ３β阻害剤がＣＨＩＲ９９０２１である、請求項１から１４の何れか一項に記載の方法。 The method according to any one of claims 1 to 14, wherein the GSK3β inhibitor is CHIR99021.
  16. 前記第一培養工程の培地がヒストン脱アセチル化酵素阻害剤を濃度１．０μｍｏｌ／Ｌ～５．０ｍｍｏｌ／Ｌの濃度で含む、請求項２から１５の何れか一項に記載の方法。 The method according to any one of claims 2 to 15, wherein the medium of the first culture step contains a histone deacetylase inhibitor at a concentration of 1.0 μmol / L to 5.0 mmol / L.
  17. 前記第一培養工程および第二培養工程の培地がＭＡＰＫ／ＥＲＫキナーゼ阻害剤を濃度０．３μｍｏｌ／Ｌ～５．０μｍｏｌ／Ｌの範囲で含む、請求項２から１６の何れか一項に記載の方法。 The medium according to any one of claims 2 to 16, wherein the medium of the first culture step and the second culture step contains a MAPK / ERK kinase inhibitor in a concentration range of 0.3 μmol / L to 5.0 μmol / L. Method.
  18. 多能性幹細胞がヒトｉＰＳ細胞である、請求項１から１７の何れか一項に記載の方法。 The method according to any one of claims 1 to 17, wherein the pluripotent stem cell is a human iPS cell.
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