Method for producing mature cardiomyocytes

Abstract

The present invention provides a method for producing mature cardiomyocytes, the method comprising a step for culturing cardiomyocytes in a culture medium containing an Src inhibitor.

Description

Method for producing mature cardiomyocytes

[0001] The present invention relates to a method for producing mature cardiomyocytes, more specifically to a method for producing mature cardiomyocytes, which comprises a step of culturing cardiomyocytes in a medium containing an Src inhibitor, and uses of the cardiomyocytes obtained by the method.

[0002] Cardiomyocytes are the cells that make up the myocardium of animals and play an important role in the contractile function of the heart. Abnormalities in the myocardium lead to heart disease, but for severe heart disease, there is no treatment other than heart transplantation, so the development of alternative therapies such as regenerative medicine is desired. For example, research is being conducted on methods for generating cardiomyocytes from pluripotent stem cells, and on the application of these generated cardiomyocytes to heart disease pathology models and regenerative medicine.

[0003] Cardiomyocytes generated in vitro from pluripotent stem cells are known to be less mature, at the fetal cardiomyocyte level. For example, cardiomyocytes induced from iPS cells express TNNI1, the fetal cardiomyocyte isoform of troponin I, but barely express TNNI3, the adult cardiomyocyte isoform. The inventors previously reported an ERRγ activator that induces TNNI3 expression, but this compound also expressed TNNI1, resulting in a maturity level comparable to that of neonatal cardiomyocytes, with both TNNI1 and TNNI3 positive (Non-Patent Document 1). Furthermore, expression of S100A1, a marker of adult cardiomyocytes, was low, highlighting the need for a method to mature cardiomyocytes to the adult cardiomyocyte level.

[0004] Miki K. et al. Nat Commun. 12(1):3596 (2021)

[0005] Therefore, an objective of the present invention is to provide a method for producing mature cardiomyocytes, and cardiomyocytes produced by the method.

[0006] The present inventors focused on the involvement of Torin1, an mTOR (mechanistic target of rapamycin) inhibitor, in cardiomyocyte maturation and conceived the idea that inhibiting upstream pathways in the mTOR (mechanistic target of rapamycin) signaling pathway might promote cardiomyocyte maturation. After extensive research, the inventors focused on Src and successfully demonstrated that treating cardiomyocytes with an Src inhibitor enhanced cardiomyocyte maturation and induced increased TNNI3 expression as well as decreased TNNI1 expression. Furthermore, cardiomyocytes treated with an Src inhibitor also showed increased expression of S100A1, a marker for adult cardiomyocytes. Based on these findings, the inventors concluded that methods using Src inhibitors can dramatically enhance cardiomyocyte maturation compared to conventional methods. Furthermore, the use of an Src inhibitor in combination with an mTOR inhibitor further enhanced cardiomyocyte maturation. Based on these findings, the inventors conducted further research and completed the present invention.

[0007] That is, the present invention provides the following: [1] A method for producing mature cardiomyocytes, comprising a step of culturing cardiomyocytes in a medium containing an Src inhibitor. [2-1] The method according to [1], wherein at least one Src inhibitor is selected from the group consisting of dasatinib, saracatinib, and NXP900. [2-2] The method according to [1], wherein at least one Src inhibitor is dasatinib. [3] The method according to any one of [1] to [2-2], wherein the medium contains an mTOR inhibitor. [4-1] The method according to [3], wherein at least one mTOR inhibitor is Torin1 or Torin2. [4-2] The method according to [3], wherein at least one mTOR inhibitor is Torin1. [5] The method according to any one of [1] to [4-2], wherein the mature cardiomyocytes are cells expressing TNNI3 and S100A1. [6-1] The method according to any one of [1] to [5], wherein the cardiomyocytes are derived from pluripotent stem cells. [6-2] The method according to any one of [1] to [6-1], wherein the cardiomyocytes are derived from a human. [7] The method according to any one of [1] to [6], wherein the cardiomyocytes are contained in cardiac organoids. [8] Mature cardiomyocytes obtained by the method according to any one of [1] to [7], or cardiac organoids comprising said cells. [9-1] A transplantation therapeutic agent comprising the mature cardiomyocytes or cardiac organoids according to [8]. [9-2] The transplantation therapeutic agent according to [9-1], for treating or preventing cardiac disease.

[10] A method for maturing cardiomyocytes, comprising culturing cardiomyocytes in a medium containing an Src inhibitor.

[11] A method for treating or preventing cardiac disease, comprising administering or transplanting an effective amount of the mature cardiomyocytes or cardiac organoids according to [8] into a mammal.

[12] The mature cardiomyocytes or cardiac organoids according to [8], for use in treating or preventing cardiac disease.

[13] Use of the mature cardiomyocytes or cardiac organoids according to [8] in the manufacture of a medicament for treating or preventing cardiac disease.

[0008] According to the present invention, a novel method is provided that can produce mature cardiomyocytes from immature cardiomyocytes.

[0009] Dasatinib mechanism of action and IC 50 Measurement results. Dasatinib has inhibitory activity against Src, and Torin1 has inhibitory activity against mTOR (left). Cell proliferation rate and IC in cardiac organoids after 96 hours of treatment with dasatinib. 50 The results of an MTT assay showing the effect of dasatinib on cardiomyocytes are shown (right panel). n=4, error bars: standard deviation. Micrographs of ventricular cardiomyocytes treated with dasatinib (days 0 and 7 of dasatinib treatment). Dasatinib was shown to enhance TNNI3 expression and reduce TNNI1 expression in cardiomyocytes. Immunoblotting results for cardiomyocyte markers (TNNI3, TNNI1), cardiomyocyte maturation markers (CD36, CAV3, S100A1), phosphorylated AKT (pAKT), a downstream marker of the Src pathway, cell cycle markers (CDK4, p53), and β-actin were also shown. Dasatinib and / or Torin1 treatment increased the expression of cardiomyocyte maturation markers. Principal component analysis (PCA) plot of gene expression changes following dasatinib and Torin1 treatment. CX49 indicates the dasatinib-treated group, and Combo indicates the dasatinib and Torin1-treated group. Measurement of the contractile force of engineered heart tissue (EHT). Combined treatment with dasatinib and Torin1 demonstrated an increase in EHT contractile force. n=3, error bars: standard deviation. *p<0.05, unpaired t-test. Cardiomyocytes generated by embryoid body formation were isolated into single cells 16 days after the start of differentiation induction and seeded onto fibronectin-coated 3mm x 7mm coverslips. After seeding onto the coverslips, 200nM Torin1 and 1μM dasatinib were added for 7 days, and intracellular action potentials were measured using the patch clamp technique (nine samples each, **p<0.01; tested by Tukey's multiple comparisons test). The whiskers in the box plots (APA, Vmax) represent the minimum to maximum values ​​(min to max). The error bars in the resting membrane potential diagram represent standard deviation.

[0010] 1. Method for Producing Cardiomyocytes The present invention provides a method for producing mature cardiomyocytes (hereinafter, sometimes referred to as the "production method of the present invention"), which comprises culturing cardiomyocytes in a medium containing an Src inhibitor. As shown in the Examples below, the combined use of an Src inhibitor and an mTOR inhibitor can promote cardiomyocyte maturation more than the use of an Src inhibitor alone. Therefore, in a preferred embodiment, the medium used in the production method of the present invention contains an mTOR inhibitor. Cardiomyocytes are broadly classified into immature (i.e., "fetal") cardiomyocytes and mature (i.e., "adult") cardiomyocytes. Hereinafter, unless otherwise specified, "cardiomyocytes" refers to immature cardiomyocytes. Furthermore, in this specification, the terms "culturing cardiomyocytes in a medium containing an Src inhibitor" and "culturing cardiomyocytes in a medium containing an Src inhibitor and an mTOR inhibitor" can be appropriately interpreted as "contacting cardiomyocytes with an Src inhibitor" and "contacting cardiomyocytes with an Src inhibitor and an mTOR inhibitor," respectively.

[0011] Cardiomyocytes are cells that constitute the myocardium of animals. As used herein, immature and mature cardiomyocytes refer to cells that repeatedly contract and relax (exhibit pulsatile activity). Immature and mature cardiomyocytes typically express at least one cardiomyocyte marker selected from the group consisting of cardiac troponin T (cTNT), αMHC (α myosin heavy chain, MYH6), and βMHC (MYH7). "Immature cardiomyocytes" refer to cardiomyocytes that express TNNI1. As used herein, "mature cardiomyocytes" refer to cells that express TNNI3 and have reduced or absent TNNI1 expression levels (in other words, "do not express TNNI1") compared to cardiomyocytes prior to the step of culturing in a medium containing an Src inhibitor. These cells typically exhibit increased expression of at least one myocardial maturation marker compared to cardiomyocytes prior to the step of culturing in a medium containing an Src inhibitor. Typically, mature cardiomyocytes are cells whose proliferation activity has been reduced or eliminated compared to cardiomyocytes before the step of culturing in a medium containing an Src inhibitor. Examples of the cardiomyocyte maturation markers include TNNI3, CD36, CAV3, S100A1, etc., and mature cardiomyocytes are typically cells in which at least TNNI3 expression is elevated, and preferably, further, S100A1 expression is elevated. In one embodiment, mature cardiomyocytes do not express TNNI1, but express TNNI3 and / or S100A1.

[0012] Typically, the proliferation activity may be evaluated based on the expression level of cell division-related genes (e.g., CDK4, MKI67, CCNA1, CCNB1, E2F1, etc.). Alternatively, it may be evaluated based on the expression level of genes associated with the resting phase of the cell cycle (e.g., TP53, CDKN2A, HES1, RB1, etc.). In one embodiment, mature cardiomyocytes are cells that have a lower expression level of CDK4 and / or a higher expression level of TP53 (p53 protein) compared to cardiomyocytes before the step of culturing in a medium containing an Src inhibitor.

[0013] By culturing cardiomyocytes in a medium containing an Src inhibitor, the cardiomyocytes can be matured, and cardiomyocytes in cardiac tissue can be matured.Therefore, in one embodiment of the present invention, the cardiomyocytes used in the present invention are contained in cardiac organoids such as cardiac tissue, and the cardiomyocytes are subjected to the method of the present invention in the form of cardiac organoids.Therefore, the present invention also provides a method for producing cardiac organoids containing mature cardiomyocytes, which comprises culturing cardiac organoids containing cardiomyocytes in a medium containing an Src inhibitor (preferably also an mTOR inhibitor).As shown in the examples below, such cardiac organoids can have enhanced contractile force compared to those not treated with an Src inhibitor.In addition, cardiac organoids can show improved action potential amplitude (APA) and / or increased rise velocity (Vmax) compared to those not treated with an Src inhibitor.

[0014] In this specification, unless otherwise specified, "cells" includes "cell populations". Furthermore, unless otherwise specified, "cells" refers to those obtained by cell culture. A cell population may be composed of one type of cell, or may be composed of two or more types of cells. Furthermore, unless otherwise specified, "cell populations" also include "organoids" such as "cell aggregates" and "artificial tissues".

[0015] In this specification, "organoid" refers to a structure (typically a spheroid) containing multiple types of cells. Also, "artificial tissue" typically refers to a structure having a structure and / or function similar to that of tissue in vivo. Whether a structure is an organoid or an artificial tissue can be determined, for example, by observing a sample stained as needed (e.g., immunostaining, hematoxylin-eosin (HE) staining, etc.) under a microscope to confirm the localization of cells and the presence or absence of layer structure formation.

[0016] In this specification, " cardiac organoid " refers to the organoid that comprises cardiomyocytes, and this organoid also includes artificial cardiac tissue. " Artificial cardiac tissue " refers to the artificial tissue that comprises cardiomyocytes. Hereinafter, unless otherwise specified, artificial cardiac tissue will simply be referred to as " cardiac tissue ". Typically, the cardiac organoid of the present invention comprises WT1 positive epicardial cells, CD31 positive endothelial cells, NFATC1 positive endocardial cells, and VIM positive cardiac fibroblasts. In addition, cardiac organoid can have the tight junction that is represented by ZO-1 expression.

[0017] The cardiomyocytes used in the present invention can be obtained by known methods. Examples include isolation from the heart using known techniques, differentiation induction of pluripotent stem cells, and procurement from companies such as ATCC. Cardiomyocytes can be isolated from the heart using, for example, flow cytometry or mass cytometry, magnetic cell separation, or affinity columns immobilized with desired antigens, using surface antigens (e.g., CD172a) as an indicator. Preferably, the cardiomyocytes used in the present invention are obtained by differentiation induction of pluripotent stem cells. The origin of the cardiomyocytes is not particularly limited, and may be from rodents such as rats, mice, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. Humans are the preferred origin.

[0018] Known methods can be used to induce differentiation of pluripotent stem cells into cardiomyocytes, including, for example, the method described in Laflamme MA & Murry CE, Nature 2011, May 19;473(7347):326-35 Review, Funakoshi, S. et al., Sci Rep 8, 19111 (2016), and Miki, K. et al., Cell Stem Cell. 2015 Jun 4;16(6):699-711.

[0019] Other examples include, but are not limited to, a method for producing cardiomyocytes by forming cell masses (embryoid bodies) from induced pluripotent stem cells in suspension culture (WO2016 / 104614), a method for producing cardiomyocytes in the presence of a substance that suppresses bone morphogenic protein (BMP) signaling (WO2005 / 033298), a method for producing cardiomyocytes by sequentially adding Activin A and BMP (WO2007 / 002136), a method for producing cardiomyocytes in the presence of a substance that promotes activation of the canonical Wnt signaling pathway (WO2007 / 126077), and a method for isolating FLk / KDR-positive cells from induced pluripotent stem cells and producing cardiomyocytes in the presence of cyclosporin A (WO2009 / 118928).

[0020] Other methods include a method using cytokines to induce cardiomyocyte differentiation in embryoid body formation (Yang L, et al., Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population., Nature., 2008 May 22;453(7194):524-8), a method using 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 U S A., 2012 July 3;109(27):E1848-57), and a method using both adherent and suspension cultures without using cytokines to induce cardiomyocyte differentiation (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) have also been proposed.

[0021] More specifically, examples of methods for producing cardiomyocytes include methods comprising the following steps (A) to (C). Thus, the production method of the present invention may comprise at least one of the following steps (A) to (C): (A) a step of suspension culture of pluripotent stem cells to form cell aggregates (embryoid bodies (EBs)); (B) a step of suspension culture of the cell aggregates obtained in step (A) in the presence of BMP4, activin A, and bFGF to induce cell aggregates containing mesodermal cells; and (C) a step of suspension culture of the cell aggregates obtained in step (B) in the presence of VEGF and a Wnt inhibitor to induce differentiation into cardiomyocytes.

[0022] The formation of cell aggregates in step (A) is typically carried out by the following method. First, pluripotent stem cells are recovered from subculture and dispersed into single cells or a state close to single cells. Dissociation of pluripotent stem cells is carried out using an appropriate cell dissociation solution. Examples of cell dissociation solutions include EDTA; trypsin, collagenase IV, metalloproteases, and other proteolytic enzymes, which can be used alone or in appropriate combinations. Commercially available cell dissociation solutions include Accutase (MILLIPORE), Dispase (EIDIA), and TrypLE (Invitrogen). The dispersed pluripotent stem cells are suspended in a medium in a low-adhesion culture vessel. The duration of step (A) can be determined appropriately by those skilled in the art and is typically 1 to 5 days.

[0023] Step (B) is a step of inducing differentiation of pluripotent stem cells into mesodermal cells, and the medium used in this step preferably contains an extracellular matrix (e.g., 0.5% Matrigel) in addition to the above-mentioned additives. The period of step (B) can be appropriately determined by those skilled in the art, and is usually 2 to 6 days, more preferably 3 to 4 days.

[0024] Step (C) is a step of inducing differentiation of mesodermal cells into cardiomyocytes, and typically involves culturing the cells under hypoxic (e.g., 5%) conditions for a predetermined period (e.g., 3 to 10 days) and then culturing them under normal oxygen conditions. Because step (C) allows cardiomyocytes to be maintained, the period of this step is not particularly limited, but is usually 3 to 20 days, and more preferably 5 to 15 days.

[0025] Examples of Wnt inhibitors used in step (C) include DKK1 protein, sclerostin, IWR-1e, IWP-2, IWP-3, IWP-4, IWP-L6, C59, ICG-001, FH535, WIKI4, KYO2111, PNU-74654, XAV939, and derivatives thereof.

[0026] Cardiac organoids can also be induced by, for example, culturing the cell aggregates obtained in step (A) above in suspension in a medium containing CHIR99021, BMP4, and Activin A, then treating the cell aggregates with Wnt-C59 for approximately 2 days, and continuing the suspension culture until the 15th day.

[0027] It is also possible to produce artificial cardiac tissue from cardiac organoids by dispersing cell aggregates into single cells or a state close to this, as in step (A), and then culturing the cells in an adhesive culture medium for artificial cardiac tissue culture.

[0028] The term "pluripotent stem cells" refers to stem cells that can differentiate into various tissues and cells with different morphologies and functions in the body and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). 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 (ntES cells), multipotent germline stem cells (mGS cells), and embryonic germ stem cells (EG cells). Preferably, iPS cells (more preferably, human iPS cells) are used. When the pluripotent stem cells are ES cells or any cells derived from human embryos, they may be produced by or without the destruction of the embryo. However, from an ethical standpoint, cells produced without the destruction of the embryo are preferred.

[0029] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by isolating 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. Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from a developmentally arrested embryo (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).

[0030] nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same properties as ES cells derived from fertilized eggs (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 blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To generate nt ES cells, nuclear transfer technology (Cibelli JB et al. (1998), Nature Biotechnol., 16:642-646) is combined with ES cell generation technology (see above) (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, and the egg is then cultured for several hours to reprogram the embryo.

[0031] Examples of ES cell lines that can be used in the present invention include mouse ES cell lines established by, for example, inGenious targeting laboratory, Inc., RIKEN (Riken), etc., and human ES cell lines established by, for example, the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, and Cellartis, Inc. Specific examples of human ES cell lines 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.

[0032] iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of iPS cells, including iPSCs 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 cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPSCs established by selecting 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 iPSCs created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), iPSCs 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.), etc. can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells produced 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., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7, 795-797), or patent publications (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) and known in the art can be used.

[0033] Available induced pluripotent stem cell lines include various iPSC lines established by the NIH, RIKEN, Kyoto University, etc. Examples of human iPSC lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 strains, and Kyoto University's 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 1390B1, 1390C1, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, and FfI-01s04 strains.

[0034] The induced pluripotent stem cells used in the production method of the present invention may be cells derived from patients with hereditary diseases (e.g., patients with hereditary heart disease). Cells induced to differentiate from pluripotent stem cells derived from patients with hereditary heart disease can serve as disease models that reflect the pathology of the disease, making them suitable for screening therapeutic or preventive drugs for the disease. Alternatively, pluripotent stem cells derived from patients with hereditary heart disease can be genetically repaired by genome editing using the CRISPR-Cas system or the like, and then differentiated into mature cardiomyocytes or cardiac organoids containing these cells, making it possible to use these cells or organoids as therapeutic agents for heart disease.

[0035] mGS cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various cell lineages, e.g., 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 similar culture conditions to ES cells (Takebayashi M. et al. (2008) Experimental Medicine, Vol. 26, No. 5 (Supplementary Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0036] EG cells are derived from embryonic primordial germ cells (PGCs) and have pluripotency similar to that of ES cells. They can be established by culturing PGCs in the presence of LIF, bFGF, stem cell factor, and other substances (Matsui Y. et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551).

[0037] The species from which the pluripotent stem cells are derived is not particularly limited, and may be cells from, for example, rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. The preferred species is human.

[0038] As used herein, the term "Src inhibitor" refers to a substance that inhibits the phosphorylation of a target protein by the tyrosine kinase Src. The Src inhibitor may be a substance that specifically inhibits the phosphorylation of a specific protein by Src, but is preferably one that inhibits the phosphorylation function of Src itself by binding to the Src binding pocket or the like. Examples of Src inhibitors used in the present invention include A419259, SU6656, PP1, 1-naphthyl PP1, PP2, PP121, indirubin-3'-(2,3-dihydroxypropyl)-oximether, TX-1123, Src Kinase Inhibitor I (CAS 179248-59-0), AZM475271, dasatinib, bosutinib, pelitinib, repotrectinib, herbimycin A, KB SRC 4, MNS, PD166285, TC-S7003, and Src Inhibitor 1 (CAS No. 179248-59-0), CCT196969, WH-4-023, saracatinib (AZD0530), NXP900, etc., with dasatinib being preferred. Saracatinib and NXP900 can also be suitably used. Only one type of Src inhibitor may be used, or multiple types may be used.

[0039] mTOR exists in cells as two complexes: mTORC1 (mTOR Complex 1) and mTORC2 (mTOR Complex 2). mTORC1 contains raptor, and mTORC2 contains rictor. These complexes are differentially regulated and have different substrate specificities and rapamycin sensitivity. For example, mTORC1 phosphorylates S6 kinase (S6K) and 4EBP1, thereby promoting increased protein translation and ribosome synthesis, thereby promoting cell growth and cell cycle progression. S6K also acts in a feedback pathway to attenuate PI3K / Akt activity. mTORC2 is thought to regulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases, such as Akt.

[0040] The mTOR inhibitor used in the present invention is not particularly limited as long as it can inhibit the phosphorylation of Akt by the mTOR complex (typically, phosphorylation at the S473 site). The mTOR inhibitor used in the present invention may be any of a substance having inhibitory activity against mTORC1, a substance having inhibitory activity against mTORC2, and a substance having inhibitory activity against both of these complexes, but is preferably a substance having inhibitory activity against at least mTORC2, and more preferably one having inhibitory activity against mTORC1. Specific examples of mTOR inhibitors used in the present invention include small molecule compounds such as Torin1, Torin2, omipalisib (GSK2126458), KU-0063794, OSI-027, XL388, Palomid 529 (P529), WYE-354, torkinib (PP242), sapanisertib (TAK-228), WYE-687, AZD8055, and vistusertib (AZD2014), with Torin1 being preferred. Torin2 may also be used. Antibodies, peptides, or aptamers that inhibit Akt phosphorylation of mTOR are also preferred. A single mTOR inhibitor may be used, or multiple mTOR inhibitors may be used. Furthermore, the mTOR inhibitor of the present invention may be used in combination with other mTOR inhibitors (for example, when the mTORC2 inhibitor used is a specific inhibitor of mTORC2, it may be used in combination with an mTORC1 inhibitor).

[0041] When the substance used in the present invention is a low molecular weight compound such as dasatinib, the compound includes not only the free form but also its pharmacologically acceptable salts and hydrates. Pharmacologically acceptable salts vary depending on the type of compound, but include, for example, inorganic base salts such as alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), aluminum salts, ammonium salts, etc., as well as base addition salts such as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc., or inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, etc., and acid addition salts such as organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, etc. In the case of dasatinib, examples of its salts include dasatinib hydrochloride, and examples of its hydrates include dasatinib monohydrate.

[0042] When the substances used in the present invention are proteins or peptides, their origin is not particularly limited, but is preferably mammalian (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.). Furthermore, the proteins or peptides used in the production methods of the present invention include not only wild-type proteins or peptides but also their mutants having similar functions. Examples of mutants include proteins or peptides that have a high degree of identity (e.g., 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence of a specific wild-type protein or peptide.

[0043] Substances capable of suppressing the expression of Src and substances capable of suppressing the expression of any protein constituting the mTOR complex are also suitable as inhibitors for use in the present invention. Hereinafter, these expression inhibitors may be collectively referred to as "gene expression inhibitors." Proteins constituting mTORC1 include mTOR, RAPTOR, PRAS40, deptor, and mLST8, while proteins constituting mTORC2 include mTOR, rictor, mSIN1, mLST8, deptor, and procto. While it is sufficient to suppress the expression of at least one of these proteins, preferred targets are mTOR, deptor, and mLST8, which are common to mTORC1, with mTOR being more preferred.

[0044] As used herein, unless otherwise specified, the term "gene expression" is used to mean at least "production of a functional protein," but preferably also "production of a transcription product." Furthermore, as used herein, transcription products typically include mRNA and pre-mRNA, but are preferably mRNA.

[0045] Furthermore, if the production of the protein encoded by the gene is detected at least by the method described in the Examples below (immunoblotting), the gene can be said to be expressed or positive. On the other hand, if the production of the protein encoded by the gene is not detected (i.e., below the detection limit) by the method described in the Examples below (immunoblotting), or if it is at background levels, the gene can be said to be not expressed or negative. Furthermore, when comparing the expression levels of each gene, the protein amount in immunoblotting is used as the standard.

[0046] Gene expression inhibitors are not limited as long as they can suppress the expression of target proteins in cardiomyocytes, but are preferably nucleic acids. Examples of such nucleic acids include antisense nucleic acids (e.g., antisense oligonucleotides (ASOs) and the like) (including nucleic acids encoding such nucleic acids), siRNA (including nucleic acids encoding such siRNA), heteroduplex oligonucleotides (HDO), shRNA (including nucleic acids encoding such shRNA), and miRNA (microRNA) (including nucleic acids encoding such miRNA). Hereinafter, when a gene expression inhibitor is a nucleic acid, such a nucleic acid may be referred to as a "gene expression inhibitory nucleic acid." A single type of gene expression inhibitor may be used, or multiple types may be used. Furthermore, a gene expression inhibitor may be used in combination with other gene expression inhibitors (e.g., a combination of an mTORC1 expression inhibitor and an mTORC2 expression inhibitor).

[0047] When the protein targeted by the gene expression-inhibiting nucleic acid has multiple isoforms, the nucleic acid is typically one that can inhibit the expression of a transcript encoding the full-length protein. Furthermore, nucleic acids targeting other isoforms can also be used as long as they can inhibit phosphorylation by Src or mTOR complex proteins. The base sequence targeted by the gene expression-inhibiting nucleic acid (target sequence) can be appropriately designed, for example, based on information from a known database (e.g., the NCBI database). The length of the target sequence is not particularly limited as long as the gene expression-inhibiting nucleic acid can specifically recognize and bind to it, but is preferably 12 nucleotides or longer, more preferably 15 nucleotides or longer, and even more preferably 17 nucleotides or longer. The upper limit of the length is also not particularly limited, but is, for example, 30 nucleotides or shorter, preferably 25 nucleotides or shorter, and more preferably 22 nucleotides or shorter.

[0048] As used herein, "complementary" refers to a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, wobble base pairs, etc.) through hydrogen bonds. Therefore, the term "complementary sequence" refers not only to sequences that are completely complementary to a target RNA sequence or target DNA sequence (i.e., hybridize without mismatches), but also to sequences containing one to several (e.g., 2, 3, 4, 5, or more) mismatches, as long as they can hybridize with the target sequence under stringent conditions or under the physiological conditions of mammalian cells. For example, the term "complementary sequence" refers to a sequence that has 80% or more (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more), and most preferably 100%, identity with a sequence that is completely complementary to a target RNA sequence or target DNA sequence.

[0049] Antisense nucleic acids are single-stranded nucleic acids that contain a sequence complementary to a target RNA sequence. Antisense nucleic acids form a double-stranded region with the target RNA sequence due to the sequence complementary to the target RNA sequence, and this double-stranded region is cleaved by ribonuclease H (RNase H), thereby suppressing the expression of a target gene. The length of the antisense nucleic acid is not particularly limited, but is typically 10 to 30 nucleotides, more preferably 13 to 30 nucleotides.

[0050] siRNA is a double-stranded RNA consisting of an RNA having a sequence complementary to a target RNA sequence (i.e., an antisense strand) and its complementary strand. Also, a preferred embodiment of siRNA is a single-stranded RNA in which a sequence complementary to the target RNA sequence (a first sequence) is linked to its complementary sequence (a second sequence) via a hairpin loop, and the first sequence forms a double-stranded structure with the second sequence by adopting a hairpin loop structure (small hairpin RNA: shRNA). Furthermore, shRNA may be in the form of a nucleic acid (e.g., an expression vector) encoding the shRNA.

[0051] HDO refers to a double-stranded nucleic acid composed of a DNA backbone (i.e., an antisense strand) and an RNA (cRNA) strand complementary to the DNA. When HDO is taken up into cells, the cRNA strand is cleaved by RNase H inside the cell. The HDO backbone DNA becomes a single-stranded antisense nucleic acid through cleavage of the cRNA strand, and it is presumed that the antisense effect is exerted by binding to the target RNA, where RNase H again cleaves the target RNA.

[0052] The length of each nucleic acid strand of the double-stranded nucleic acid is not particularly limited as long as it can exert an antisense effect, but is, for example, 10 to 50 nucleotides, preferably 15 to 30 nucleotides, more preferably 20 to 27 nucleotides.

[0053] As used herein, "miRNA" refers to a single-stranded or double-stranded RNA (e.g., miRNA / miRNA) that does not cleave target RNA like siRNA, but recognizes the 3' untranslated region (UTR) of target RNA and controls translation. * miRNA refers to endogenous non-coding RNA (ncRNA) of about 20 to 25 bases that is originally encoded on the genome, and pri-miRNA is expressed from the miRNA gene, followed by the generation of pre-miRNA, which then produces mature-miRNA. The mature-miRNA is then incorporated into RISC to produce single-stranded miRNA. The miRNA used in the present invention may be in the form of pri-miRNA or pre-miRNA, and may be in the form of mature-miRNA (miRNA / miRNA * ) or even in the form of single-stranded RNA. Furthermore, miRNA may be in the form of a nucleic acid (e.g., an expression vector) that encodes the miRNA. The length of the miRNA (when the miRNA is double-stranded, the length of each nucleic acid strand) is not particularly limited as long as it can exert an antisense effect, but is, for example, 10 to 50 nucleotides long, preferably 15 to 30 nucleotides long, and more preferably 20 to 27 nucleotides long.

[0054] Gene expression-inhibiting nucleic acids can be obtained by chemical synthesis using conventionally known techniques or by production using genetic recombination technology. Alternatively, commercially available nucleic acids can be used. For example, nucleic acids can be appropriately designed based on the target RNA sequence using commercially available software (e.g., RNAiDesigner; Invitrogen). In the case of double-stranded nucleic acids, the sense and antisense strands of the target RNA sequence can be synthesized using a commercially available automated DNA / RNA synthesizer (Applied Biosystems, Beckman, etc.), denatured in an appropriate annealing buffer at about 90°C to about 95°C for about 1 minute, and then annealed at about 30°C to about 70°C for about 1 to about 8 hours. HDO can also be prepared, for example, by the method described in WO2013 / 089283.

[0055] The basal medium used in the present invention is not particularly limited, but includes RPMI-1640 medium, EagleMEM (EMEM), Dulbecco's modified MEM, Glasgow's MEM (GMEM), α-MEM, 199 medium, IMDM, DMEM, Hybridoma serum-free medium, KnockOut TMDMEM, Advanced TM medium (e.g. Advanced MEM, Advanced RPMI, Advanced DMEM / F-12), Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, DMEM / F-12, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's Medium (e.g. Waymouth's MB752 / 1), CMRL medium (e.g. CMRL-1066), Williams' medium E, Brinster's BMOC-3 Medium, Essential 8(E8) Medium, Essential 8 Flex Medium, StemPro 34, MesenPRO RS (Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (ReproCELL Co., Ltd.), ProculAD (Rohto Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (Lonza), EX-CELL302 medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMed TM Examples of suitable media include, but are not limited to, iPSC Medium (ReproCELL Corporation) and mixtures thereof. In the method of the present invention, DMEM is preferred. StemPro 34 is also preferred for inducing differentiation of pluripotent stem cells into cardiomyocytes.

[0056] In particular, for culture under feeder-free and xeno-free conditions, StemFit (registered trademark) AK02 medium (Ajinomoto Co., Inc.), StemFit (registered trademark) AK03 medium (Ajinomoto Co., Inc.), StemFit (registered trademark) Basic03 medium, CTS (registered trademark) KnockOut SR XenoFree Medium (Gibco), mTeSR1 medium, TeSR1 medium (Stem Cell Technologies), Iscove's modified Dulbecco's medium (GE Healthcare), Improved MEM (Thermo Fisher Scientific), and the like can also be used.

[0057] If necessary, the medium may contain serum, such as fetal bovine serum, horse serum, or human serum, or may contain one or more serum substitutes such as Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, or 3'-thiolglycerol, or may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone, and putrescine.

[0058] As used herein, "suspension culture" refers to culture performed under conditions that maintain cells or cell clumps suspended in a culture medium, i.e., culture under conditions that do not allow the formation of strong cell-substratum junctions between the cells or cell clumps and the culture vessel. Furthermore, when performing suspension culture, cells typically remain in the form of cell clumps before and after suspension culture. As used herein, "adhesion culture" refers to culture under conditions that allow the formation of strong cell-substratum junctions between the cells and the cultureware, etc.

[0059] The culture vessel used for suspension culture is not particularly limited as long as it is capable of "suspension culture," and can be appropriately determined by one skilled in the art. Examples of such culture vessels include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. Furthermore, bioreactors are an example of vessels for suspension culture. These culture vessels are preferably non-cell-adhesive to enable suspension culture. Examples of non-cell-adhesive culture vessels include those whose surfaces have not been artificially treated (e.g., coated with extracellular matrix) to improve cell adhesion. The well bottom shape of these culture vessels is not particularly limited, and examples include flat-bottomed, U-shaped, and V-shaped vessels.

[0060] Culture vessels used in adhesion culture include those whose surfaces have been artificially treated to improve cell adhesion (e.g., coating with basement membrane preparations, extracellular matrices such as fibronectin, laminin or fragments thereof, entactin, collagen, gelatin, synthemax, vitronectin, etc., or polymers such as polylysine and polyornithine, or surface treatments such as positive charge treatment). Commercially available basement membrane preparations include, for example, Matrigel and Geltrex.

[0061] Alternatively, from the viewpoint of xeno-free, laminin or a fragment thereof is also preferred. Examples of laminin or a fragment thereof include laminin-111 or a fragment thereof comprising its E8 region, laminin-211 or a fragment thereof comprising its E8 region (e.g., iMatrix-211), laminin-121 or a fragment thereof comprising its E8 region, laminin-221 or a fragment thereof comprising its E8 region, laminin-332 or a fragment thereof comprising its E8 region, laminin-3A11 or a fragment thereof comprising its E8 region, laminin-411 or a fragment thereof comprising its E8 region (e.g., iMatrix-411), laminin-421 or a fragment thereof comprising its E8 region, and laminin-511 or a fragment thereof comprising its E8 region (e.g., iMatrix-511, iMatrix-511). silk), laminin-521 or a fragment thereof containing its E8 region, laminin-213 or a fragment thereof containing its E8 region, laminin-423 or a fragment thereof containing its E8 region, laminin-523 or a fragment thereof containing its E8 region, laminin-212 / 222 or a fragment thereof containing its E8 region, and laminin-522 or a fragment thereof containing its E8 region.

[0062] In at least one of the steps of the production method of the present invention and other steps (e.g., steps (A) to (C) above), cells may be cultured under feeder-free conditions and / or xeno-free conditions. In the production method of the present invention, all steps may be performed under feeder-free and xeno-free conditions. As used herein, "feeder-free" refers to a medium or culture conditions that do not contain other cell types (i.e., feeder cells) that play a supporting role and are used to establish culture conditions for the cells to be cultured. Furthermore, "xeno-free" refers to a medium or culture conditions that do not contain components derived from organisms other than the biological species of the cells to be cultured.

[0063] The culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being about 2 to 5%.

[0064] The seeding density of cells is not particularly limited as long as the cells can grow. Typically, the seeding density is 1.0 × 10 2 ~1.0×10 7 cells / cm2 , preferably 1.0 x 10 3 ~1.0×10 6 cells / cm 2 , more preferably 1.0 × 10 4 ~1.0×10 5 cells / cm 2 is.

[0065] When dasatinib is used, the concentration of the Src inhibitor used in the present invention in the medium is typically 5 nM to 50 μM, preferably 50 nM to 10 μM, and more preferably 200 nM to 5 μM (particularly 1 μM). When an Src inhibitor other than dasatinib is used, the concentration of the mTOR inhibitor in the medium is appropriately selected.

[0066] When Torin1 is used, the concentration of the mTOR inhibitor used in the present invention in the medium is typically 1 nM to 10 μM, preferably 10 nM to 2 μM, and more preferably 50 nM to 600 nM (particularly 200 nM). When an mTOR inhibitor other than Torin1 is used, the concentration of the mTOR inhibitor in the medium is selected appropriately.

[0067] The culture period in the steps of the production method of the present invention is not particularly limited, but is typically 1 to 20 days, preferably 3 to 15 days, and more preferably 5 to 10 days (particularly 7 days).

[0068] The method of the present invention may include a step of isolating mature cardiomyocytes obtained by the method of the present invention. The isolated cells may be cryopreserved using a cell cryopreservation solution. The collected cells may be counted using a cell counter, or may be labeled with an antibody against a cell surface marker and selected by flow cytometry, mass cytometry, magnetic cell sorting, or the like.

[0069] In yet another embodiment, the present invention provides a method for maturing cardiomyocytes, comprising culturing cardiomyocytes in a medium containing an Src inhibitor. In a preferred embodiment, the medium used in the method for maturing cardiomyocytes contains an mTOR inhibitor. The definitions, specific examples, and culturing methods of cardiomyocytes, Src inhibitors, and mTOR inhibitors are all incorporated by reference in the description of the production method of the present invention.

[0070] 2. Mature cardiomyocytes, cardiac organoids containing said cells, and their uses By the method of the present invention, mature cardiomyocytes or cardiac organoids such as cardiac tissue containing said cells can be obtained.Therefore, in another aspect of the present invention, the method of the present invention ("obtained" can be appropriately read as "obtained") also provides mature cardiomyocytes or cardiac organoids containing said cells (hereinafter, sometimes referred to as "cardiomyocytes of the present invention").

[0071] Since the cardiomyocytes of the present invention can be used in medical treatments such as regenerative medicine, in another aspect, a transplantation therapeutic agent (hereinafter, sometimes referred to as the "transplantation therapeutic agent of the present invention") containing the cardiomyocytes of the present invention is provided. The present invention also encompasses a method for treating or preventing heart disease, in which an effective amount of the cardiomyocytes of the present invention is administered or transplanted into a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) that is the target of treatment or prevention. Unless otherwise specified, herein, therapeutic or preventive agents (or therapeutic or preventive methods) for heart disease also encompass pharmaceuticals (or methods) that can treat and prevent the heart disease.

[0072] Examples of cardiac diseases include heart failure, chronic heart failure, severe cardiac failure, ischemic heart disease, myocardial infarction, acute myocardial infarction, chronic myocardial infarction, cardiomyopathy, ischemic cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, dilated cardiomyopathy, amyloid cardiomyopathy, alcoholic cardiomyopathy, viral cardiomyopathy, stress-induced cardiomyopathy, myocarditis, congenital disorders, genetic disorders, left ventricular noncompaction, mitral valve prolapse, arrhythmogenic right ventricular dysplasia, various valvular stenosis diseases, various valvular regurgitation diseases, etc. The cardiomyocytes of the present invention exert a therapeutic or preventive effect on cardiac diseases by, for example, transplanting them into the affected area.

[0073] The transplantation therapy agent of the present invention can be administered or transplanted into the body of a subject in need thereof. The cells or organoids to be transplanted should be administered in a therapeutically or prophylactically effective amount, which may vary depending on factors such as the age, weight, size of the transplant site, and severity of the disease of the transplant subject. The number of cells is not particularly limited, but for example, 10 × 10 4 Cell ~10×10 11It can be as small as a cell.

[0074] When using the cardiomyocytes of the present invention as a transplantation therapy agent, it is desirable to use cells derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, in order to avoid 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). If sufficient cells cannot be obtained due to age, constitution, or other reasons, they can be transplanted in a state that avoids rejection by embedding them in capsules made of polyethylene glycol or silicone, or in porous containers.

[0075] The cardiomyocytes of the present invention are prepared as parenteral preparations such as injections, suspensions, and infusions by mixing with a pharmaceutically acceptable carrier according to conventional methods. Therefore, in one embodiment, a method for producing a transplantation therapy agent is also provided, which includes a step of formulating the cardiomyocytes of the present invention. Such a method may include a step of preparing the cardiomyocytes of the present invention. Furthermore, the method may include a step of preserving the cardiomyocytes of the present invention.

[0076] Pharmaceutically acceptable carriers that can be contained in such parenteral formulations 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.), etc. The cardiomyocytes of the present invention may be formulated with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0077] The transplantation therapy agent of the present invention is provided in a cryopreserved state under conditions typically used for cryopreserving cells and can be thawed immediately before use. In this case, it may further contain serum or a serum substitute, an organic solvent (e.g., DMSO), etc. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0078] The cardiomyocytes of the present invention can also be used in methods for screening candidate drugs that are useful for treating or preventing cardiac disease. Thus, in yet another aspect of the present invention, a method for screening therapeutic or preventive drugs for cardiac disease is provided, comprising culturing the cardiomyocytes of the present invention in the presence or absence of a test substance. Examples of such cardiac diseases include those similar to the cardiac diseases targeted for treatment or prevention by the transplantation therapy agents of the present invention. Using cells derived from patients with cardiac disease as the cardiomyocytes of the present invention used for screening may enable more accurate screening.

[0079] Test substances used in the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low molecular weight compounds, and natural compounds.

[0080] The test substance can also be obtained using any of the many approaches to combinatorial library technology known in the art, including (1) biological libraries, (2) synthetic library technology using deconvolution, (3) "one-bead one-compound" library technology, and (4) synthetic library technology using affinity chromatography selection. While the biological library technology using affinity chromatography selection is limited to peptide libraries, the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12:145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al. (1994) J. Med. Chem. 37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al. (1994) J. Med. Chem. 37:1233-51).Compound libraries can be stored in solution (see Houghten (1992) Bio / Techniques 13:412-21) or on beads (Lam (1991) Nature 354:82-4), chips (Fodor (1993) Nature 364:555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-9), or phage (Scott and Smith (1990) Science 249:386-90; Devlin (1990) Science 249:404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-82; Felici (1991) J. Mol. Biol. 222:301-10; U.S. Patent Application No. 2002103360).

[0081] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0082] Example 1: Evaluation of Cardiotoxicity of Dasatinib Derivation of Human Cardiac Organoids (hHOs) from hiPSCs Human cardiac organoids were generated from PSCs according to established protocols. Briefly, monolayers of the feeder-free iPS cell line 1390C1 were disrupted using Accumax and then suspended in Essential 8 Flex medium (Gibco) containing 10 μM of the ROCK inhibitor Y-27632. On day -2, 10,000 cells were dispensed into 100 μl volumes of HEMA-coated 96-well plates to form EBs. The following day, fresh Essential 8 Flex medium was added. By day 0, this medium was replaced, and differentiation was initiated using insulin-free RPMI 1640 / B-27 (Gibco) containing CHIR99021, BMP4, and Activin A. Periodic medium changes were performed over the following days. Specifically, a fresh version of RPMI 1640 / B-27 was introduced on days 1, 4, and 6, with Wnt-C59 included in the medium on day 2. On day 7, organoids underwent a short-term treatment with CHIR99021 in RPMI 1640 / B-27. From this point until day 15, the medium was changed every two days.

[0083] Cardiotoxicity Assay in iPSC-Derived Human Cardiac Organoids (hHOs) For each experiment, dasatinib was freshly prepared in DMSO. After differentiation, hHOs were individually seeded into low-binding 96-well plates. On day 15 of organoid development, they were treated with decreasing concentrations of dasatinib, consistently maintaining a 1:2 dilution ratio. An MTT assay was employed to measure cell proliferation 96 hours after treatment. To measure cytotoxicity, absorbance at 595 nm was read using a microplate reader. IC was then calculated using GraphPad Prism 7 software. 50 The value was calculated.

[0084] The results are shown in Figure 1. IC of dasatinib 50 The concentration of 1 μM was 8.7±0.2 μM. This result suggests that 1 μM is a sufficiently low concentration and that the toxicity to cells is negligible.

[0085] Example 2: Verification of the Effect of Dasatinib on Promoting Cardiomyocyte Maturation Differentiation of hiPSCs into Ventricular Cardiomyocytes To differentiate into ventricular cardiomyocytes, human iPS cell line 201B7-luc-FUCCI-MYH6-iRFP670 cells (Kasamoto, M. et al., Stem Cell Reports 18, 1672-1685 (2023)) were first dissociated into individual cells using Accumax dilution solution. These single cells were cultured in 1.5 ml / well of StemPro-34 medium (obtained from Invitrogen) enriched with additional components such as L-glutamine, monothioglycerol (MTG), ascorbic acid (AA), transferrin, ROCK inhibitor Y-27632, Corning Matrigel, and BMP4. This combination was used at a concentration of 1 x 10 6 The goal was to generate embryoid bodies (EBs) at a density of 100 cells / well. The next day, another 1.5 ml of StemPro-34 medium supplemented with bFGF, activin A, and BMP4 was added. By day 3, the EBs were washed with IMDM (Invitrogen's version) and then exposed to StemPro-34 medium enriched with VEGF and IWP-3. On day 7, the medium was replaced with fresh StemPro-34 mixture and then every 2–3 days thereafter. Culture plates were first maintained in a hypoxic environment (5% O2) for 10 days before transitioning to standard oxygen levels.

[0086] Image acquisition for monitoring the developmental switch between fetal and adult isoforms of cardiac troponin I: Image processing and analysis. To monitor the transition switch from fetal to adult isoforms of cardiac troponin I (a crucial developmental switch that can distinguish fetal, neonatal, or adult cardiomyocytes), we visualized TNNI1 (eGFP) and TNNI3 (mCherry) (Miki, K. et al., Nat Commun 12, 3596 (2021)) using a reporter cell line constructed based on the feeder-free cell line 1390D4. To observe the expression of TNNI3 after 7 days of treatment with small molecules, we used reporter cell lines constructed based on the 1390D4 feeder-free cell line 1390D4. We then visualized TNNI1 (eGFP) and TNNI3 (mCherry) (Miki, K. et al., Nat Commun 12, 3596 (2021)). We then visualized TNNI3 expression in ventricular cardiomyocytes (TNNI1) at day 16.+ ) beating EBs were differentiated. Images from the microscope were captured using a BZ-X710 and processed with Keyence BZ-X Analyzer software. Color representations are specified in the figure captions.

[0087] The results are shown in Figure 2. Figure 2 shows that dasatinib increases the expression of TNNI3 and decreases the expression of TNNI1 in cardiomyocytes. In other words, treatment of cardiomyocytes with dasatinib led to the maturation of immature fetal cardiomyocytes.

[0088] <Combined treatment of dasatinib and the mTOR kinase inhibitor Torin1 on ectopic cardiac maturation in HiPSC-CM monolayers> Beating hiPSC-CM monolayers differentiated from the on-feeder human iPS cell line 201B7-luc-FUCCI-MYH6-iRFP670 were treated with cTNT on day 16. + Embryoid bodies (EBs) were dissociated and plated onto fibronectin-coated dishes. After dissociation, they were cultured in induction media 3 (IM3; 50 μg / mL ascorbic acid, 2 mM L-glutamine, 150 μg / mL transferrin, 4 x 10 -4 Cells were maintained in StemPro-34 medium supplemented with 100 mM MTG and 5 ng / mL VEGFA. To assess maturation, dasatinib (CST #9052) and Torin1 (Sigma 475991) were prepared freshly and separately in DMSO for each experiment. To monitor the acquisition of cardiac maturation-related markers, hiPSC-CMs were treated with a combination regimen of 1 μM dasatinib and 200 nM Torin1 for 7 days, with medium changes every other day. Maturation was determined by increased expression of adult isoforms of sarcomere-associated proteins and decreased expression of the fetal isoform of cardiac troponin I (TNNI1). Functionally, maturation was confirmed by increased contractile force, improved action potential amplitude (APA), increased rise velocity (Vmax), and resting membrane potential derived from engineered heart tissue (EHT).

[0089] Immunoblotting: hiPSC-CMs were lysed using M-PER Mammalian Protein Extraction Reagent Buffer (78501) from Thermo Scientific. Protein content was measured using a Bradford assay with BSA as the standard. Primary antibodies were commercially available and included anti-phospho-AKT (9271, CST), anti-β-actin (A5441, Sigma), anti-cTnI TNNI3 (ab10231), anti-S100A1 (Invitrogen; PA1-932), anti-CD36 (ab13362), anti-p53 (12790S, D963E, CST), anti-CAV3 (Sigma; AV09021), anti-CDK4 (9282S, CST), and anti-TNNI1 (ab203515), all at a dilution of 1:1000. The secondary antibodies applied were goat anti-rabbit IgG with HRP (sc-2054, Santa Cruz) and anti-mouse IgG with HRP (7076, CST), both diluted 1:5000. To control for endogenous protein loading, we included images of a 0.2 μm nitrocellulose membrane stained with Ponceau S (Sigma, 6226-79-5) after methanol wet transfer.

[0090] The results are shown in Figure 3. Figure 3 shows that treatment with dasatinib alone, Torin1 alone, and the combination of dasatinib and Torin1 (particularly the combination treatment) decreased the protein expression of the fetal cardiomyocyte marker TNNI1, while increasing the protein expression of cardiomyocyte maturation markers (TNNI3, CD36, CAV3, and S100A1). Furthermore, treatment with dasatinib alone, Torin1 alone, and the combination treatment of dasatinib and Torin1 (particularly the combination treatment) decreased the protein expression of CDK4, while increasing the protein expression of p53, indicating a decrease in cardiomyocyte proliferation (i.e., cardiomyocyte maturation). Furthermore, the expression level of β-actin is known to decrease during cardiac maturation (Skwarek-Maruszewska A. et al., J Cell Sci, 122(Pt 12):2119-2126 (2009)). These results suggest that both dasatinib and Torin1 have a maturation effect on cardiomyocytes, but that their combined use enhances this effect.

[0091] Example 3: Principal Component Analysis of Cardiomyocyte Maturation <Creating a Principal Component Analysis Plot from Total RNA Sequencing Reads> The R-based pipline (Lucena-Cacace, A., and Yoshida, Y., Methods Mol Biol 2320, 219-232 (2021)) was used to generate the principal component analysis. Principal component analysis (PCA) of the RNA-seq data was performed using the DESeq2 package. First, the raw count data was normalized and variance was stabilized using the vst function in DESeq2. Following normalization, PCA was calculated using the plotPCA function, which takes the transformed data and visualizes the main sources of variance within the dataset. The hiPSC-CM monolayers used in Example 2 were used in this example.

[0092] The resulting PCA plot is shown in Figure 4. Sample clustering was examined based on the top principal components of the single-treatment (dasatinib, Torin1) and combination (combo) samples. Figure 4 shows the main patterns of gene expression variation between samples, demonstrating that dasatinib treatment increases cardiomyocyte maturation, and that this effect is further enhanced by the combination of Torin1.

[0093] Example 4: Preparation of cardiac tissue and evaluation of its function <Preparation of artificial cardiac tissue (EHT)> Beating EBs (60-80% cTNT) on day 16 differentiated from the on-feeder human iPS cell line 201B7-luc-FUCCI-MYH6-iRFP670 were used. + ) were dissociated overnight with collagenase 1. The next day, single-cell suspensions were prepared by a second enzymatic digestion using Accumax at 37°C for 15 minutes. After dissociation, single cells were resuspended in DMEM supplemented with 1% P / S (penicillin / streptomycin) and 10% fetal bovine serum (FBS). EHTs were generated using an EHT silicone rack (DiNAQOR Deutschland GmbH) and a device (Sumitomo Bakelite Co., Ltd.) in a 24-well plate (Breckwoldt, K. et al., Nat Protoc 12, 1177-1197 (2017)). 1x10 6 The cells were mixed with EHT master mix (NCM medium, fibrinogen, Matrigel basement membrane matrix, 2x DMEM, and Y-27632). One EHT was generated by mixing 97 μl of the master mix with 3 μl of thrombin (100 U / ml) and pipetting it into a device placed in an EHT silicone rack at 37°C. After 2 hours of incubation, the EHT silicone rack with the attached EHT was transferred to a 24-well plate containing EHT medium (DMEM, heat-inactivated bovine serum, penicillin / streptomycin, aprotinin, and insulin). Two days after EHT generation, the combined treatment for ectopic maturation of EHT was performed.

[0094] <Measurement of EHT contractile force> Each EHT was formed with two pillars at both ends. The movement of the artificial heart tissue and pillars during contraction was recorded on video, and the contractile force of the EHT (unit: Newton) was calculated based on the movement of the pillars.

[0095] Cardiomyocytes were seeded on a cover glass and action potentials were measured using the patch clamp technique. A glass electrode was placed close to the beating cardiomyocyte, forming a gigaseal between the glass electrode and the cell membrane, and the intracellular potential was measured.

[0096] The results are shown in Figures 5 and 6. Figure 5 shows that combined treatment with dasatinib and Torin1 enhanced EHT contractile force. Figure 6 shows significant improvements in action potential amplitude and rise rate, but no significant differences in resting membrane potential. From the perspectives of contractile force and electrophysiology, Torin1 treatment and combined treatment with dasatinib and Torin1 led to EHT maturation.

[0097] The present invention provides a novel method for producing mature cardiomyocytes from immature cardiomyocytes. The cardiomyocytes produced by this method are similar to adult cardiomyocytes and can be used in adult-level two-dimensional and three-dimensional cardiac cell models, drug discovery screening, regenerative medicine, and the like.

[0098] This application is based on patent application No. 2024-017532 filed in Japan (filing date: February 7, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing mature cardiomyocytes, comprising culturing cardiomyocytes in a medium containing an Src inhibitor.

2. The method of claim 1, wherein at least one of the Src inhibitors is dasatinib.

3. The method according to claim 1 or 2, wherein the culture medium contains an mTOR inhibitor.

4. The method of claim 3, wherein at least one of the mTOR inhibitors is Torin1.

5. The method according to any one of claims 1 to 4, wherein the mature cardiomyocytes are cells that express TNNI3 and S100A1.

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

7. The method according to any one of claims 1 to 6, wherein the cardiomyocytes are contained in cardiac organoids.

8. A mature cardiomyocyte obtained by the method according to any one of claims 1 to 7, or a cardiac organoid comprising said cell.

9. A transplantation therapy comprising the mature cardiomyocytes or cardiac organoids described in claim 8.

10. A method for maturing cardiomyocytes, comprising culturing cardiomyocytes in a medium containing an Src inhibitor.

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