Method for purifying cardiomyocytes

Abstract

The present invention provides a method for producing a cell population containing cardiomyocytes, the method comprising the steps of: (1) bringing a histone deacetylase inhibitor into contact with a cell population which contains cardiomyocytes and cells other than the cardiomyocytes, and are obtained by culturing pluripotent stem cells in media for differentiating cardiomyocytes; and (2) culturing the cell population.

Description

[Technical Field]

[0001] The present invention relates to a method for producing and purifying cardiomyocytes, and more particularly to a method for producing and purifying cardiomyocytes using a histone deacetylase inhibitor.

[0002] BACKGROUND OF THE INVENTION Although the incidence of myocardial infarction has decreased in recent years, heart disease, including myocardial infarction, remains a leading cause of death worldwide. Heart transplantation is currently the only treatment for patients with severe heart failure, but heart transplantation is plagued by a shortage of donor donors. Cell therapy using cardiomyocytes has therefore attracted attention as a potential treatment for improving heart disease. Attention has also been focused on establishing in vitro drug efficacy and safety testing using cardiomyocytes. Therefore, a stable supply of uniform cardiomyocytes suitable for cell therapy and in vitro testing is needed.

[0003] One method for stably providing uniform cardiomyocytes is to induce the differentiation of stem cells or cardiac progenitor cells into cardiomyocytes, and various efforts have been made to establish an efficient method for inducing differentiation into cardiomyocytes. Examples of such differentiation methods include a method in which pluripotent stem cells are cultured in a medium containing an EGFR inhibitor to promote the differentiation of pluripotent stem cells into cardiomyocytes (Patent Document 1), a method in which induced pluripotent stem cells are differentiated into cardiomyocytes and then the cardiomyocytes are contacted with a Neuregulin 1 antagonist or an ErbB antagonist to mature the cardiomyocytes (Patent Document 2), a method in which undifferentiated progenitor cells such as myoblasts are contacted with a deacetylase inhibitor to promote the differentiation of the undifferentiated progenitor cells (Patent Document 3), and a method in which adult progenitor cells such as cardiac progenitor cells are contacted with a histone deacetylase (HDAC) inhibitor to promote the differentiation of the progenitor cells (Patent Document 4). Methods that do not involve the process of inducing differentiation from stem cells have also been reported. For example, Patent Document 5 discloses a method for producing cardiac progenitor cells or cardiac cells from somatic cells such as fibroblasts by direct reprogramming. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2014 / 136519 [Patent Document 2] U.S. Publication No. 2010 / 0183565 [Patent Document 3] International Publication No. 2003 / 033678 [Patent Document 4] International Publication No. 2009 / 073618 [Patent Document 5] International Publication No. 2015 / 038704 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for producing a cell population containing highly pure cardiomyocytes by a means different from the above-mentioned conventional methods, and also to provide a method for purifying highly pure cardiomyocytes from a cell population containing cardiomyocytes. [Means for solving the problem]

[0006] The present inventors conducted extensive research to solve the above-mentioned problems. Instead of promoting the differentiation of undifferentiated cells into cardiomyocytes, they came up with the idea that adding a compound that inhibits the proliferation of cells other than cardiomyocytes or reduces the number of cells other than cardiomyocytes to a cell population already containing cardiomyocytes could increase the proportion of cardiomyocytes in the cell population, i.e., purify cardiomyocytes. Therefore, they first added a compound library to iPS cells and cardiomyocytes induced to differentiate from iPS cells, and screened for compounds that significantly reduce the number of iPS cells but have little effect on cardiomyocytes. As a result, several HDAC inhibitors were identified as candidate compounds with cardiomyocyte purification properties. They then contacted embryoid bodies containing cardiomyocytes with these candidate compounds and found that cardiomyocyte purification was successful. Based on these findings, the present inventors conducted further research and completed the present invention.

[0007] That is, the present invention provides the following. [1] A method for producing a cell population containing cardiomyocytes, comprising: (1) contacting a cell population containing cardiomyocytes and cells other than cardiomyocytes obtained by culturing pluripotent stem cells in a medium for cardiomyocyte differentiation with a histone deacetylase inhibitor; (2) culturing the cell population; A method comprising: [2] The method according to [1], wherein the contact of the cell population with the histone deacetylase inhibitor in step (1) is carried out on or after day 7 from the start of the induction of differentiation of the pluripotent stem cells. [3] The method according to [1] or [2], wherein the inhibitor is an inhibitor of class I histone deacetylase. [4] The method according to any one of [1] to [3], wherein the inhibitor is at least one selected from the group consisting of FK228, Entinostat, Trichostatin A, and Panobinostat. [5] The method according to any one of [1] to [4], wherein the pluripotent stem cells are induced pluripotent stem cells. [6] A method for removing or reducing undifferentiated cells from a cell population containing cardiomyocytes contaminated with undifferentiated cells, the method comprising the step of contacting the cell population with a histone deacetylase inhibitor. [7] The method according to [6], wherein the undifferentiated cells are pluripotent stem cells. [8] A cell population containing cardiomyocytes obtained by the method according to any one of [1] to [7]. [9] A cell transplantation therapy agent comprising the cell population described in [8].

[10] A method for purifying cardiomyocytes, comprising: (1) contacting a cell population containing cardiomyocytes and cells other than cardiomyocytes obtained by culturing pluripotent stem cells in a medium for cardiomyocyte differentiation with a histone deacetylase inhibitor; (2) culturing the cell population; A method comprising: [Effects of the Invention]

[0008] According to the present invention, a cell population containing highly purified cardiomyocytes is provided. Such a cell population can be suitably used in cell transplantation therapy for cardiac diseases. Also provided are a method for purifying highly purified cardiomyocytes from a cell population containing cardiomyocytes, and a method for reducing cells other than cardiomyocytes (i.e., non-cardiomyocytes) from a cell population containing cardiomyocytes. [Brief explanation of the drawings]

[0009] [Figure 1]1 shows the intracellular ATP levels of undifferentiated iPS cells (myocardial reporter iPS cell line) and purified cardiomyocytes derived from the same iPS cells after treatment with a histone deacetylase inhibitor in the compound screening of Test Example 1. [Figure 2] 1 shows the intracellular ATP levels of iPS cells and iPS cell-derived cardiomyocytes in Test Example 2 after treatment with a histone deacetylase inhibitor. [Figure 3] 1 shows the rate of sarcomeric α-actinin positive cells determined by flow cytometry analysis of CiRA clinical iPS cell-derived cardiomyocytes after treatment with a histone deacetylase inhibitor in Test Example 3. [Figure 4] 1 shows the rate of sarcomeric α-actinin positive cells determined by flow cytometer analysis of iPS cell-derived cardiomyocytes after treatment with a histone deacetylase inhibitor in Test Example 4. [Figure 5] 1 shows the non-cardiomyocyte rate after compound treatment in Test Example 5 (END: endodermal lineage cells, SMC: smooth muscle-like cells, EC: endothelial-like cells).

[0010] (Detailed Description of the Invention) 1. Method for producing a cell population containing cardiomyocytes The present invention provides a method for producing a cell population containing cardiomyocytes (hereinafter also referred to as the "production method of the present invention"), which comprises the steps of (1) contacting a cell population containing cardiomyocytes and cells other than cardiomyocytes with a histone deacetylase (HDAC) inhibitor, and (2) culturing the cell population.

[0011] As used herein, "cardiomyocytes" refer to cells that are positive for at least one of sarcomeric α-actinin, cardiac troponin T, and troponin I type 1 (TNNI1), and are preferably sarcomeric α-actinin-positive cells. Typically, these are cardiac muscle cells that have the ability to self-pulsate. "Cells other than cardiac muscle cells" refer to cells that do not fall under the category of cardiac muscle cells, and specific examples include smooth muscle cells, endothelial cells, stem cells (e.g., pluripotent stem cells), and cardiac progenitor cells.

[0012] As used herein, "positive" means that a protein or gene is expressed in a detectable amount by at least one method known in the art. Protein detection can be performed using antibody-based immunological assays, such as ELISA, immunostaining, and flow cytometry. Furthermore, in the case of proteins that are expressed intracellularly but not on the cell surface (e.g., transcription factors or their subunits), the target protein can be detected by expressing a reporter protein together with the protein and detecting the reporter protein. Gene detection can be performed using nucleic acid amplification and / or nucleic acid detection methods, such as RT-PCR, biochips (e.g., microarrays), and RNAseq. Protein or gene expression can be determined by common methods. For example, when flow cytometry is used, it can be determined that a protein is detectably expressed if the expression level is relatively high compared to the expression level in a negative control group.

[0013] As used herein, "negative" means that the expression level of a protein or gene is below the lower limit of detection by all or any of the above-mentioned known techniques. The lower limit of detection for protein or gene expression may vary depending on the technique, but can be determined by a general technique.

[0014] As used herein, the term "cell population" refers to a population consisting of two or more cells of the same or different types. The term "cell population" also refers to a mass of cells of the same or different types. The cell population containing cardiomyocytes and cells other than cardiomyocytes used in step (1) can be produced by culturing pluripotent stem cells in a medium for cardiomyocyte differentiation. Therefore, the production method of the present invention may include step (0) of inducing differentiation of pluripotent stem cells into cardiomyocytes prior to step (1).

[0015] Examples of pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (nuclear transfer Embryonic stem cells: ntES cells), multipotent germline stem cells (mGS cells), embryonic germ stem cells (EG cells), and Muse cells (multi-lineage differentiating stress enduring cells), with iPS cells (more preferably human iPS cells) being preferred. When the pluripotent stem cells are ES cells or any cells derived from human embryos, the cells may be produced by or without embryo destruction, but are preferably produced without embryo destruction. The pluripotent stem cells are preferably derived from mammals (e.g., mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, and humans), and more preferably from humans. Therefore, the most preferred pluripotent stem cells for use in the present invention are human iPS cells.

[0016] "Induced pluripotent stem cells (iPS cells)" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells (iPS cells)." These include iPS cells established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPS cells established by selecting cells using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPS cells created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), and iPS cells established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) can also be used. Other examples that can be used include induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, as developed by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920), induced pluripotent stem cells developed by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells developed by Sakurada et al. (JP Patent Publication No. 2008-307007). In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7, Any of the induced pluripotent stem cells known in the art and described in the literature (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008-2336610 A, US 2009-047263 A, WO 2007-069666 A, WO 2008-118220 A, WO 2008-124133 A, WO 2008-151058 A, WO 2009-006930 A, WO 2009-006997 A, WO 2009-007852 A) can be used. Various iPS cell lines established by the NIH, RIKEN, Kyoto University, etc. can be used as induced pluripotent stem cell lines. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and NiPS-B2 strain; Kyoto University's 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, and 648A1 strain; and iPS cell stocks for regenerative medicine.

[0017] As used herein, the term "somatic cells" refers to any animal cell (preferably a mammalian cell, including a human cell) excluding germline cells such as eggs, oocytes, and embryonic stem cells, or totipotent cells. Somatic cells include, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature, healthy or diseased somatic cells. They also include primary cultured cells, passaged cells, and established cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue progenitor cells; and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (e.g., skin cells), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (e.g., exocrine pancreatic cells), brain cells, lung cells, kidney cells, and adipocytes.

[0018] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (for example, blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Methods for establishing and maintaining human and monkey ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc. Natl. Acad. Sci. USA 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; Suemori H. et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; Ueno M. et al. (2006), Proc. Natl. Acad. Sci. USA 103:9554-9559; Suemori H. et al. (2001), Dev. Dyn., 222:273-279; Kawasaki H. et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I. et al. (2006), Nature. 444:481-485, etc.Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage before the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or can be established using developmentally arrested embryos (Zhang X. et al. (2006), Stem Cells 24: 2669-2676). Regarding "ES cells," various mouse ES cell lines established by inGenious targeting laboratory, Inc., RIKEN (Riken), and other institutions are available, while various human ES cell lines established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, and other institutions are available. For example, human ES cell lines that can be used include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 strains distributed by ESI Bio, H1 and H9 strains distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 strains distributed by RIKEN.

[0019] Nuclear transfer ES cells (nt ES cells) are cloned embryonic stem cells (ES cells) derived from a cloned embryo using nuclear transfer technology. They have almost the same properties as ES cells derived from a fertilized egg (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). Specifically, nt ES (nuclear transfer ES) cells are established from the inner cell mass of a blastocyst derived from a cloned embryo, which was obtained by replacing the nucleus of an unfertilized egg with that of a somatic cell. To generate nt ES cells, nuclear transfer technology (Cibelli JB et al. (1998), Nature Biotechnol., 16:642-646) is combined with ES cell generation technology (mentioned above) (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, and the egg can be reprogrammed by culturing it for several hours.

[0020] mGS cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various cell lineages. For example, when transplanted into mouse blastocysts, chimeric mice can be generated (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004), Cell, 119:1001-1012). They are capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, germline stem cells can be obtained by repeated passage under culture conditions similar to those for ES cells (Takebayashi M. et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0021] EG cells are derived from primordial germ cells (PGCs) during the fetal stage and possess pluripotency similar to that of ES cells. They can be established by culturing PGCs in the presence of substances such as LIF, bFGF, and stem cell factor (Matsui Y. et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551).

[0022] Muse cells are non-tumorigenic pluripotent stem cells present in living organisms and can be produced, for example, by the method described in WO 2011 / 007900. Specifically, fibroblasts or bone marrow stromal cells are trypsinized for a long period of time, preferably 8 or 16 hours, followed by suspension culture to obtain pluripotent cells, which are SSEA-3 and CD105 positive.

[0023] The step (0) is not particularly limited as long as it can induce the differentiation of pluripotent stem cells into cardiomyocytes. For example, pluripotent stem cells can be induced to differentiate into cardiomyocytes by culturing them in a cardiomyocyte differentiation medium. In one embodiment of the present invention, the step (0) may include (0-1) a step of inducing the differentiation of pluripotent stem cells into mesodermal cells, and (0-2) a step of inducing the differentiation of the mesodermal cells into cardiomyocytes. As used herein, the term "cardiomyocyte differentiation medium" refers to a medium containing a factor that promotes differentiation into cardiomyocytes, such as a cytokine (hereinafter sometimes referred to as "cardiomyocyte differentiation inducer"), and a basal medium. The cardiomyocyte differentiation inducer also encompasses factors necessary for inducing differentiation into intermediate cells (e.g., mesodermal cells) during the process of inducing differentiation of pluripotent stem cells into cardiomyocytes.

[0024] Basal media used in the present invention include, for example, StemFit (e.g., StemFit AK03N, StemFit AK02N) (Ajinomoto Co.), StemPro-34 (Thermo Fisher Scientific), PECM (Primate ES Cell Medium), GMEM (Glasgow Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof.

[0025] The basal medium may be supplemented with, as appropriate, ROCK inhibitors (e.g., Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152, Wf-536, etc.), serum (e.g., fetal bovine serum (FBS), human serum, horse serum, etc.) or serum substitutes, insulin, various vitamins (e.g., vitamin C (e.g., ascorbic acid)), L-glutamine, various amino acids such as non-essential amino acids, 2-mercaptoethanol, thioglycerol (e.g., α-monothioglycerol (MTG)), various cytokines, stem cell factor (SCF), activin, etc.), various hormones, various growth factors (e.g., leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, etc.), various extracellular matrices, various cell adhesion molecules, antibiotics such as penicillin / streptomycin and puromycin, pH indicators such as phenol red, and the like. Serum replacements include albumin, transferrin, fatty acids, insulin, collagen precursors, trace elements, Knockout Serum Replacement (KSR), ITS-supplements, and mixtures thereof.

[0026] In the present invention, vitamin C refers to L-ascorbic acid and its derivatives, and L-ascorbic acid derivatives refer to those that become vitamin C through an enzymatic reaction in vivo. Examples of ascorbic acid derivatives used in the present invention include vitamin C phosphate (e.g., ascorbic acid 2-phosphate), ascorbic acid glucoside, ascorbyl ethyl, vitamin C ester, ascorbyl tetrahexyldecanoate, ascorbyl stearate, and ascorbic acid 2-phosphate-6 palmitate. Vitamin C phosphate (e.g., ascorbic acid 2-phosphate) is preferred, including L-ascorbate phosphates such as sodium L-ascorbate phosphate and magnesium L-ascorbate phosphate.

[0027] Induced pluripotent stem cells or embryoid bodies may be cultured in either adherent or suspension culture. Adherent culture may be performed using a culture vessel coated with an extracellular matrix component, or co-culture with feeder cells. Examples of feeder cells include, but are not limited to, fibroblasts (mouse embryonic fibroblasts (MEF) and mouse fibroblasts (STO)). Feeder cells are preferably inactivated by known methods, such as irradiation (e.g., gamma rays) or treatment with anticancer drugs (e.g., mitomycin C). Examples of extracellular matrix components include Matrigel (Niwa A, et al., PLoS One. 6(7):e22261, 2011), fibrous proteins such as gelatin, collagen, and elastin, glycosaminoglycans and proteoglycans such as hyaluronic acid and chondroitin sulfate, and cell adhesive proteins such as fibronectin, vitronectin, and laminin.

[0028] The culture temperature is not particularly limited, but is, for example, about 37° C. to 42° C., preferably about 37° C. to 39° C. Culture may also be performed under hypoxic conditions, and in the present invention, hypoxic conditions are exemplified by oxygen concentrations of 15%, 10%, 9%, 8%, 7%, 6%, 5%, or less.

[0029] Suspension culture refers to the cultivation of cells in a non-adherent state in a culture vessel. This can be performed using, but is not limited to, a culture vessel that has not been artificially treated to improve cell adhesion (e.g., coated with an extracellular matrix, etc.) or a culture vessel that has been artificially treated to inhibit adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA) or a nonionic surface-active polyol (e.g., Pluronic F-127)). Suspension culture can also be performed using a culture vessel equipped with a stirring blade, such as a single-use bioreactor (Bio-t Inc.), a single-use bioreactor (Thermo Fisher), a single-use bioreactor (Sartorius Stedium), or a single-use bioreactor (GE Healthcare Life Sciences). The type of culture vessel and stirring speed can be appropriately selected by those skilled in the art depending on the type of cells being cultured. Examples of stirring speeds include, but are not limited to, 0-100 rpm, 20-80 rpm, or 45-65 rpm.

[0030] Furthermore, in the case of suspension culture, it is preferable to culture the cells after forming embryoid bodies (EBs). Therefore, the step (0) may include a step of forming embryoid bodies from pluripotent stem cells. In such a step, it is preferable to dissociate pluripotent stem cells that have formed colonies into single cells and then form embryoid bodies. In the step of dissociating pluripotent stem cells, cells that have adhered to each other to form a cluster are dissociated (separated) into individual cells. Methods for dissociating pluripotent stem cells include, for example, mechanical dissociation and dissociation methods using a dissociation solution having both protease and collagenase activity (e.g., Accutase™ and Accumax™) or a dissociation solution having only collagenase activity. Preferably, a method of dissociating pluripotent stem cells using a dissociation solution having both protease and collagenase activity (particularly preferably, Accumax™) is used. The medium used in the above step preferably contains thioglycerol, L-glutamine, and / or ascorbic acid.

[0031] Examples of cardiomyocyte differentiation-inducing factors used in the above step (0-1) include Wnt signal activators, activin A, BMP4, and bFGF, which may be used alone or in combination. In one embodiment of the present invention, a combination of activin A, BMP4, and bFGF is used. In addition, the medium used in the above step (0-1) preferably contains thioglycerol, L-glutamine, and / or ascorbic acid.

[0032] As used herein, the term "Wnt signal activator" refers to a substance that activates the Wnt signal pathway. Examples of Wnt signal activators include Wnt proteins and GSK3β inhibitors (e.g., BIO, CHIR99021, etc.). These may be used alone or in combination. When a Wnt signal activator is used, its concentration in the medium is not particularly limited. When BIO or CHIR99021 is used as the Wnt signal activator, it is preferably used at a final concentration in the medium of 100 nM to 100 μM, preferably 1 μM to 10 μM.

[0033] When activin A is used, its concentration in the culture medium is preferably 1 ng / mL to 100 ng / mL, and examples include 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL and 100 ng / mL.

[0034] When BMP4 is used, its concentration in the medium is preferably 1 ng / mL to 1 μg / mL, and may be 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 51 ng / mL, 52 ng / mL, 53 ng / mL, 54 ng / mL, 55 ng / mL, 56 ng / mL, 57 ng / mL, 58 ng / mL, 59 ng / mL, 60 ng / mL, 61 ng / mL, Examples include 1 μg / mL, 19 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL and 1 μg / mL.

[0035] When bFGF is used, its concentration in the culture medium is preferably 1 ng / mL to 100 ng / mL, and examples include 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL and 100 ng / mL.

[0036] The duration of step (0-1) is not particularly limited as long as mesodermal cells are obtained, but is preferably 12 hours or more (e.g., 1 day, 2 days or more), and can be 6 days or less (e.g., 5 days, 4 days, 3 days or less). Whether or not mesodermal cells have been obtained can also be monitored, and in this case, this can be determined by the expression of mesodermal marker genes. Examples of mesodermal marker genes include T, MIXL1, and NODAL.

[0037] Examples of cardiomyocyte differentiation inducers used in step (0-2) include Wnt inhibitors and VEGF, which may be used alone or in combination. The medium used in step (0-2) preferably contains thioglycerol, L-glutamine, and / or ascorbic acid.

[0038] As used herein, the term "Wnt inhibitor" refers to a substance that inhibits signal transduction, which continues from Wnt binding to its receptor to the accumulation of β-catenin. It may be a substance that inhibits binding to the Frizzled family of receptors, or a substance that promotes β-catenin degradation. Examples of Wnt inhibitors include DKK1 protein (e.g., in humans, NCBI accession number: NM_012242), sclerostin (e.g., in humans, NCBI accession number: NM_025237), IWR-1 (Merck Millipore), IWP-2 (Sigma-Aldrich), IWP-3 (Sigma-Aldrich), IWP-4 (Sigma-Aldrich), PNU-74654 (Sigma-Aldrich), XAV939 (Sigma-Aldrich), and derivatives thereof. Among these, IWP-3 or IWP-4 is preferred. A single Wnt inhibitor may be used, or multiple Wnt inhibitors may be used in combination.

[0039] When a Wnt inhibitor is used, its concentration in the medium is preferably 1 nM to 50 μM, for example, but not limited to, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, and 50 μM, and more preferably 1 μM.

[0040] When VEGF is used, the concentration thereof in the culture medium is preferably 1 to 100 ng / mL, and examples thereof include 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL and 100 ng / mL.

[0041] In the above step (0-2), a BMP inhibitor and / or a TGFβ inhibitor may be further added to the basal medium as a cardiomyocyte differentiation inducer. As used herein, the term "BMP inhibitor" refers to proteinaceous inhibitors such as Chordin, Noggin, and Follistatin, as well as Dorsomorphin (6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine) and its derivatives (PB Yu et al. (2007), Circulation, 116:II#60; PB Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904, LDN-193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), etc. Among these, Dorsomorphin is preferred. BMP inhibitors and TGFβ inhibitors may be used alone or in combination.

[0042] When a BMP inhibitor is used, the concentration thereof in the culture medium is preferably 1 nM to 50 μM, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, and 50 μM, but is not limited to these.

[0043] As used herein, a TGFβ inhibitor refers to a substance that inhibits signal transduction that continues from the binding of TGFβ to its receptor to SMAD, and may be a substance that inhibits binding to the ALK family receptor, or a substance that inhibits the phosphorylation of SMAD by the ALK family. Examples of TGFβ inhibitors include Lefty-1 (NCBI Accession No.: mouse: NM_010094, human: NM_020997), SB431542, SB202190 (RK Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO 2009146408), and derivatives thereof. Among these, SB431542 is preferred.

[0044] When a TGFβ inhibitor is used, the concentration thereof in the culture medium is preferably 1 nM to 50 μM, and examples thereof include, but are not limited to, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 5.2 μM, 5.4 μM, 5.6 μM, 5.8 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, and 50 μM.

[0045] The duration of step (0-2) is not particularly limited as long as cardiomyocytes can be obtained, but may be one day or more (e.g., one day, two days, three days, four days, five days, six days, seven days, or more). Since long-term culturing does not affect the establishment of cardiomyocytes, no upper limit is particularly set, but the duration is typically 40 days or less. Whether or not cardiomyocytes have been obtained may be monitored, and in this case, confirmation can be obtained by the number of beating cardiomyocytes, expression of cardiomyocyte markers, expression of ion channels, response to electrophysiological stimuli, etc.

[0046] Step (0) may further comprise step (0-3) of culturing the cardiomyocytes obtained in step (0-2) in the presence or absence of VEGF and / or bFGF. The medium used in this step preferably contains thioglycerol, L-glutamine and / or ascorbic acid. The medium used in this step may also contain a myocardial maturation compound (e.g., N-(1,1-dioxo-2,3-dihydro-1H-1-benzothiophen-5-yl)-2-(4-{5-[1-oxo-5-(piperidin-1-yl)-1,3-dihydro-2H-isoindol-2-yl]-1H-benzimidazol-2-yl}phenoxy)acetamide) and / or a multikinase inhibitor (e.g., 2-(4-{3-[3-(2-amino-2-phenylethoxy)-4-cyanophenyl]pyrazolo[1,5-a]pyrimidin-6-yl}-1H-pyrazol-1-yl)-N-(2-methoxyethyl)acetamide).

[0047] When VEGF is used in step (0-3), the concentration thereof in the medium is preferably 1 to 100 ng / mL, and examples thereof include 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, and 100 ng / mL.

[0048] When bFGF is used in step (0-3), its concentration in the medium is preferably 1 to 100 ng / mL, and examples thereof include 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, and 100 ng / mL. More preferably, it is 5 ng / mL.

[0049] The duration of step (0-3) is not particularly limited, but may be one day or more (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more). Since long-term culturing does not affect the establishment of cardiomyocytes, no upper limit is particularly set, but the duration is typically 60 days or less. By performing step (0-3) above, the efficiency of differentiation into cardiomyocytes can be improved.

[0050] Furthermore, before carrying out the above step (0-2) or step (0-3), the embryoid bodies may be dissociated by the same method as described above.

[0051] The methods specifically described above are merely illustrative and are not limited to these. Examples include a method of co-culturing pluripotent stem cells with END2 cells, which are mouse-derived support cells (Mummery, C., et al., Circulation. 107(21), 2733-40 (2003)), and a method of inducing cardiomyocytes by culturing embryoid bodies using BMP4, FGF2, insulin, and serum (Paul, W B., et al., PLoSone. 6(4), e18293 (2011)). Alternatively, a method of inducing differentiation of cardiomyocytes in adherent culture without using cytokines (Lian X, et al., Proc Natl Acad Sci U S A., 2012 July 3;109(27):E1848-57) or a method of inducing differentiation of cardiomyocytes in a combination of adherent culture and suspension culture without using cytokines (Minami I, et al., Cell Rep., 2012 Nov 29;2(5):1448-60) may be used.

[0052] By contacting a cell population containing cardiomyocytes obtained as described above with an HDAC inhibitor and then culturing the cell population, a cell population with a higher purity of cardiomyocytes can be produced compared to the cell population before contact with the HDAC inhibitor. That is, cardiomyocytes can be purified using an HDAC inhibitor. Therefore, in another aspect of the present invention, there is provided a method for purifying cardiomyocytes (hereinafter also referred to as the "purification method of the present invention"), comprising: (1') contacting a cell population containing cardiomyocytes and cells other than cardiomyocytes, obtained by culturing pluripotent stem cells in a cardiomyocyte differentiation medium, with an HDAC inhibitor; and (2') culturing the cell population. In yet another aspect, there is also provided a method for reducing non-cardiomyocytes from a cell population containing cardiomyocytes and cells other than cardiomyocytes (hereinafter also referred to as the "method for reducing non-cardiomyocytes of the present invention"), comprising the steps (1') and (2').

[0053] As used herein, purifying cardiomyocytes means that the rate of decrease in the number of cells other than cardiomyocytes ("cell number" means the number of viable cells; the same applies hereinafter) exceeds the rate of decrease in cardiomyocytes due to an HDAC inhibitor, or that the proliferation of cells other than cardiomyocytes is inhibited, causing the proliferation rate of cardiomyocytes to exceed the proliferation rate of cells other than cardiomyocytes, thereby increasing the proportion of cardiomyocytes in a cell population (number of cardiomyocytes in a cell population / total number of cells in a cell population). Therefore, an increase in the proportion of cardiomyocytes in purified cardiomyocytes is distinct from an increase in the proportion of cardiomyocytes due to promoting the induction of differentiation of cardiac progenitor cells into cardiomyocytes or inhibiting the induction of differentiation of cardiac progenitor cells into cells other than cardiomyocytes.

[0054] As shown in the Examples below, treatment with an HDAC inhibitor resulted in a greater reduction in the number of pluripotent stem cells than in the number of cardiomyocytes, and further demonstrated an increase in the proportion of cardiomyocytes in a cell population. Without wishing to be bound by any theory, it is speculated that the reduction in cell number caused by an HDAC inhibitor is the result of the HDAC inhibitor inducing cell apoptosis. It is also speculated that the proportion of undifferentiated cells, such as pluripotent stem cells, contained in a cell population is reduced by the HDAC inhibitor, or that the proportion of cardiomyocytes in the cell population is increased by removing undifferentiated cells from the cell population. Therefore, in another aspect of the present invention, there is provided a method for removing or reducing undifferentiated cells from a cell population (hereinafter also referred to as the "method for removing undifferentiated cells of the present invention"), comprising the steps of: (I) contacting a cell population containing cardiomyocytes contaminated with undifferentiated cells with a histone deacetylase inhibitor; and (II) culturing the cell population.

[0055] As used herein, the term "undifferentiated cells" refers to cells that retain differentiation potential, i.e., pluripotency, multipotency, oligopotency, or unipotency. Specific examples of undifferentiated cells include the above-mentioned pluripotent stem cells, mesodermal cells with differentiation potential, and cardiac progenitor cells.

[0056] In steps (1), (1'), and (I), the period for contacting the cardiomyocyte-containing cell population with the HDAC inhibitor is not particularly limited, but is preferably, for example, 1 hour or more (e.g., 2 hours, 3 hours, 5 hours, 12 hours, 1 day, 2 days, 3 days, or more). Furthermore, since long-term culture does not affect the establishment of cardiomyocytes or cardiac progenitor cells, no upper limit is particularly set, but typically, 60 days or less (e.g., 50 days, 40 days, 30 days, 20 days, 14 days, 13 days, 12 days, 11 days, or less) is preferred. Contacting the cardiomyocyte-containing cell population with the HDAC inhibitor may be carried out by adding the HDAC inhibitor to a medium containing the cell population, or by seeding the cell population in a medium to which the HDAC inhibitor has been added in advance. The timing of contact with the HDAC inhibitor is not particularly limited as long as it is the timing at which cardiomyocytes are included in the cell population. For example, contact is preferably carried out in step (0-2) or (0-3) above, and is preferably carried out on or after the seventh day from the start of differentiation induction of pluripotent stem cells, based on the day of initiation of differentiation induction.

[0057] The HDACs inhibited by the HDAC inhibitors used in the present invention may be any of class I HDACs (e.g., HDAC1, HDAC2, HDAC3, HDAC8), class II HDACs (e.g., HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, HDAC10), class III HDACs (e.g., SirT1, SirT2, SirT3, SirT4, SirT5, SirT6, SirT7), and class IV HDACs (e.g., HDAC11), but are preferably class I or II HDACs, and more preferably inhibitors of class I HDACs, particularly HDAC1 or HDAC2. As used herein, inhibitors of class I HDACs may have other activities, such as inhibitory activity against HDACs of classes other than class I, as long as they have inhibitory activity against class I HDACs, or may have inhibitory activity specific to class I HDACs. The same applies to inhibitors of other classes of HDACs.

[0058] Examples of HDAC1 inhibitors used in the present invention include Trichostatin A, CI994 (Tacedinaline), Quisinostat (JNJ-26481585), CUDC-907, PCI-24781 (Abexinostat), RG2833 (RGFP109), Romidepsin (FK228, Depsipeptide), Resminostat, Pracinostat (SB939), Rocilinostat (ACY-1215), Mocetinostat (MGCD0103), CAY10603, Entinostat (MS-275), 4SC-202, HPOB, PCI-34051, and Tubastatin A HCl.

[0059] Examples of HDAC2 inhibitors include Trichostatin A, Quisinostat (JNJ-26481585), CUDC-907, CUDC-101, PCI-24781 (Abexinostat), Romidepsin (FK228, Depsipeptide), Rocilinostat (ACY-1215), Pracinostat (SB939), Mocetinostat (MGCD0103), 4SC-202, and HPOB.

[0060] Examples of HDAC3 inhibitors include Trichostatin A, RGFP966, CUDC-907, Quisinostat (JNJ-26481585), RG2833 (RGFP109), PCI-24781 (Abexinostat), CUDC-101, Pracinostat (SB939), Resminostat, Rocilinostat (ACY-1215), 4SC-202, Mocetinostat (MGCD0103), HPOB, Entinostat (MS-275), and Droxinostat.

[0061] Examples of inhibitors against HDAC4 include Trichostatin A, Tasquinimod, Quisinostat (JNJ-26481585), LMK-235, CUDC-101, Pracinostat (SB939), TMP269, CUDC-907, and Rocilinostat (ACY-1215).

[0062] Examples of inhibitors against HDAC5 include quisinostat (JNJ-26481585), LMK-235, CUDC-101, pracinostat (SB939), TMP269, CUDC-907, and rocilinostat (ACY-1215).

[0063] Examples of inhibitors of HDAC6 include Trichostatin A, CAY10603, Tubacin, Rocilinostat (ACY-1215), Nexturastat A, Tubastatin A HCl, Tubastatin A, HPOB, CUDC-101, PCI-24781 (Abexinostat), CUDC-907, Resminostat, Quisinostat (JNJ-26481585), Pracinostat (SB939), Droxinostat, and PCI-34051.

[0064] Examples of inhibitors against HDAC7 include TMP269, Quisinostat (JNJ-26481585), Pracinostat (SB939), CUDC-101, CUDC-907, Rocilinostat (ACY-1215), and the like.

[0065] Examples of inhibitors of HDAC8 include PCI-34051, Quisinostat (JNJ-26481585), CUDC-101, Rocilinostat (ACY-1215), Pracinostat (SB939), CUDC-907, PCI-24781 (Abexinostat), Tubastatin A HCl, Droxinostat, and HPOB.

[0066] Examples of HDAC9 inhibitors include TMP269, Quisinostat (JNJ-26481585), CUDC-101, Pracinostat (SB939), and CUDC-907.

[0067] Examples of inhibitors of HDAC10 include Trichostatin A, Quisinostat (JNJ-26481585), CUDC-907, PCI-24781 (Abexinostat), CUDC-101, Pracinostat (SB939), HPOB, and PCI-34051.

[0068] Examples of inhibitors against HDAC11 include Quisinostat (JNJ-26481585), CUDC-907, Pracinostat (SB939), and Mocetinostat (MGCD0103).

[0069] Inhibitors of class III HDACs include sirtinol, SirReal2, nicotinamide (Vitamin B3), selisistat (EX 527), thiomyristoyl, salermide, OSS_128167, AK 7, 3-TYP, tenovin-1, rocilinostat (ACY-1215), and inauhzin.

[0070] Alternatively, non-selective HDAC inhibitors may be used, such as vorinostat (SAHA), panobinostat (LBH589), and belinostat (PXD101). Among these, FK228, entinostat, trichostatin A, and panobinostat are preferred. A single HDAC inhibitor may be used, or multiple HDAC inhibitors may be used in combination.

[0071] The concentration of the HDAC inhibitor in the medium can be appropriately selected by those skilled in the art, but is preferably 0.1 nM to 10 μM, for example, and specific examples include 0.5 nM, 1 nM, 2 nM, 3 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM. The concentration can also be changed appropriately depending on the type of compound. For example, when Trichostatin A is used, 0.1 μM to 10 μM is preferable, when Panobinostat is used, 0.1 to 3 μM is preferable, when FK228 is used, 1 nM to 1 μM is preferable, and when Entinostat is used, 0.1 μM to 10 μM is preferable, but is not limited to these concentrations.

[0072] The cell population culture methods in steps (2), (2'), and (II) are the same as those in (0-3) above. The culture period is also the same, and the culture should be continued at least while the cell population is in contact with the HDAC inhibitor.

[0073] 2. Cardiomyocyte-containing cell population The present invention also provides a cell population containing cardiomyocytes (hereinafter also referred to as the "cell population of the present invention") obtained by the production method, purification method, method for reducing non-cardiomyocytes, or method for removing undifferentiated cells of the present invention. As described above, the cell population of the present invention contains cardiomyocytes with high purity. "High purity" specifically means that the percentage of cardiomyocytes in the cell population (number of cardiomyocytes in the cell population / total number of cells in the cell population) is 80% or higher (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher). Naturally, when the cell population is used in combination with other cells or cell populations, such as mesenchymal stem cells, the above percentage of cardiomyocytes refers to the percentage before mixing with the other cells or cell populations. In a preferred embodiment, the cell population of the present invention contains a higher percentage of cardiomyocytes than cell populations obtained by conventional methods of inducing cardiomyocytes from pluripotent stem cells. Such a cell population may be further purified by cell sorting or the like, and such purified cell populations are also encompassed by the "cell population of the present invention."

[0074] 3. Cell transplantation therapy The present invention also provides a cell transplantation therapeutic agent (hereinafter also referred to as the "cell transplantation therapeutic agent of the present invention") comprising the cell population of the present invention. The cell transplantation therapeutic agent of the present invention may be used for autologous or allogeneic transplantation. It may also be used in combination with other drugs, such as immunosuppressants. As described above, the cell population of the present invention contains highly purified cardiomyocytes, making it suitable for use as a source of a cell transplantation therapeutic agent. The cell population of the present invention or the cell transplantation therapeutic agent of the present invention is useful for treating or preventing cardiac disease. Therefore, the present invention also encompasses a method for treating or preventing cardiac disease, in which an effective amount of the cell population or cell transplantation therapeutic agent of the present invention is administered or transplanted into a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) to be treated or prevented. Cardiac diseases to be treated or prevented include, but are not limited to, defects caused by diseases or disorders such as heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated phase hypertrophic cardiomyopathy, and dilated cardiomyopathy.

[0075] When the cell population of the present invention is used in a cell transplantation therapy, it is desirable to use a cell population containing cells derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, from the viewpoint of preventing rejection. Here, "substantially the same" means that the HLA genotype is identical to that of the transplanted cells to an extent that immune responses can be suppressed with immunosuppressants, for example, somatic cells with an HLA type that matches the three HLA loci (HLA-A, HLA-B, and HLA-DR) or the four HLA loci (HLA-C plus HLA-C). Alternatively, the cells can be transplanted in a state where they are embedded in a capsule or porous container made of polyethylene glycol or silicone to avoid rejection.

[0076] The cell population of the present invention is prepared as a parenteral preparation such as an injection, suspension, or infusion by mixing with a pharmaceutically acceptable carrier according to conventional methods. Pharmaceutically acceptable carriers that can be included in such parenteral preparations include aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). The cell transplantation therapy of the present invention may be formulated with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc. When the transplantation therapy of the present invention is formulated as an aqueous suspension, approximately 1 × 10 cells are added to the aqueous solution. 6 ~Approx. 1×10 8 The cell population containing cardiomyocytes can be suspended to a concentration of cells / mL. It can also be administered together with a scaffold that promotes engraftment. Examples of scaffolds include, but are not limited to, bio-derived components such as collagen and synthetic polymers such as polylactic acid.

[0077] Alternatively, cardiac disease may be treated by forming the obtained cardiomyocytes into a sheet and applying it to the patient's heart. When administering a myocardial sheet, it is achieved by placing it so that it covers the desired area. This placement can be achieved using techniques well known in the art. If the desired area is large, the sheet may be placed so that it surrounds the tissue. Furthermore, administration can be performed multiple times to the same area to achieve the desired effect. When placing multiple times, it is desirable to allow sufficient time for the desired cells to engraft into the tissue and induce angiogenesis. The mechanism of such cardiac disease treatment may be an effect resulting from the engraftment of the myocardial sheet, or an indirect effect unrelated to cell engraftment (e.g., the effect of mobilizing recipient-derived cells to the damaged site by secreting an attractant). When using a myocardial sheet to treat cardiac disease, it may contain a cell scaffold material (scaffold) such as collagen, fibronectin, or laminin in addition to cardiomyocytes. Alternatively, it may contain any cell type(s) in addition to cardiomyocytes. The number of cardiomyocytes used to treat cardiac disease is not particularly limited as long as the administered myocardial sheet is effective in treating cardiac disease, and can be adjusted appropriately depending on the size of the affected area and the size of the body.

[0078] In yet another embodiment, the cell population of the present invention can be used for drug screening for the treatment of cardiac diseases or for evaluating the cardiotoxicity of drugs. For example, the efficacy and toxicity of a test drug can be evaluated by administering the test drug to the cell population of the present invention and examining the response of cardiomyocytes.

[0079] The present invention will be explained in more detail in the following examples, but the scope of the present invention is not limited to these examples. [Example]

[0080] Test Example 1: Screening for compounds with purifying effects on cardiomyocytes Assuming that there are differences in the sensitivity to compounds targeting cell proliferation and the cell cycle due to the differences in the proliferative properties between cardiomyocytes and non-cardiomyocytes, we performed screening for compounds having a purification effect on cardiomyocytes using a research-use iPS cell line.

[0081] <iPS cell line> To detect the maturation of cardiomyocytes, a double knock-in human iPS cell line (reporter iPS cell line) was prepared by inserting the sequences of reporter proteins EmGFP (SEQ ID NO: 1) at the TNNI1 locus and mCherry (SEQ ID NO: 2) at the TNNI3 locus. The maintenance culture of the above reporter iPS cell line was performed by the conventional method (Okita K, et al. Stem Cells. 2012 Nov 29. doi: 10.1002 / stem.1293). The human iPS cells were established using PBMC (LP_167, Sample ID: 20130318) purchased from CTL by an episomal vector (carried genes; OCT3 / 4, KLF4, SOX2, L-MYC, LIN28, mouse p53DD) (reference; Okita K, et al. Stem Cells. 2012 Nov 29. doi: 10.1002 / stem.1293).

[0082] <Induction of differentiation into cardiomyocytes> Induction of differentiation into cardiomyocytes was performed in a 6-well plate according to the method described in the paper (Miki et al, Cell Stem Cell. 2015 Jun 4;16. doi: 10.1016 / j.stem.2015.04.005.). Briefly, for the induction of differentiation into cardiomyocytes, the reporter iPS cell line was treated with TrypLE select (Life Technologies) diluted 1 / 2 with 0.5 mM EDTA / PBS for 4 - 5 minutes, then the cells were detached with a cell scraper (IWAKI) and dissociated into single cells by pipetting. The medium was removed by centrifugation at 1,000 rpm for 5 minutes, and the obtained cells were seeded at 2×10 per well of a 6-well plate. 610 cells were seeded in StemPro34 medium containing 1% L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid (Sigma), and 4 × 10 monothioglycerol. -4 The cells were cultured in 1.5 mL / well of medium supplemented with M, 10 μM Y-27632, 2 ng / mL BMP4 (R&D), and 0.5% Growth Factor Reduced Matrigel at 37°C under 5% oxygen conditions to form embryoid bodies (day 0). The next day (day 1), StemPro34 medium was supplemented with 1% L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid (Sigma), and 4 × 10 monothioglycerol. -4 1.5 mL of medium containing 2 ng / mL BMP4 (R&D), activin A 12 ng / mL, bFGF 5 ng / mL, and BMP4 18 ng / mL was added to each well, and the cells were cultured at 37°C and 5% oxygen for another 2 days. On the third day, the 6-well plate was tilted to allow the embryoid bodies to settle, and after removing 80-90% of the medium, 1.5 mL of IMDM was added to each well. After removing 80-90% of the medium, the plate was tilted again to allow the embryoid bodies to settle, and the plate was then resuspended in StemPro34 medium containing 1% L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid (Sigma), and 4 × 10 monothioglycerol. -4 The cells were cultured in a medium supplemented with 10 ng / mL VEGF, 1 μM IWP-3, 0.6 μM Dorsomorphin, and 5.4 μM SB431542 at 37°C under 5% oxygen for 3 days. On the sixth day, the 6-well plate was tilted and left to settle, and 80-90% of the medium was removed. After that, the embryoid bodies were cultured in a medium containing 1% L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid (Sigma), and 4 × 10 monothioglycerol. -4 StemPro34 medium supplemented with 5 ng / mL VEGF was added. The cells were cultured for 10 days at 37°C under 5% oxygen. During this period, the medium was replaced with the same medium every 2-3 days. From the 10th day onwards, the cells were cultured at 37°C under 21% oxygen.

[0083] <Single-cell generation of embryoid bodies and compound screening> On day 22 after the start of differentiation, the 6-well plate containing the embryoid bodies was tilted and left for 1-2 minutes until the embryoid bodies settled to the edge of the well. The supernatant was aspirated, taking care not to aspirate the embryoid bodies. Then, 2 mL of PBS was added, tilted as above, and left for 1-2 minutes. The PBS was aspirated, taking care not to aspirate the embryoid bodies. EBs were dissociated into single cells using a Papain Dissociation System (Worthington), centrifuged (70 g, 6 minutes), suspended in 2% FBS / PBS, and GFP-positive cells were isolated using a flow cytometer. After centrifugation, the supernatant was removed and the cells were resuspended in cardiac differentiation medium based on StemPro34 medium (Stempro34 with ascorbic acid 50 μg / mL, L-glutamine 2 mM, transferrin 150 mg / mL, and monothioglycerol 4 × 10 -4 The resuspended cells were then resuspended in 100 mL of 5% CO₂ containing 5 ng / mL VEGF. The resuspended cells were plated onto a CellCarrier-384 plate Ultra Microplate pre-coated with fibronectin, with 2.0 × 10 cells. 3 The cell population obtained by this culture is hereinafter referred to as cardiomyocytes. In addition, iPS cell lines were treated with 0.5x TrypLE select (Life Technologies, diluted 1 / 2 with 0.5mM EDTA / PBS) for 4-5 minutes, then detached with a cell scraper (IWAKI) and dissociated into single cells by pipetting. The medium was removed by centrifugation (1,000 rpm, 5 minutes), and the cells were resuspended in StemFit AK02 medium supplemented with 10µM Y-27632. 2.0x10 cells were plated onto a CellCarrier-384 Ultra Microplate (PerkinElmer / 6057300) coated with iMatrix-511 (Nippi). 3 Cells were seeded at 10 cells / well. For cardiomyocytes and single-celled iPS cells, the evaluation compound was added 2 days after seeding and cultured for 2 days. 48 hours after compound treatment, the number of viable cells was evaluated by measuring the intracellular ATP level using CellTiter-Glo (PerkinElmer). As a result of the screening, the results of the compound (HDAC inhibitor) that significantly decreased the number of viable cells (ATP level) of iPS cells compared to that of cardiomyocytes are shown in Fig. 1. In Fig. 1, the relative values are shown as when the ATP levels of the DMSO controls for iPS cells and cardiomyocytes were set to 100% (n = 2).

[0084] Test Example 2: Verification of purification effect in clinical iPS cell lines Next, it was confirmed by an ATP assay whether the HDAC inhibitor found in the screening of Test Example 1 also has a purifying effect on cardiomyocytes derived from clinical iPS cell lines.

[0085] <iPS cell line> A clinical iPS cell line prepared by CiRA was used. The maintenance culture of the iPS cell line was performed according to the conventional method (Okita K, et al. Stem Cells. 2012 Nov 29. doi: 10.1002 / stem.1293).

[0086] <Induction of differentiation into cardiomyocytes> The induction of differentiation into cardiomyocytes was performed in the same manner as the method described in <Induction of differentiation into cardiomyocytes> of Test Example 1 above.

[0087] <Single-cell formation of embryoid bodies and screening of compounds> On day 20, the 10 cm dish containing the embryoid bodies was tilted and allowed to settle. The supernatant was aspirated, taking care not to aspirate the embryoid bodies. 3 mL of IMDM containing 10 μg / mL DNase and 100 μg / mL Liberase was added per dish and incubated at 37°C under normal oxygen conditions for 1 hour. After 1 hour, the tube was left to stand for 1–2 minutes until the embryoid bodies settled. The supernatant was aspirated, taking care not to aspirate the embryoid bodies. 2 mL of TrypLE select containing 10 μg / mL DNase was added per dish and incubated at 37°C under normal oxygen conditions for 15 minutes. 2 mL of IMDM containing 10 μg / mL DNase was then added per dish. The cells were separated into single cells by pipetting and centrifuged at 100 g for 5 minutes at 4°C. After centrifugation, the supernatant was removed and the cells were suspended in I3 medium (the same cardiac differentiation medium as in Test Example 1), and then 2 x 10 cells were plated on a fibronectin-coated 96-well plate. 4 The cells were seeded at a seeding density of 100 cells / well. The cell population obtained by this culture is hereinafter referred to as cardiomyocytes. At this time, some of the cells were fixed and stained with anti-sarcomeric α-actinin antibody, and sarcomeric α-actinin expression was analyzed using a flow cytometer. The sarcomeric α-actinin positive rate was 99.4%. In addition, iPS cells cultured in a 10 cm dish were washed with 5 mL of PBS, then 5 mL of Accutase was added and the dish was left to stand at 37°C under normal oxygen conditions for 7 minutes. After 7 minutes, the cells were detached by pipetting to form single cells, which were then centrifuged (1,000 rpm, 5 minutes). After centrifugation, the supernatant was removed and the cells were suspended in undifferentiated maintenance medium supplemented with 10 μM Y-27632. 5 × 10 cells were then plated onto a 96-well plate coated with iMatrix-511 (Nippi). 3 The cells were seeded at a seeding density of 100 cells / well. For cardiomyocytes and single-cell iPS cells, on the day after seeding, the medium was removed, and 100 μL of a new medium diluted with a compound (0.1 nM to 10 μM) or DMSO (0.1%) was added. The next day, the supernatant was removed, the cells were washed with PBS, and intracellular ATP was measured using the ATPlite 1step ATP detection system (PerkinElmer). The results are shown in Figure 2 (mean value ± standard deviation, n = 4). The effect of compound treatment on intracellular ATP levels was expressed as a relative value when the DMSO-treated cells of each of the iPS cells and cardiomyocytes were set to 100%.

[0088] From the results of Test Examples 1 and 2 above, regardless of the type of iPS cell line, the viable cell count of undifferentiated iPS cells was significantly decreased by treatment with an HDAC inhibitor, but in cardiomyocytes differentiated from iPS cells, the decrease in viable cell count was low.

[0089] Test Example 3: Verification of the effect of HDAC inhibitors on embryoid bodies containing a mixture of cardiomyocytes and non-cardiomyocytes 1 From the above results, the inventors considered that cardiomyocytes might be purified in embryoid bodies in which cardiomyocytes and non-cardiomyocytes such as iPS cells coexist by using an HDAC inhibitor. Therefore, the effect of an HDAC inhibitor on embryoid bodies was verified by the following procedure.

[0090] <iPS cell line> A clinical-grade iPS cell line prepared by CiRA was used. The maintenance culture of the iPS cell line was performed according to the conventional method (Okita K, et al. Stem Cells. 2012 Nov 29. doi: 10.1002 / stem.1293).

[0091] <Induction of differentiation into cardiomyocytes> Induction of differentiation into cardiomyocytes was performed in the same manner as the method described in <Induction of differentiation into cardiomyocytes> of Test Example 1 above.

[0092] <Compound treatment of embryoid bodies, single-cell formation of embryoid bodies, and cell sorting> Embryoid bodies on day 20 were collected in a centrifuge tube and allowed to settle. The supernatant was aspirated to avoid absorbing the embryoid bodies. The medium was replaced with fresh I3 medium (the same cardiac differentiation medium as in Test Example 1), and the medium was divided into 6-well plates at 3 mL / well to ensure that each well contained an equal amount of embryoid bodies. The evaluation compounds (1 nM to 10 μM) listed in Table 1 or DMSO (0.1%) were then added. The concentrations of each compound (final concentration in the medium) are listed in Table 1. The cells were then cultured at 37°C under normal oxygen conditions for 3 days. Three days after compound addition, the 6-well plate was left to settle the embryoid bodies, and the embryoid bodies were collected into a 1.5 mL tube using a pipette equipped with a wide-bore tip. The cells were centrifuged at 500 g for 1 minute. The supernatant was removed, and 500 μL of IMDM containing 10 μg / mL DNase and 100 μg / mL Liberase was added per tube. The tubes were then incubated at 37°C under normoxic conditions for 1 hour. After 1 hour, the tubes were centrifuged at 500×g for 1 minute, and the supernatant was removed, taking care not to aspirate the embryoid bodies. 500 μL of TrypLE select containing 10 μg / mL DNase was then added per tube. The tubes were incubated at 37°C under normoxic conditions for 15 minutes. Single cells were then obtained by pipetting, and 500 μL of IMDM containing 10 μg / mL DNase was added per tube. The cells were then mixed by inversion and centrifuged at 700×g for 5 minutes. After centrifugation, the supernatant was removed, and the cells were resuspended in Cytofix / Cytoperm Fixation / Permeabilization Solution (BD) and fixed at room temperature for 15 minutes. The fixed cells were stained with anti-sarcomeric α-actinin antibody, and the expression of sarcomeric α-actinin was analyzed using a flow cytometer. The results are shown in Figure 3 (mean ± standard deviation of n = 3 for DMSO-treated cells only, n = 1 for compound-treated cells).

[0093] [Table 1]

[0094] Test Example 4: Verification of the effect of HDAC inhibitors on embryoid bodies containing a mixture of cardiomyocytes and non-cardiomyocytes 2 Next, a similar verification was carried out using the same cell line as in Test Example 3, and it was confirmed that the cardiomyocyte rate was also improved with Entinostat, and that the cardiomyocyte rate was also improved with a lower concentration of FK228.

[0095] <Induction of differentiation into cardiomyocytes> The induction of differentiation into cardiomyocytes was carried out in the same manner as described in Test Example 1 above under <Induction of differentiation into cardiomyocytes>.

[0096] <Chemical treatment of embryoid bodies, isolation of embryoid bodies into single cells, and cell sorting> Embryoid bodies on day 20 were collected in a centrifuge tube and allowed to settle. The supernatant was aspirated to avoid absorbing the embryoid bodies. The medium was replaced with fresh I3 medium (the same cardiac differentiation medium as in Test Example 1), and the medium was divided into 6-well plates at 3 mL per well to ensure that each well contained an equal amount of embryoid bodies. The test compound (1 nM–10 μM) or DMSO (0.1%) was then added. The concentrations of each compound (final concentration in the medium) are listed in Table 2. The plates were then cultured at 37°C under normoxic conditions for 4 days. Four days after compound addition, the 6-well plate was left to settle the embryoid bodies, and the embryoid bodies were collected into 1.5 mL tubes using a pipette with a wide tip. The tubes were centrifuged at 500 g for 1 minute. The supernatant was removed, and 500 μL of IMDM solution containing 10 μg / mL DNase and 100 μg / mL Liberase was added per tube. The tubes were then incubated at 37°C under normoxic conditions for 1 hour. After 1 hour, the tubes were centrifuged at 500g for 1 minute, and the supernatant was removed, taking care not to aspirate the embryoid bodies. Then, 500µL of TrypLE select supplemented with 10µg / mL DNase was added per tube and incubated at 37°C under normal oxygen conditions for 15 minutes. After pipetting, single cells were obtained. Then, 500µL of IMDM supplemented with 10µg / mL DNase was added per tube, mixed by inversion, and centrifuged (700g for 5 minutes). After centrifugation, the supernatant was removed, and the cells were resuspended in Cytofix / Cytoperm Fixation / Permeabilization Solution (BD) and incubated at room temperature for 15 minutes for fixation. The fixed cells were stained with anti-sarcomeric α-actinin antibody, and the percentage of sarcomeric α-actinin-positive cells was measured by flow cytometry. The results are shown in FIG. 4 (mean values ​​(standard deviations) of n=3 for DMSO-treated cells only, and n=1 for compound-treated cells).

[0097] [Table 2]

[0098] Test Example 5. Verification of the effect of HDAC inhibitors on embryoid bodies containing a mixture of cardiomyocytes and non-cardiomyocytes 3 Next, verification was carried out using the same cell line as in Test Example 3, and it was confirmed that the rate of non-cardiomyocytes was reduced by FK-228 treatment.

[0099] <Induction of differentiation into cardiomyocytes> The induction of differentiation into cardiomyocytes was carried out in the same manner as described in Test Example 1 above under <Induction of differentiation into cardiomyocytes>.

[0100] <Treatment of embryoid bodies with compounds and conversion of embryoid bodies into single cells> Embryoid bodies on day 21 were collected in a centrifuge tube and allowed to settle. The supernatant was aspirated to avoid absorbing the embryoid bodies. The medium was replaced with fresh I3 medium (the same cardiac differentiation medium as in Test Example 1). The medium was then divided into 6-well plates at 3 mL per well to ensure that each well contained an equal amount of embryoid bodies. FK-228 (0.1 nM, 1 nM) or DMSO (0.1%) was added. The plates were then cultured at 37°C under normal oxygen conditions for 3 days. Three days after compound addition, the 6-well plates were allowed to settle, and the embryoid bodies were collected into 1.5 mL tubes using a pipette equipped with a wide-bore tip. The plates were centrifuged at 500 g for 1 minute. The supernatant was removed, and 3 mL of a solution containing 1 mL of IMDM (Iscove's Modified Dulbecco's Media) supplemented with 10 μg / mL DNase and 100 μg / mL Liberase was added to each tube. The tubes were then incubated at 37°C under normal oxygen conditions for 1 hour. After 1 hour, the tubes were centrifuged at 500 xg for 1 minute, and the supernatant was removed without absorbing the embryoid bodies. 500 μL of TrypLE select (Thermo) supplemented with 10 μg / mL DNase was added to each tube, and the tubes were incubated at 37°C under normal oxygen conditions for 10 minutes. After incubation, the cells were separated into single cells by pipetting, and 500 μL of IMDM supplemented with 10 μg / mL DNase was added to each tube and mixed by inversion to prepare a single-cell suspension.

[0101] The single-cell suspension was centrifuged at 400g for 3 minutes, the supernatant removed, and the cells were resuspended in Bambanker and frozen at -80°C. The frozen cells were thawed in a 37°C water bath, centrifuged at 400g for 5 minutes, and the supernatant removed. After tapping the cell pellet, PBS containing 1% BSA was added, and the cells were centrifuged at 300g for 1 minute. The supernatant was removed. The cells were stained with APCFire750-labeled anti-CD326 antibody, PE-labeled anti-CD49a antibody, BV605-labeled anti-CD31 antibody, and DAPI in 1% BSA-containing PBS. After removing DAPI-positive dead cells, the signal intensity of each fluorescent dye was measured to separate the cell populations and determine their proportions.

[0102] Cardiomyocytes induced to differentiate from iPS cell lines into cardiac muscle were separated into four main cell populations based on the differences in the expression of these three surface markers: CD326-positive cells (endodermal lineage cells), CD326-negative CD31-positive cells (vascular endothelial-like cells), CD326-negative CD31-negative CD49a-positive cells (smooth muscle-like cells), and CD326-negative CD31-negative CD49a-negative cells.

[0103] The results are shown in FIG. 5 (mean values ​​(standard deviations) of n=3 for DMSO-treated cells only, and n=1 for compound-treated cells).

[0104] These results demonstrate that cardiomyocytes in embryoid bodies can be purified by inhibiting HDAC. [Industrial Applicability]

[0105] The present invention provides a cell population containing highly purified cardiomyocytes. Such a cell population is useful because it can be suitably used in cell transplantation therapy for heart disease and in screening for therapeutic agents for heart disease.

[0106] This application is based on patent application No. 2020-050269 (filing date: March 19, 2020) and patent application No. 2020-145097 (filing date: August 28, 2020) filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing a cell population comprising human cardiomyocytes, comprising: (1) contacting a cell population containing cardiomyocytes and cells other than cardiomyocytes, obtained by culturing human pluripotent stem cells in a medium for cardiomyocyte differentiation, with a histone deacetylase inhibitor; and (2) culturing the cell population; A method comprising:

2. The method according to claim 1, wherein the contacting of the cell population with the histone deacetylase inhibitor in step (1) is carried out on or after day 7 from the start of the differentiation induction of human pluripotent stem cells.

3. The method of claim 1 or 2, wherein the inhibitor is an inhibitor of class I histone deacetylase.

4. The method according to any one of claims 1 to 3, wherein the inhibitor is at least one selected from the group consisting of FK228, Entinostat, Trichostatin A, and Panobinostat.

5. The method according to any one of claims 1 to 4, wherein the human pluripotent stem cells are induced pluripotent stem cells.

6. A method for purifying human cardiomyocytes, comprising: (1) contacting a cell population containing cardiomyocytes and cells other than cardiomyocytes, obtained by culturing human pluripotent stem cells in a medium for cardiomyocyte differentiation, with a histone deacetylase inhibitor; and (2) culturing the cell population; A method comprising:

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