Method for purifying cardiomyocytes

Abstract

To provide a method for producing a cardiac muscle cell by using a receptor type tyrosine kinase inhibitor, and to provide a method for purifying the cardiac muscle cell.SOLUTION: A method for producing a cell population comprising cardiomyocytes, comprising the steps of: (1) culturing pluripotent stem cells in a medium for cardiomyocyte differentiation to obtain a cell population comprising cardiomyocytes or myocardial progenitor cells and other cells; and (2) culturing the cell population in the presence of a receptor tyrosine kinase inhibitor (excluding an EGF receptor inhibitor). And (2) a step of culturing the cell population.SELECTED DRAWING: None

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 receptor tyrosine kinase 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, by suppressing the proliferation of non-cardiomyocyte cells or reducing the number of non-cardiomyocyte cells in a cell population that already contains cardiomyocytes, cardiomyocytes could be purified. Therefore, they first performed single-cell RNA sequencing analysis on a cell population containing cardiomyocytes induced to differentiate from iPS cells, and clustered the cells in the cell population. The clustering results revealed differences in the expression of receptor tyrosine kinases between cardiomyocytes and other cells. They then examined the change in the proportion of cardiomyocytes in the cell population using a receptor tyrosine kinase inhibitor and found that receptor tyrosine kinase inhibitors could successfully purify cardiomyocytes. 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 or cardiac progenitor cells and other cells obtained by culturing pluripotent stem cells in a medium for cardiomyocyte differentiation with a receptor tyrosine kinase inhibitor (excluding EGF receptor inhibitors); and (2) culturing the cell population; A method comprising: [2] The method according to [1], wherein the contact of the cell population with the receptor tyrosine kinase inhibitor in step (1) is carried out on or after the fourth day from the start of the differentiation induction of the pluripotent stem cells. [3] The method according to [1] or [2], wherein the cell population in step (1) is contacted with the receptor tyrosine kinase inhibitor for one day or more. [4] The method according to any one of [1] to [3], wherein the inhibitor is an inhibitor of at least one receptor tyrosine kinase selected from the group consisting of a VEGF receptor, a PDGF receptor, an HGF receptor, and an FGF receptor. [5] The method according to any one of [1] to [4], wherein the inhibitor is at least one selected from the group consisting of N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide, N-{4-[(6,7-dimethoxyquinolin-4-yl)oxy]-3-fluorophenyl}-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide, AMG337, ASP5878, BGJ398, foretinib, ZM323881, CP-673451, crenolanib, and crizotinib. [6] The method according to any one of [1] to [5], wherein the pluripotent stem cells are induced pluripotent stem cells. [7] A cell population containing cardiomyocytes obtained by the method according to any one of [1] to [6]. [8] A cell transplantation therapy agent comprising the cell population described in [7]. [9] A method for purifying cardiomyocytes, comprising: (1) contacting a cell population containing cardiomyocytes or cardiac progenitor cells and other cells obtained by culturing pluripotent stem cells in a medium for cardiomyocyte differentiation with a receptor tyrosine kinase inhibitor; and (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 is a method for purifying highly purified cardiomyocytes from a cell population containing cardiomyocytes and cardiac progenitor cells. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows the t-SNE plot of clustering based on single-cell RNA sequencing data of cardiomyocytes differentiated from iPS cells, and the expression levels of sarcomeric-α-actinin and cTnT (Cardiac Troponin T) in each cluster. The circled area in the upper panel indicates the non-cardiomyocyte population. In the middle and lower panels, the vertical axis represents gene expression levels, and the "identity" label on the horizontal axis indicates the cluster number in Figure 1. Each dot represents an individual cell, and the boxed area represents the non-cardiomyocyte cluster. [Figure 2-1] Figure 2-1 shows the t-SNE plot of the clustering results of iPS cell-derived cardiomyocytes based on single RNA sequencing data, along with the expression of the VEGFR1 and VEGFR2 genes in each cell. In the panel showing gene expression, dark gray dots represent cells with high expression levels, while light gray dots represent cells with low expression levels. Cell populations with high expression levels are circled, and the population name is indicated in the figure (CM: cardiomyocytes (CM), SMC: smooth muscle-like cells (SMC), END: endodermal lineage cells (END), EC: endothelial-like cells). [Figure 2-2] Figure 2-2 shows the t-SNE plot of the clustering results of iPS cell-derived cardiomyocytes based on single RNA sequencing data, along with the expression of the VEGFR3, PDGFRA, and PDGFRB genes in each cell. In the panel showing gene expression, dark gray dots represent cells with high expression levels, while light gray dots represent cells with low expression levels. Cell populations with high expression levels are circled, and the population name is indicated in the figure (SMC: smooth muscle-like cells (hereafter SMC), EC: endothelial-like cells). [Figure 2-3]Figure 2-3 shows the t-SNE plot of the clustering results of iPS cell-derived cardiomyocytes based on single RNA sequencing data, along with the expression of the FGFR4, HGFR (c-Met), and EGFR1 genes in each cell. In the gene expression panel, dark gray dots represent cells with high expression levels, while light gray dots represent cells with low expression levels. Cell populations with high expression levels are circled, and the population name is indicated in the figure (END: endoderm lineage cells (hereafter referred to as END)). [Figure 2-4] Figures 2-4 show t-SNE plots of the clustering results of iPS cell-derived cardiomyocytes based on single RNA sequencing data, along with the EGFR3 gene expression of each cell. Dark gray dots in the gene expression panels represent cells with high expression levels, while light gray dots represent cells with low expression levels. Cell populations with high expression levels are circled, and the population name is indicated in the figure (END: endoderm lineage cells (hereafter referred to as END)). [Figure 3] Figure 3 shows the separation of iPS cell-derived cardiomyocytes by their expression of CD326, CD31, and CD49a. Using flow cytometry, TNNI1 reporter iPS cell-derived cardiomyocytes, with a cardiomyocyte rate (TNNI1 positivity) of 89.6% (leftmost panel), were separated into CD326-positive cells (END, cells boxed in the center left panel), CD326-negative CD31-positive cells (EC, cells boxed in the center right panel), CD326-negative CD31-negative CD49a-positive cells (SMC, cells boxed in the center right panel), and CD326-negative CD31-negative CD49a-negative cells (Triple Negative: TN, cells located at the bottom left of the center right panel). More than 99% of the TN cells were positive for the cardiac marker TNNI1 (cardiomyocytes) (rightmost panel). The percentages of EC and SMC are shown based on the CD326-negative cells, not the cells within the panel. [Figure 4] Figure 4 shows the cardiomyocyte rate after compound treatment. *: Measurement not performed due to small cell numbers. The vertical axis shows the experiment number, and the horizontal axis shows the actinin-positive cell rate (cardiomyocyte rate). [Figure 5]5 shows the non-cardiomyocyte rate after compound treatment (END: endodermal lineage cells, SMC: smooth muscle-like cells, EC: endothelial-like cells). The vertical axis indicates the experiment number, and the horizontal axis indicates the non-cardiomyocyte rate. [Figure 6] 6 shows the average (standard deviation) of cardiomyocyte percentage after compound treatment (n=4). The vertical axis indicates the experiment number, and the horizontal axis indicates the cardiomyocyte percentage. [Figure 7] 7 shows the non-cardiomyocyte rate after compound treatment (END: endodermal lineage cells, SMC: smooth muscle-like cells, EC: endothelial-like cells). The vertical axis shows the experiment number, and the horizontal axis shows the non-cardiomyocyte rate. [Figure 8] 8 shows the average value (standard deviation) (n=3) of the cardiomyocyte percentage after compound treatment. The vertical axis indicates the experiment number, and the horizontal axis indicates the cardiomyocyte percentage. [Figure 9] 9 shows the average value (standard deviation) (n=3) of the relative ratio of the number of recovered cells after compound treatment, when the number of recovered cells without compound treatment was set to 1. The vertical axis indicates the experiment number, and the horizontal axis indicates the relative ratio of the number of recovered cells. [Figure 10] 10 shows the average value (standard deviation) of the cardiomyocyte percentage after compound treatment (n=4). The vertical axis indicates the experiment number, and the horizontal axis indicates the cardiomyocyte percentage. [Figure 11] 11 shows the average value (standard deviation) (n=4) of the relative ratio of the number of recovered cells after compound treatment, when the number of recovered cells without compound treatment was set to 1. The vertical axis indicates the experiment number, and the horizontal axis indicates the relative ratio of the number of recovered cells. [Figure 12] 12 shows the average value (standard deviation) of the cardiomyocyte percentage after compound treatment (n=4). The vertical axis indicates the experiment number, and the horizontal axis indicates the cardiomyocyte percentage. [Figure 13] 13 shows the mean (standard deviation) (n=4) of the non-cardiomyocyte rate after compound treatment (END: endodermal lineage cells, SMC: smooth muscle-like cells, EC: endothelial-like cells). The vertical axis indicates the experiment number, and the horizontal axis indicates the non-cardiomyocyte rate. [Figure 14] 14 shows the average value (standard deviation) (n=4) of the relative ratio of the number of recovered cells after compound treatment, when the number of recovered cells without compound treatment was set to 1. The vertical axis indicates the experiment number, and the horizontal axis indicates the relative ratio of the number of recovered cells. [Figure 15] 15 shows the cardiomyocyte rate after compound treatment (experiment number 1 is the average of n=2, others are n=1). The vertical axis shows the experiment number, and the horizontal axis shows the cardiomyocyte rate. [Figure 16] Figure 16 shows the non-cardiomyocyte rate after compound treatment (END: endodermal lineage cells, SMC: smooth muscle-like cells, EC: endothelial-like cells). The vertical axis shows the experiment number, and the horizontal axis shows the non-cardiomyocyte rate. *: Measurement not performed due to small number of cells. [Figure 17] Figure 17 shows the cardiomyocyte percentage after compound treatment (experiment number 1: n=4, experiments 2 and 3: n=3, mean value (standard deviation); experiments 4 and 5: n=1). The vertical axis shows the experiment number, and the horizontal axis shows the cardiomyocyte percentage. [Figure 18] Figure 18 shows the non-cardiomyocyte rate after compound treatment (experiment number 1: n = 4, experiment number 2: n = 3, mean value (standard deviation); experiment number 3: n = 2, experiment numbers 4 and 5: n = 1) (END: endoderm lineage cells, SMC: smooth muscle-like cells, EC: endothelial-like cells). The vertical axis indicates the experiment number, and the horizontal axis indicates the non-cardiomyocyte rate. [Figure 19] 19 shows the average value (standard deviation) of cardiomyocyte cell ratio after compound treatment (n=4, n=3 for experiment number 2). The vertical axis indicates the experiment number, and the horizontal axis indicates the cardiomyocyte cell ratio. [Figure 20] 20 shows the mean (standard deviation) (n=4) of the non-cardiomyocyte rate after compound treatment (END: endodermal lineage cells, SMC: smooth muscle-like cells, EC: endothelial-like cells). The vertical axis indicates the experiment number, and the horizontal axis indicates the non-cardiomyocyte rate. [Figure 21] 21 shows the average value (standard deviation) (n=4) of the relative ratio of the number of recovered cells after compound treatment, when the number of recovered cells without compound treatment was set to 1. The vertical axis indicates the experiment number, and the horizontal axis indicates the relative ratio of the number of recovered 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"). The production method of the present invention comprises: (1) a step of contacting a cell population containing cardiomyocytes or cardiac progenitor cells and other cells with a receptor tyrosine kinase inhibitor; and (2) a step of culturing the cell population. In the production method of the present invention, the "receptor tyrosine kinase inhibitor" in (1) above excludes EGF receptor inhibitors.

[0011] As used herein, "cardiomyocytes" refer to cells positive for at least one of sarcomeric α-actinin, cardiac troponin T (cTnT), and troponin I type 1 (TNNI1), preferably sarcomeric α-actinin-positive cells. Typically, these cells are cardiac muscle cells with self-pulsating activity. "Myocardial progenitor cells" refer to precursor cells of the aforementioned cardiomyocytes that are positive for at least one of Nkx2.5, GATA4, MEF2C, and MESP1. The above "other cells" refer to cells that do not fall into either the cardiomyocyte or myocardial progenitor cell category (hereinafter also referred to as "non-cardiomyocytes"). Specific examples of such cells include smooth muscle cells, endothelial cells, and stem cells (e.g., pluripotent stem 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 or cardiac progenitor cells and non-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 or cardiac progenitor cells 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)," including 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. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318:1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451:141-146), and induced pluripotent stem cells created by Sakurada et al. (JP Patent Publication No. 2008-307007) can also be used. 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., Schooler HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No. 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 ES cell lines were subsequently 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. 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). As for "ES cells," various mouse ES cell lines established by inGenious targeting laboratory, RIKEN (Riken Institute), and others 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 others 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 produced by nuclear transfer technology and have almost the same properties as fertilized egg-derived ES cells (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 obtained by replacing the nucleus of an unfertilized egg with that of a somatic cell. To generate nt ES cells, a combination of nuclear transfer technology (Cibelli JB et al. (1998), Nature Biotechnol., 16:642-646) and ES cell generation technology (mentioned above) is used (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 for several hours.

[0020] mGS cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to embryonic stem 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 embryonic stem 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 embryonic primordial germ cells (PGCs) and have similar pluripotency to ES cells. They can be established by culturing PGCs in the presence of LIF, bFGF, and stem cell factor (Matsui Y. et al. (1992), Cell, 70:841-847; JL Reisnick 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] Step (0) is not particularly limited as long as it can induce the differentiation of pluripotent stem cells into cardiomyocytes or cardiac progenitor cells. For example, pluripotent stem cells can be induced to differentiate into cardiomyocytes or cardiac progenitor cells by culturing them in a cardiomyocyte differentiation medium. In one embodiment of the present invention, 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 or cardiac progenitor cells. As used herein, "cardiomyocyte differentiation medium" refers to a medium containing a factor that promotes differentiation into cardiomyocytes, such as cytokines (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 differentiation induction process from pluripotent stem cells into cardiomyocytes or cardiac progenitor cells.

[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 stromal 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 form embryoid bodies (EBs) and then culture them. 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 the 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 group are dissociated (separated) into individual cells. Methods for dissociating pluripotent stem cells include, for example, mechanical dissociation, and the use of a dissociation solution having protease activity and collagenase activity (e.g., Accutase TM and Accumax TM Dissociation methods using a dissociation solution having only collagenase activity are preferred. Dissociation solutions having both protease activity and collagenase activity (particularly preferred are Accumax TMThe 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 ng / ml, 19ng / ml, 20ng / ml, 30ng / ml, 40ng / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml, 500ng / ml, 600ng / ml, 700ng / ml, 800ng / ml, 900ng / ml and 1µg / ml.

[0035] When bFGF is used, its concentration in the 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 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, "BMP inhibitors" include 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 (PBYu et al. (2007), Circulation, 116:II_60; PBYu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904), and LDN-193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). Among these, Dorsomorphin is preferred. The BMP inhibitor and the TGFβ inhibitor may be used alone or in combination of two or more types.

[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 or cardiac progenitor cells are obtained, but may be one day or more (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more). Since long-term culturing does not affect the establishment of cardiomyocytes or cardiac progenitor cells, no upper limit is particularly set, but the duration is typically 40 days or less. Whether or not cardiomyocytes or cardiac progenitor cells have been obtained may be monitored, and in this case, confirmation can be obtained by the number of beating cardiomyocytes, the expression of cardiomyocyte or cardiac progenitor cell markers, the expression of ion channels, the response to electrophysiological stimuli, etc.

[0046] Step (0) may further comprise step (0-3) of culturing the cardiomyocytes or cardiac progenitor cells 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. 5 ng / ml is more preferred.

[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 culture does not affect the establishment of cardiomyocytes or cardiac progenitor cells, 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 or cardiac progenitor cells 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., Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. 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., A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability. PLoSone. 6(4), e18293(2011)). Alternatively, a method of inducing cardiomyocyte differentiation in adherent culture without using cytokines (Lian X, et al., Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling., Proc Natl Acad Sci USA., 2012 July 3;109(27):E1848-57), or a method of inducing cardiomyocyte differentiation in a combination of adherent culture and suspension culture without using cytokines (Minami I, et al., A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions., Cell Rep., 2012 Nov 29;2(5):1448-60), may be used.

[0052] By contacting a cell population containing cardiomyocytes or cardiac progenitor cells obtained as described above with a receptor tyrosine kinase 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 receptor tyrosine kinase inhibitor. That is, cardiomyocytes can be purified using a receptor tyrosine kinase 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 or cardiac progenitor cells and non-cardiomyocytes, obtained by culturing pluripotent stem cells in a cardiomyocyte differentiation medium, with a receptor tyrosine kinase inhibitor; and (2') culturing the cell population.

[0053] As used herein, purifying cardiomyocytes means that the rate of decrease in the number of non-cardiomyocytes ("cell number" means the number of viable cells; the same applies hereinafter) by a receptor tyrosine kinase inhibitor exceeds the rate of decrease in cardiomyocytes, or that the rate of proliferation of cardiomyocytes exceeds the rate of proliferation by inhibiting the proliferation of non-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, this is distinct from an increase in the proportion of cardiomyocytes due to promotion of differentiation of cardiac progenitor cells into cardiomyocytes or inhibition of differentiation of cardiac progenitor cells into cells other than cardiac myocytes. Without wishing to be bound by any theory, it is speculated that the decrease in cell number due to a receptor tyrosine kinase inhibitor is the result of apoptosis induced in cells by the receptor tyrosine kinase inhibitor.

[0054] In steps (1) and (1'), the period for which the cell population containing cardiomyocytes or cardiac progenitor cells is contacted with the receptor tyrosine kinase 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. The contact of the cell population containing cardiomyocytes or cardiac progenitor cells with the receptor tyrosine kinase inhibitor may be carried out by adding the receptor tyrosine kinase inhibitor to a medium containing the cell population, or by seeding the cell population in a medium to which the receptor tyrosine kinase inhibitor has been added in advance. The timing of contacting the receptor tyrosine kinase inhibitor is not particularly limited as long as the cell population contains cardiomyocytes or cardiac progenitor cells, but for example, it is preferable to contact the cells in step (0-2) or (0-3) above, and based on the day when differentiation induction of pluripotent stem cells is initiated, it is preferable to contact the cells on or after the fourth day (e.g., the fifth, sixth, seventh day or later) from the start of differentiation induction.

[0055] Receptor tyrosine kinases that are inhibited by the receptor tyrosine kinase inhibitors used in the present invention include EGF receptors (also called ErbB or HER) (e.g., ErbB1 (EGFR), ErbB2 (HER2), ErbB3 (HER3), ErbB4 (HER4)), insulin receptors (e.g., IR-A, IR-B), insulin-like growth factor 1 receptors, VEGF receptors (e.g., VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), VEGFR-3 (Flt-4)), PDGF receptors (e.g., PDGFRα, PDGFRβ), HGF receptors (also called c-Met), FGF receptors (e.g., FGF R1, FGFR2, FGFR3, FGFR4), CCK, NGF receptor (also called Trk receptor) (e.g., TrkA, TrkB, TrkC), Eph (Ephrin) receptor (e.g., EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6), AXL (also called TAM receptor), TIE (e.g., TIE-1, TIE-2), RYK, DDR (e.g., DDR1), RET, ROS, LTK, ROR (e.g., ROR1, ROR2), MuSK, and LMR. Among these, VEGF receptor, PDGF receptor, HGF receptor, and FGF receptor are preferred targets of receptor tyrosine kinase inhibitors. Furthermore, although EGF receptor inhibitors are excluded from the receptor tyrosine kinase inhibitors used in the step (1), EGF receptor inhibitors can be used as the receptor tyrosine kinase inhibitors used in the step (1').

[0056] As used herein, a receptor tyrosine kinase inhibitor may have other activities, such as inhibitory activity against other receptor tyrosine kinases, as long as it has inhibitory activity against at least one of the above receptor tyrosine kinases, or may have inhibitory activity specific to one type of receptor tyrosine kinase. Furthermore, receptor tyrosine kinase inhibitors other than EGF receptor inhibitors do not exclude those that have inhibitory activity against receptor tyrosine kinases other than the EGF receptor and also have inhibitory activity against the EGF receptor. In this specification, the term "EGF receptor inhibitor" refers to a substance whose EGF receptor inhibitory activity is superior to other activities, and specifically refers to a substance that inhibits the EGF receptor by at least 90% or more, or a substance whose 50% inhibitory concentration for the EGF receptor is 10 μM or less.

[0057] Cetuximab, Erlotinib, and EGF-induced anti-inflammatory drugs HCl(OSI-744)、Gefitinib(ZD1839)、Lapatinib(GW-572016)Ditosylate、Afatinib(BIBW2992)、Canertinib( CI-1033, TAS6417, PD153035, MTX-211, HS-10296, Theliatinib (HMPL-309), Lapatinib, AG-490 (Tyrphostin). B42, CP-724714, Dacomitinib (PF-00299804), WZ4002, Sapitinib (AZD8931), CUDC-101, AG-1478 (Tyrphostin AG-1478), PD153035 HCl, Pelitinib(EKB-569), AC480(BMS-599626), AEE788(NVP-AEE788), AP26113-analogue(ALK-IN-1), OSI-420, WZ3146, HER2-Inhibitor-2. 1, WZ8040, AST-1306, Rociletinib(CO-1686), Genistein, Varlitinib, Icotinib, TAK-285, WHI-P154, Daphnetin, PD168393, Tyrphostin 9. CNX-2006, AG-18, AZ5104, Lazertinib, AZD9291, CL-387785(EKI-785), Olmutinib (BI 1482694), (-)-Epigallocatechin Gallate, Erlotinib, Gefitinib hydrochloride、Afatinib(BIBW2992)Dimaleate、AZD3759、Poziotinib(HM781-36B)、Brigatinib(AP26113)、Osimertinib mesylate、Naquotinib(ASP8273)Chrysophanic Acid, nazartinib (EGF816), norcantharidin, lifirafenib (BGB-283), lidocaine hydrochloride, butene, EAI045, cyasterone, and avitinib (AC0010).

[0058] Examples of inhibitors of insulin receptor or insulin-like growth factor 1 receptor include HNMPA, GSK1904529A, Dovitinib (TKI-258) Dilactic Acid, Dovitinib (TKI258) Lactate, NVP-AEW541, Dovitinib (TKI-258), NVP-ADW742, Linsitinib (OSI-906), GSK1904529A, BMS-754807, TAE226 (NVP-TAE226), and Ceritinib (LDK378).

[0059] Inhibitors of PDGFRα include N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide (compound of formula (I))

[0060] [ka]

[0061] , Ponatinib (AP24534), Telatinib, Amuvatinib (MP-470), Ki8751, KRN 633, Crenolanib (CP-868596), Axitinib, CP-673451, Tivozanib (AV-951), Nintedanib (BIBF 1120), Dovitinib (TKI-258, CHIR-258), Dovitinib (TKI258) Lactate, Dovitinib (TKI-258) Dilactic Acid, Masitinib (AB1010), etc. PDGFRβ inhibitors include N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide, CP-673451, sunitinib malate, sunitinib, TSU-68 (SU6668, orantinib), MK-2461, sorafenib tosylate, linifanib (ABT-869), axitinib, crenolanib (CP-868596), dovitinib (TKI-258) dilactic acid, dovitinib (TKI-258, CHIR-258), dovitinib (TKI258) lactate, tivozanib (AV-951), and nintedanib (BIBF). 1120), Masitinib (AB1010), KRN 633, etc.

[0062] VEGFR-1 inhibitors include N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide, ZM 306416, axitinib, motesanib diphosphate (AMG-706), linifanib (ABT-869), MGCD-265, cediranib (AZD2171), foretinib (GSK1363089), OSI-930, dovitinib (TKI-258, CHIR-258), pazopanib HCl (GW786034 HCl), and dilactic acid bacteria. Acid, Dovitinib (TKI258) Lactate, Cabozantinib (XL184, BMS-907351), Cabozantinib malate (XL184), Regorafenib (BAY 73-4506), Lenvatinib (E7080), Tivozanib (AV-951), Nintedanib (BIBF 1120), AEE788 (NVP-AEE788), Vatalanib (PTK787) 2HCl, KRN 633, Brivanib (BMS-540215), Brivanib Alaninate (BMS-582664), etc. Examples of inhibitors for VEGFR-2 include N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide, N-{4-[(6,7-dimethoxyquinolin-4-yl)oxy]-3-fluorophenyl}-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound of formula (II))

[0063] [ka]

[0064] , Cabozantinib (XL184, BMS-907351), Cabozantinib malate (XL184), Ki8751, Apatinib, Ponatinib (AP24534), ZM323881, LY2874455, Sorafenib Tosylate, BMS-794833, Golvatinib (E7050), RAF265 (CHIR-265), SKLB1002, Vandetanib (ZD6474), CYC116, Sunitinib Malate, Sunitinib, PD173074, Semaxanib (SU5416), TSU-68 (SU6668, Orantinib), Axitinib, Cediranib (AZD2171), Foretinib (GSK1363089), MGCD-265, Motesanib Diphosphate (AMG-706), Linifanib (ABT-869), Lenvatinib (E7080), Regorafenib (BAY 73-4506), Telatinib, Tivozanib (AV-951), OSI-930, Dovitinib (TKI-258) Dilactic Acid, Nintedanib (BIBF 1120), Dovitinib (TKI258) Lactate, Dovitinib (TKI-258, CHIR-258), Brivanib Alaninate (BMS-582664), Brivanib (BMS-540215), Pazopanib, Pazopanib HCl (GW786034 HCl), Vatalanib (PTK787) 2HCl, ENMD-2076 L-(+)-Tartaric acid, ENMD-2076 L-(+)-Tartaric acid, AEE788 (NVP-AEE788), KRN 633, etc.VEGFR-3 inhibitors include N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide, SAR131675, axitinib, foretinib (GSK1363089), cediranib (AZD2171), telatinib, MGCD-265, lenvatinib (E7080), cabozantinib (XL184, BMS-907351), cabozantinib malate (XL184), motesanib diphosphate (AMG-706), dovitinib (TKI-258, CHIR-258), and dovitinib (TKI-258) dilactic acid. Acid, Dovitinib(TKI258)Lactate, Nintedanib(BIBF 1120), Tivozanib(AV-951), ENMD-2076 L-(+)-Tartaric acid, ENMD-2076, Regorafenib(BAY 73-4506), Pazopanib HCl(GW786034 HCl), Pazopanib, KRN 633, Linifanib (ABT-869), AEE788 (NVP-AEE788), Vatalanib (PTK787)2HCl, etc.

[0065] Examples of inhibitors of HGF receptors include N-{4-[(6,7-dimethoxyquinolin-4-yl)oxy]-3-fluorophenyl}-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide, crizotinib (PF-02341066), cabozantinib (BMS-907351), foretinib (GSK1363089), PHA-665752, SU11274, JNJ-38877618 (OMO-1), gluteninib (SCC244), altiratinib, SAR125844, SGX-523, BMS-777607, tivantinib (ARQ 197), JNJ-38877605, PF-04217903, and MGCD-265. Analogs include capmatinib (INCB28060), BMS-754807, BMS-794833, AMG-208, MK-2461, golvatinib (E7050), AMG-458, NVP-BVU972, AMG 337, merestinib (LY2801653), S49076, pulsatilla saponin D, norcantharidin, NPS-1034, and savolitinib (AZD6094).

[0066] Inhibitors for FGFR1 include, for example, ASP5878, Ponatinib (AP24534), PD173074, Danusertib (PHA-739358), Brivanib Alaninate (BMS-582664), Brivanib (BMS-540215), TSU-68 (SU6668, Orantinib), SSR128129E, AZD4547, BGJ398 (NVP-BGJ398), LY2874455, Dovitinib (TKI-258, CHIR-258), Dovitinib (TKI258) Lactate, Dovitinib (TKI-258) Dilactic Acid, CH5183284 (Debio-1347), MK-2461, and Nintedanib (BIBF 1120). Examples of inhibitors for FGFR2 include ASP5878, BGJ398 (NVP-BGJ398), AZD4547, LY2874455, CH5183284 (Debio-1347), Nintedanib (BIBF 1120), MK-2461, etc. Examples of inhibitors for FGFR3 include ASP5878, BGJ398 (NVP-BGJ398), AZD4547, LY2874455, Dovitinib (TKI-258) Dilactic Acid, Dovitinib (TKI258) Lactate, Dovitinib (TKI-258, CHIR-258), CH5183284 (Debio-1347), MK-2461, Nintedanib (BIBF 1120), etc. Examples of inhibitors against FGFR4 include ASP5878, LY2874455, AZD4547, CH5183284 (Debio-1347), and Nintedanib (BIBF 1120).

[0067] Examples of inhibitors against the NGF receptor include BMS-754807, GW441756, DS-6051b, GNF-5837, CH7057288, Altiratinib, Selitrectinib (LOXO-195), BMS-935177, Entrectinib (RXDX-101), Sitravatinib (MGCD516), PF-06273340, Belizatinib (TSR-011), and Larotrectinib (LOXO-101) sulfate.

[0068] Examples of inhibitors against Eph receptors include NVP-BHG712 and Sitravatinib (MGCD516).

[0069] Examples of inhibitors of AXL include BMS-777607, Bemcentinib (R428), Cabozantinib malate (XL184), UNC2250, Dubermatinib (TP-0903), UNC-2025, LDC1267, UNC2881, RXDX-106 (CEP-40783), S49076, Sitravatinib (MGCD516), 2-D08, Gilteritinib (ASP2215), and NPS-1034.

[0070] Examples of TIE inhibitors include MGCD-265 analog, Tie2 kinase inhibitor, altiratinib, and pexmetinib (ARRY-614).

[0071] Other receptor tyrosine kinase inhibitors can also be selected appropriately. Preferred receptor tyrosine kinase inhibitors include N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide, N-{4-[(6,7-dimethoxyquinolin-4-yl)oxy]-3-fluorophenyl}-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide, AMG337, ASP5878, BGJ398, foretinib, ZM323881, CP-673451, crenolanib, and crizotinib. Only one type of receptor tyrosine kinase inhibitor can be used, or multiple types can be used in combination. In addition, in order to obtain cardiomyocytes of higher purity, other drugs (such as histone deacetylase inhibitors) may be used simultaneously with the receptor tyrosine kinase inhibitor or in a step before or after the receptor tyrosine kinase inhibitor.

[0072] The inhibitor may contain one or more of the above compounds or salts thereof. The above compounds or salts thereof can be produced according to methods known per se.

[0073] When the compound is in the form of a salt, a pharmacologically acceptable salt is preferred, and examples of such salts include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Suitable examples of salts with inorganic bases include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts; ammonium salts; and the like. Suitable examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, and the like. Suitable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Suitable examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Suitable examples of salts with basic amino acids include salts with arginine, lysine, ornithine, etc. Suitable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, and the like.

[0074] The above compound may be any of a hydrate, a non-hydrate, a solvate, and a non-solvate. In addition, the above compounds may contain isotopes (e.g., 2 H, 3 H, 11 C. 14 C. 18 F, 35 S, 125 The compound may be a compound labeled or substituted with, for example, I. 1 H 2 Deuterium-converted compounds converted to H(D) are also included in the above compounds. Tautomers are also encompassed within the scope of the compounds. The compound may be a pharmaceutically acceptable cocrystal or cocrystal salt. Here, a cocrystal or cocrystal salt refers to a crystalline substance composed of two or more distinct solids at room temperature, each of which has different physical properties (e.g., structure, melting point, heat of fusion, hygroscopicity, solubility, stability, etc.). A cocrystal or cocrystal salt can be prepared by a known cocrystallization method.

[0075] The concentration of the receptor tyrosine kinase inhibitor in the medium can be appropriately selected by those skilled in the art, but for example, 1 nM to 10 μM is preferable, and 10 nM to 3 μM is more preferable, and specific examples include 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 N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide is used, the concentration is preferably 20 nM to 10 μM. When N-{4-[(6,7-dimethoxyquinolin-4-yl)oxy]-3-fluorophenyl}-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide is used, the concentration is preferably 0.2 μM to 10 μM. When AMG337 is used, the concentration is preferably 0.2 μM to 10 μM. When using ASP5878, the concentration is preferably 0.2 μM to 10 μM, when using ASP5878, the concentration is preferably 1 nM to 1 μM, when using BGJ398, the concentration is preferably 5 nM to 5 μM, when using Foretinib, the concentration is preferably 5 nM to 5 μM, when using ZM323881, the concentration is preferably 0.1 μM to 10 μM, when using CP-673451, the concentration is preferably 0.1 μM to 10 μM, when using Crenolanib, the concentration is preferably 0.1 μM to 10 μM, and when using Crizotinib, the concentration is preferably 0.1 μM to 10 μM, but is not limited to these concentrations.

[0076] The method for culturing the cell population in steps (2) and (2') is the same as in (0-2) or (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 receptor tyrosine kinase inhibitor.

[0077] 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 or purification method 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 cardiomyocytes at a higher percentage than cell populations obtained by conventional methods of inducing cardiomyocytes from pluripotent stem cells. Such cell populations may be further purified by cell sorting or other methods, and cell populations purified in this manner are also encompassed by the "cell population of the present invention."

[0078] 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.

[0079] 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.

[0080] 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, a buffer (e.g., phosphate buffer, sodium acetate buffer), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, etc. When the transplantation therapy of the present invention is formulated as an aqueous suspension, approximately 1 × 10 cells / ml of the cell population are added to the aqueous solution. 6 ~Approx. 1×10 8 The cell population containing cardiomyocytes can be suspended to a concentration of 100 cells / mL. The cell population can also be administered together with a scaffold that promotes engraftment. Examples of scaffolds include, but are not limited to, bioderived components such as collagen and synthetic polymers such as polylactic acid.

[0081] 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.

[0082] 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.

[0083] 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]

[0084] Example 1. Single-cell RNA sequencing analysis of cells induced to differentiate from iPS cells into cardiomyocytes We used clinical iPS cell lines generated at the Center for iPS Cell Research and Application (CiRA) at Kyoto University. iPS cell lines were maintained and cultured according to conventional methods (Okita K, et al. Stem Cells. 2012 Nov 29. doi:10.1002 / stem.1293). Cardiomyocyte differentiation was performed according to the method described in a paper (Miki et al. Cell Stem Cell. 2015 Jun 4;16. doi:10.1016 / j.stem.2015.04.005). Cardiomyocytes were analyzed 22 days after the start of differentiation induction using single-cell RNA sequencing with Chromium from 10X Genomics. Cell clustering and analysis of each cluster were performed using software (Cellranger, Seurat, Loupe Cell Browser). In the cell population induced to differentiate from iPS cells into cardiomyocytes, we found that in addition to the cardiomyocyte population expressing sarcomeric-α-actinin and cardiac troponin T, there were also three non-cardiomyocyte populations that did not express sarcomeric-α-actinin or cardiac troponin T (based on gene expression, they were thought to be smooth muscle-like cells (SMCs), endothelial-like cells (ECs), and cells of the endodermal lineage (ENDs)) (Figure 1).

[0085] Example 2: Analysis of non-cardiomyocytes present in a cell population induced to differentiate into cardiac muscle from iPS cells We analyzed the gene expression of receptor tyrosine kinases (PDGFRA, PDGFRB, VEGFR1, VEGFR2, c-Met (HGFR), and FGFR4 using single-cell RNA sequencing data with Loupe Cell Browser. We found that gene expression of receptor tyrosine kinases (RTKs), such as PDGFRA, PDGFRB, VEGFR1, VEGFR2, c-Met (HGFR), and FGFR4, was elevated in the non-cardiomyocyte population but not in the cardiac myocyte population (Figure 2).

[0086] Example 3. Staining of non-cardiomyocytes with cell surface markers We generated a double knock-in human iPS cell line by inserting the reporter protein sequences EmGFP (SEQ ID NO: 1) into the TNNI1 locus and mCherry (SEQ ID NO: 2) into the TNNI3 locus. The human iPS cell line was generated using PBMCs (LP_167, Sample ID: 20130318) purchased from CTL, Inc., and an episomal vector (carrying 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).

[0087] iPS cell lines were maintained in culture according to conventional methods (Okita K, et al. Stem Cells. 2012 Nov 29. doi:10.1002 / stem.1293). Cardiomyocyte differentiation was performed in a 6-well plate according to the method described in a paper (Miki et al., Cell Stem Cell. 2015 Jun 4;16. doi:10.1016 / j.stem.2015.04.005.). Briefly, to induce cardiomyocyte differentiation, reporter iPS cell lines were treated with TrypLE select (Life Technologies) diluted 1 / 2 with 0.5 mM EDTA / PBS for 4-5 minutes, after which 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 resulting cells were cultured at 2 x 10 per well of a 6-well plate. 6 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 M, 10 μM Y-27632, 2 ng / mL BMP4 (R&D), and 0.5% Growth Factor Reduced Matrigel were added to the medium (1.5 mL / well) and cultured at 37°C under 5% oxygen conditions to form embryoid bodies (day 0). The next day (day 1), the cells were cultured in StemPro34 medium 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 and left 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 and left to settle, and the embryoid bodies were cultured 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 at 37°C under 5% oxygen for 8 days. During this period, the medium was replaced with the same medium every 2-3 days.

[0088] On day 15 of differentiation induction, the cell culture medium containing the embryoid bodies was centrifuged at 200 x g for 1 minute, and the supernatant was removed using a aspirator. PBS was added, and the tubes were centrifuged at 200 x g for 1 minute. The supernatant was then removed using a aspirator. 3 mL of a solution containing 10 μg / mL DNase and 100 μg / mL Liberase in Iscove's Modified Dulbecco's Media (IMDM) was added to each tube and incubated at 37°C under normal oxygen conditions for 1 hour. After 1 hour, the tubes were centrifuged at 400 x g for 5 minutes, and the supernatant was removed without aspirating the embryoid bodies. 2 mL of a solution containing 10 μg / mL DNase in Accutase (Thermo) was added to each tube and incubated at 37°C under normal oxygen conditions for 10 minutes. After standing, the cells were separated into single cells by pipetting, and 2 mL of a medium prepared by adding 10 μg / mL of DNase to IMDM was added to each tube and mixed by inversion to prepare a single cell suspension. The single cell suspension was centrifuged at 200xg for 5 minutes, the supernatant was removed, and the cells were frozen and stored at -80°C. The frozen stored cells were thawed in a 37°C hot bath and centrifuged at 400 xg for 5 minutes, after which the supernatant was removed. After tapping the cell pellet, 1 mL of 1% BSA in PBS was added and the mixture was centrifuged at 300 x g for 3 minutes. The supernatant was removed. Cells were then stained with APCFire750-conjugated anti-CD326 antibody, PE-conjugated anti-CD49a antibody, BV605-conjugated anti-CD31 antibody, and DAPI in 1% BSA PBS. After removing DAPI-positive dead cells, the signal intensity of each fluorescent dye in nuclear cells was measured to separate the cell populations and determine their proportions. The cardiomyocytes induced to differentiate from the reporter iPS cells were separated into four main cell populations based on the expression of these three surface markers: CD326-positive cells, CD326-negative CD31-positive cells, CD326-negative CD31-negative CD49a-positive cells, and CD326-negative CD31-negative CD49a-negative cells. The four cell populations were comprised of a population primarily composed of non-cardiomyocytes expressing one or more of these three surface markers (a population containing many cells that do not express the cardiac reporter gene), and a cardiac cell population negative for all three markers (a cell population expressing the cardiac reporter gene). More than 99% of the cell population negative for all three markers was cardiomyocyte (cells expressing the cardiac reporter gene) (Figure 3).

[0089] Next, focusing on the receptor tyrosine kinase discovered in Example 2, we examined under various conditions whether or not a receptor tyrosine kinase inhibitor can purify cardiomyocytes with relatively low expression of receptor tyrosine kinase (Test Examples 1 to 9). In the following Test Examples 1 to 9, Compound A represents N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide, and Compound B represents N-{4-[(6,7-dimethoxyquinolin-4-yl)oxy]-3-fluorophenyl}-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide.

[0090] Test Example 1: Verification of the cardiomyocyte purification effect of receptor tyrosine kinase inhibitors <Induction of differentiation into cardiomyocytes> The human iPS cell line used was a cell line (parental line of reporter cells used in staining non-cardiomyocytes with cell surface markers) generated using an episomal vector (carrying genes: OCT3 / 4, KLF4, SOX2, L-MYC, LIN28, mouse p53DD) using PBMCs (LP_167, Sample ID: 20130318) purchased from CTL (Reference: Okita K, et al. Stem Cells. 2012 Nov 29. doi:10.1002 / stem.1293). iPS cell lines were maintained in culture according to conventional methods (Okita K, et al. Stem Cells. 2012 Nov 29. doi:10.1002 / stem.1293). Differentiation into cardiomyocytes was performed in 6-well plates according to the method described in a paper (Miki et al. Cell Stem Cell. 2015 Jun 4;16. doi:10.1016 / j.stem.2015.04.005). VEGF was not added after day 8. On day 8 of differentiation induction, the embryoid bodies were collected into a single centrifuge tube, left at room temperature for several minutes to settle, the supernatant was removed with an aspirator, and medium was added for medium exchange. The embryoid body suspension was transferred to a 6-well plate, and 1 / 100th of the final concentration of the evaluation compound (compounds and concentrations for Experiments 2 to 8 shown in Table 1 below) was added to each well. The plate was then agitated and cell culture was carried out.

[0091] [Table 1]

[0092] On day 10 of differentiation induction, the 6-well plate was tilted and left to stand for several minutes to allow the embryoid bodies to settle. The supernatant was removed using an aspirator, and medium was added to each well to change the medium. Compounds were then added as on day 8 by adding 1 / 100th the final concentration of the test compound. On day 13 of differentiation induction, the 6-well plate was tilted to allow the embryoid bodies to settle, and the embryoid bodies were transferred to a 1.5 mL tube using a pipette equipped with a wide-bore 1 mL pipette tip. Cells were dissociated into single cells in the same manner as for staining non-cardiomyocytes with cell surface markers, and the single cell suspension was used to measure the percentage of cardiomyocyte marker-positive cells and non-cardiomyocyte marker-positive cells.

[0093] <Measurement of cardiac marker positive cell rate (cardiomyocyte rate)> The single-cell suspension was dispensed into 1.5 mL tubes and centrifuged at 400 g for 3 minutes, followed by removal of the supernatant. The cell pellets were resuspended in Cytofix / Cytoperm Fixation / Permeabilization Solution (BD) and fixed at room temperature for 15 minutes. 1 mL of 1x Perm / Wash Buffer was added to each tube, and the tubes were centrifuged at 1500 g for 3 minutes. The supernatant was then removed to wash the cells. After tapping, the cell pellets were washed again by adding 1 mL of 1x Perm / Wash Buffer and repeating the same procedure. After tapping, the cell pellets were stained with anti-sarcomeric α-actinin antibody in 1x Perm / Wash Buffer containing 1% BSA, followed by fluorescent dye Alexa647 staining, followed by DNA staining with DAPI (4',6-Diamidino-2-phenylindole Dihydrochloride). The percentage of sarcomeric α-actinin-positive cells was analyzed by measuring the Alexa647 fluorescence signal in the cell population excluding dead cells (cells with DNA content in sub-G1) using a flow cytometer. Treatment with compound A (N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide), AMG337, ASP5878, and BGJ398 increased the percentage of cardiomyocytes (actinin-positive cells) compared to untreated cells (Figure 4).

[0094] <Measurement of non-cardiomyocyte marker positive cell rate (non-cardiomyocyte rate)> Unfrozen single-cell suspensions were stained, measured, and analyzed using cell surface markers similar to those used for staining non-cardiomyocytes. As a result, treatment with Compound A, AMG337, ASP5878, and BGJ398 reduced the rate of non-cardiomyocytes (the rate of cells positive for either CD326, CD31, or CD49a) compared to untreated control (Figure 5).

[0095] Test Example 2: Verification of the cardiomyocyte purification effect of receptor tyrosine kinase inhibitors <Induction of differentiation into cardiomyocytes> The same cell line as in Test Example 1 was used for human iPS cells, and differentiation into cardiomyocytes was induced in the same manner. Medium changes were performed on days 8, 10, and 13 of differentiation induction, with the same method as in Test Example 1 on days 8 and 10 of differentiation induction and the same method as in Test Example 1 on day 13 of differentiation induction. On days 8, 10, and 13 of differentiation induction, the compounds to be evaluated (compounds and concentrations of Experiment Nos. 2 and 3 shown in Table 2 below) were added in the same manner as in Test Example 1. However, ASP5878 in Experiment No. 3 was added only on days 8 and 10 of differentiation induction. Each experiment was performed in four wells.

[0096] [Table 2]

[0097] On day 16 of differentiation induction, the cells were dissociated into single cells, fixed, and the cardiomyocyte rate (sarcomeric α-actinin positive cell rate) was measured in the same manner as in Test Example 1. As a result, treatment with foretinib and ASP5878 increased the cardiomyocyte rate compared to untreated cells (Figure 6).

[0098] Test Example 3: Verification of the cardiomyocyte purification effect of receptor tyrosine kinase inhibitors <Induction of differentiation into cardiomyocytes> The human iPS cell line used was generated using PBMCs (LP_140, Sample ID: 20120808) purchased from CTL, Inc., and an episomal vector (carrying 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). Maintenance culture of iPS cell lines was performed according to conventional methods (Okita K, et al. Stem Cells. 2012 Nov 29. doi:10.1002 / stem.1293). Differentiation into cardiomyocytes was induced in the same manner as in Example 1. VEGF was not added from Day 10 onwards. The medium was changed on days 8, 10, and 13 after differentiation induction, in the same manner as in Test Example 1 on days 8 and 10, and on day 13 on day 10 of Test Example 1. On days 8, 10, and 13 after differentiation induction, the compounds to be evaluated (compounds and concentrations of Experiment Nos. 2 to 6 shown in Table 3 below) were added in the same manner as in Test Example 1.

[0099] [Table 3]

[0100] On day 17 of differentiation induction, the cells were dissociated into single cells, and the non-cardiomyocyte rate (the rate of cells positive for any of CD326, CD31, and CD49a) was measured in the same manner as in Test Example 1. As a result, treatment with Compound A, Foretinib, ZM323881, Crenolanib, and Crizotinib reduced the non-cardiomyocyte rate compared to untreated cells (Figure 7).

[0101] Test Example 4: Verification of the cardiomyocyte purification effect of receptor tyrosine kinase inhibitors <Induction of differentiation into cardiomyocytes> The same cell line as in Test Example 3 was used as the human iPS cells, and differentiation into cardiomyocytes was also induced in the same manner. The medium was changed on days 8, 10, and 13 after differentiation induction, using the same method as in Test Example 1 on days 8 and 10, and on day 13 on day 10 of Test Example 1. On days 8, 10, and 13 after differentiation induction, the compounds to be evaluated (compounds and concentrations of Experiment Nos. 2 and 3 shown in Table 4 below) were added using the same method as in Test Example 1. Each experiment was performed in four wells.

[0102] [Table 4]

[0103] On day 17 of differentiation induction, the cells were dissociated into single cells, and the cell count and cardiomyocyte ratio were measured. Fixation and measurement of cardiomyocyte ratio (sarcomeric α-actinin positive cell ratio) were performed in the same manner as in Test Example 1. As a result, treatment with Compound A and Foretinib increased the cardiomyocyte ratio compared to untreated cells (Figure 8).

[0104] The single cell suspension was added to a 1.5 mL tube in IMDM medium to make a 5-fold dilution, and the mixture was mixed by inversion. The cell count was measured using a cell counter NC-200 (Chemometec). The results showed that the number of cells recovered in the Foretinib treatment was approximately 70% of the untreated control, and that the number of cells recovered in the Compound A treatment was almost the same (Figure 9).

[0105] Test Example 5. Verification of the cardiomyocyte purification effect of receptor tyrosine kinase inhibitors <Induction of differentiation into cardiomyocytes> The same cell line as in Test Example 3 was used as the human iPS cells, and differentiation into cardiomyocytes was also induced in the same manner. The medium was changed on days 8, 10, and 13 of differentiation induction, with the medium being changed on days 8 and 10 in the same manner as in Test Example 1, and on day 13 in the same manner as in day 10 of Test Example 1. On days 8, 10, and 13 of differentiation induction, the compounds to be evaluated (compounds and concentrations of Experiment Nos. 2 to 4 shown in Table 5 below) were added in the same manner as in Test Example 1. However, ASP5878 in Experiment No. 4 was added only on days 8 and 10 of differentiation induction. Each experiment was performed in four wells.

[0106] [Table 5]

[0107] On day 16 of differentiation induction, the cells were dissociated into single cells, fixed, and the cardiomyocyte rate (sarcomeric α-actinin-positive cell rate) was determined using the same methods as in Test Example 1. As a result, compared to untreated cells, treatment with crenolanib, compound B (N-{4-[(6,7-dimethoxyquinolin-4-yl)oxy]-3-fluorophenyl}-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide), and ASP5878 increased the cardiomyocyte rate (sarcomeric α-actinin-positive cell rate) (Figure 10).

[0108] Using the single cell suspension, cell count was measured by the method described in Test Example 4. As a result, no significant difference was observed in the number of cells recovered with or without compound treatment (FIG. 11).

[0109] Test Example 6: Verification of the cardiomyocyte purification effect of receptor tyrosine kinase inhibitors <Induction of differentiation into cardiomyocytes> We used clinical iPS cell lines generated at CiRA. iPS cell lines were maintained and cultured according to conventional methods (Okita K, et al. Stem Cells. 2012 Nov 29. doi:10.1002 / stem.1293). Cardiomyocyte differentiation was performed according to the method described in a paper (Miki et al. Cell Stem Cell. 2015 Jun 4;16. doi:10.1016 / j.stem.2015.04.005). The medium was changed on days 8, 10, 14, 17, and 21 of differentiation induction. On days 8 and 10 of differentiation induction, the medium was changed in the same manner as in Test Example 1, and on days 14 and after differentiation induction, the medium was changed in the same manner as in Test Example 1 on day 10. On days 14, 17, and 21 of differentiation induction, Compound A, one of the compounds to be evaluated, was added in the same manner as in Test Example 1. Each experiment was carried out in four wells.

[0110] [Table 6]

[0111] Cell isolation into single cells, fixation, measurement of the cardiomyocyte rate, and measurement of the non-cardiomyocyte rate were performed in the same manner as in Test Example 1, and cell count was measured in the same manner as in Test Example 4. As a result, compared to untreated cells, treatment with Compound A increased the cardiomyocyte rate (Figure 12) and decreased the non-cardiomyocyte rate (Figure 13). There was no significant difference in the number of cells recovered with or without compound treatment (Figure 14).

[0112] Test Example 7: Verification of the cardiomyocyte purification effect of receptor tyrosine kinase inhibitors <Induction of differentiation into cardiomyocytes> The human iPS cells used were a clinical iPS cell line produced by CiRA. The same cell line as in Test Example 6 was used, and differentiation into cardiomyocytes was also induced in the same manner. The medium was changed on days 8, 10, and 13 after differentiation induction. On days 8 and 10, the medium was changed in the same manner as in Test Example 1, and on day 13, the medium was changed in the same manner as in Test Example 1. On days 8, 10, and 13 after differentiation induction, the compounds to be evaluated (compounds and concentrations of Experiment Nos. 2 to 9 shown in Table 7 below) were added in the same manner as in Test Example 1 (Experiment No. 1 was performed in two wells, and the others in one well).

[0113] [Table 7]

[0114] On day 17 after differentiation induction, the cells were dissociated into single cells, fixed, and the cardiomyocyte rate (sarcomeric α-actinin-positive cell rate) and non-cardiomyocyte rate were measured in the same manner as in Test Example 1. Compared to untreated cells, treatment with Compound A, Foretinib, ZM323881, CP-67351, and Crizotinib increased the cardiomyocyte rate (Figure 15) and decreased the non-cardiomyocyte rate (Figure 16).

[0115] Test Example 8: Verification of the cardiomyocyte purification effect of receptor tyrosine kinase inhibitors <Induction of differentiation into cardiomyocytes> The same cell line as in Test Example 6 was used as the human iPS cells, and differentiation into cardiomyocytes was also induced in the same manner. Medium changes were performed on days 8, 10, 13, 17, and 20 of differentiation induction; on day 8 of differentiation induction, the method was the same as that for day 8 of Test Example 1, and on days 10 and after, the method was the same as that for day 10 of Test Example 1. On days 8, 10, 13, 17, and 20 of differentiation induction, the evaluation compound crenolanib was added in the same manner as Test Example 1 under the conditions listed in Table 8. In Experiment No. 4, the compound was added from day 8 of differentiation induction onwards, in Experiment Nos. 2 and 3 from day 10 of differentiation induction onwards, and in Experiment No. 5 from day 13 of differentiation induction onwards. Experiment No. 1 was performed in 4 wells, Experiment Nos. 2 and 3 from 3 wells, and Experiment Nos. 4 and 5 from 1 well.

[0116] [Table 8]

[0117] On day 23 of differentiation induction, the cells were dissociated into single cells, fixed, and the cardiomyocyte rate (sarcomeric α-actinin-positive cell rate) and non-cardiomyocyte rate were measured in the same manner as in Test Example 1. Crenolanib treatment increased the cardiomyocyte rate (Figure 17) and decreased the non-cardiomyocyte rate (Figure 18).

[0118] Test Example 9. Verification of the cardiomyocyte purification effect of receptor tyrosine kinase inhibitors <Induction of differentiation into cardiomyocytes> The same cell line as in Test Example 6 was used as the human iPS cells, and differentiation into cardiomyocytes was also induced in the same manner. The medium was changed on day 8 of differentiation induction in the same manner as on day 8 of Test Example 1, and on days 10 and 13 of differentiation induction in the same manner as on day 10 of Test Example 1. On days 8, 10, and 13 of differentiation induction, the compounds to be evaluated (compounds and concentrations of Experiment Nos. 2 and 3 shown in Table 9 below) were added in the same manner as in Test Example 1. This was performed in 4 wells for each experiment number.

[0119] [Table 9]

[0120] On day 17 of differentiation induction, the cells were dissociated into single cells, fixed, and the cardiomyocyte rate (sarcomeric α-actinin positive cell rate) and non-cardiomyocyte rate were measured using the same methods as in Test Example 1, and the cell count was measured using the same method as in Test Example 4. Compared to untreated cells, treatment with Foretinib and Crizotinib increased the cardiomyocyte rate (Figure 19) and decreased the non-cardiomyocyte rate (Figure 20). Compared to untreated cells, treatment with Crizotinib recovered 80% of the number of cells recovered, and treatment with Foretinib recovered almost the same number of cells (Figure 21).

[0121] These results demonstrate that cardiomyocytes can be purified from embryoid bodies in various cell lines by a simple process involving the addition of various types of receptor tyrosine kinase inhibitors to the culture medium.

[0122] The receptor tyrosine kinases targeted by the compounds used in Test Examples 1 to 9 above are shown in Table 10.

[0123] [Table 10] [Industrial Applicability]

[0124] 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.

[0125] This application is based on patent application No. 2020-050268 filed in Japan (filing date: March 19, 2020), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing a cell population comprising cardiomyocytes, comprising: (1) contacting a cell population containing cardiomyocytes or cardiac progenitor cells and other cells obtained by culturing pluripotent stem cells in a medium for cardiomyocyte differentiation with a receptor tyrosine kinase inhibitor (excluding EGF receptor inhibitors); 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 receptor tyrosine kinase inhibitor in step (1) is carried out on or after day 4 from the start of the differentiation induction of the pluripotent stem cells.

3. The method according to claim 1 or 2, wherein the contact of the cell population with the receptor tyrosine kinase inhibitor in step (1) is carried out for one or more days.

4. The method according to any one of claims 1 to 3, wherein the inhibitor is an inhibitor of at least one receptor tyrosine kinase selected from the group consisting of a VEGF receptor, a PDGF receptor, an HGF receptor, and an FGF receptor.

5. The method according to any one of claims 1 to 4, wherein the inhibitor is at least one selected from the group consisting of N-[5-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide, N-{4-[(6,7-dimethoxyquinolin-4-yl)oxy]-3-fluorophenyl}-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide, AMG337, ASP5878, BGJ398, foretinib, ZM323881, CP-673451, crenolanib, and crizotinib.

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

7. 1. A method for purifying cardiomyocytes, comprising: (1) contacting a cell population containing cardiomyocytes or cardiac progenitor cells and other cells obtained by culturing pluripotent stem cells in a medium for cardiomyocyte differentiation with a receptor tyrosine kinase inhibitor; and (2) culturing the cell population; A method comprising:

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