Method for maturing cardiomyocytes, and method for producing matured cardiomyocytes

Abstract

A method for producing matured cardiomyocytes, the method comprising: a preparation step for preparing a sample containing immature cardiomyocytes; and a pressure loading step for continuously or intermittently applying hydrostatic pressure to the sample.

Description

Method for maturing cardiomyocytes and method for producing mature cardiomyocytes

[0001] The present invention relates to a method for maturing cardiomyocytes and a method for producing mature cardiomyocytes.

[0002] Because adult cardiomyocytes hardly proliferate, cardiomyocyte loss due to ischemic heart disease or other conditions results in irreversible damage. Currently, no clinically used drug treatments have demonstrated efficacy in replacing myocardial scars with functional contractile tissue. Therefore, novel treatments for regenerating normal cardiomyocytes are desired, and replacement therapy or transplantation therapy has been proposed, in which cardiomyocytes separately produced through differentiation induction from pluripotent stem cells (e.g., human iPS cells, etc.) are administered.

[0003] For example, JP 2016-536975 A (Patent Document 1) discloses a method for producing bioengineered cardiac muscle (BHM) from pluripotent stem cells, which generally includes steps of inducing mesodermal differentiation, cardiac differentiation, and cardiac maturation through directed tissue formation.

[0004] Furthermore, International Publication No. 2017 / 073794 (Patent Document 2) discloses a method for producing myocardial tissue, comprising: (a) a step of producing cardiomyocytes from pluripotent stem cells; (b) a step of producing endothelial cells from pluripotent stem cells; (c) a step of producing mural cells from pluripotent stem cells; (d) a step of mixing the mural cells produced in step (c) with the cardiomyocytes produced in step (a) and the endothelial cells produced in step (b) at a ratio of less than 30%; and (e) a step of culturing the cell mixture obtained in step (d) in the presence of an extracellular matrix to form a three-dimensional structure.

[0005] JP 2016-536975 A, International Publication No. 2017 / 073794

[0006] Methods Mol. Biol. 2021;2320:81-88.

[0007] Cardiomyocytes (CM) differentiated from human pluripotent stem cells (hPSCs) (hereinafter sometimes referred to as "hPSC-CM") share many similarities with early human CM (immature human CM during the embryonic or fetal stage). However, the hPSC-CM differs functionally and structurally from mature human CM. For clinical applications (e.g., replacement therapy, transplantation, etc.), hPSC-CM must be functionally and structurally identical or similar to mature human CM.

[0008] Various methods have been proposed to generate mature human CM, but their effectiveness in restoring myocardial function has been limited.

[0009] The present invention has been made in view of the above circumstances, and aims to provide a method for efficiently maturing cardiomyocytes in vitro and a method for producing mature cardiomyocytes in vitro.

[0010] As a result of intensive research, the present inventors have found that applying a predetermined hydrostatic pressure to a sample containing immature cardiomyocytes leads to efficient maturation of the cardiomyocytes, and have completed the present invention.

[0011] [1] A method for maturing cardiomyocytes, comprising: a preparation step of preparing a sample containing immature cardiomyocytes; and a pressure loading step of continuously or intermittently loading hydrostatic pressure onto the sample. [2] The method for maturing cardiomyocytes according to [1], wherein the hydrostatic pressure loaded in the pressure loading step is 10 kPa or more and 100 kPa or less. [3] The method for maturing cardiomyocytes according to [1] or [2], wherein the hydrostatic pressure is loaded intermittently at a frequency of 1 hour or more and 12 hours or less per day in the pressure loading step. [4] The method for maturing cardiomyocytes according to any of [1] to [3], wherein the pressure loading step is performed for a period of 3 days or more and 8 days or less. [5] The method for maturing cardiomyocytes according to any of [1] to [4], wherein the sample further contains vascular wall cells. [6] The method for maturing cardiomyocytes according to any of [1] to [5], wherein the sample further contains vascular endothelial cells. [7] The method for maturing cardiomyocytes according to any one of [1] to [6], wherein the sample is an artificial cardiac tissue derived from iPS cells. [8] An artificial cardiac tissue comprising cardiomyocytes, wherein the cardiomyocytes comprise TNNI3-positive cells.

[0012] [9] A method for producing mature cardiomyocytes, comprising: a preparation step of preparing a sample containing immature cardiomyocytes; and a pressure loading step of continuously or intermittently loading hydrostatic pressure on the sample.

[10] The method for producing mature cardiomyocytes according to [9], wherein the hydrostatic pressure in the pressure loading step is 10 kPa or more and 100 kPa or less.

[11] The method for producing mature cardiomyocytes according to [9] or

[10] , wherein the hydrostatic pressure is loaded intermittently at a frequency of 1 hour or more and 12 hours or less per day in the pressure loading step.

[12] The method for producing mature cardiomyocytes according to any of [9] to

[11] , wherein the pressure loading step is carried out for a period of 3 days or more and 8 days or less.

[13] The method for producing mature cardiomyocytes according to any of [9] to

[12] , wherein the sample further contains vascular wall cells.

[14] The method for producing mature cardiomyocytes according to any of [9] to

[13] , wherein the sample further contains vascular endothelial cells.

[15] A method for producing mature cardiomyocytes described in any of [9] to

[14] , wherein the sample is an artificial cardiac tissue derived from iPS cells.

[0013] According to the present invention, it is possible to provide a method for efficiently maturing cardiomyocytes in vitro and a method for producing mature cardiomyocytes in vitro.

[0014] FIG. 1 is a graph (A) showing the cellular composition of artificial cardiac tissue (ECT) and a photograph (B) of the ECT. FIG. 2 is a graph showing the "cycle" when hydrostatic pressure is applied. FIG. 3 is a schematic diagram showing the schedule when hydrostatic pressure is applied. FIG. 4 is a photograph showing histological staining of the ECT. FIG. 5 is a photograph showing histological staining of the ECT. FIG. 6 is a graph showing the expression level of genes in the ECT. FIG. 7A is a photograph showing histological staining of the ECT. FIG. 7BC is a graph showing the cell number, cTnT positive ratio, and thickness of the ECT. FIG. 7DE is a graph showing the FFR of the ECT. FIG. 7FG is a graph showing the expression level of genes in the ECT. FIG. 7H is a photograph showing the results of fluorescence analysis of the ECT. FIG. 8AB is a graph showing the FFR of the ECT. FIG. 8CEF is a graph showing the evaluation results of calcium transients in the ECT. Figure 9AB is a graph showing the results of mitochondrial function (maximum oxygen consumption rate) in ECT. Figures 9C, 9D, and 9E are photographs of mitochondria in ECT (C), a graph showing gene expression levels in ECT (D), and a photograph showing mitochondrial DNA levels (E). Figures 9FG and 9F are graphs showing TOMM20 expression levels in ECT (F), and graphs showing cell size, cell perimeter, and the ratio of cell perimeter to cell area (G). Figure 10AB is an image showing the results of single-cell RNA-seq in ECT. Figure 10CD is a graph showing the results of single-cell RNA-seq in ECT. Figure 11ABC is a graph showing the cellular composition of ECT (A), a photograph of ECT (B), and a graph showing the pulsation rate and thickness of ECT (C). Figure 11DEF is a graph showing FFR in ECT (D), a graph showing gene expression levels (E), and a photograph showing ECT tissue staining (F). Fig. 12 is an image showing the results of confocal imaging of ECT. Fig. 13 is an image showing the results of confocal imaging of ECT. Fig. 14 is a graph showing FFR of ECT. Fig. 15 is a graph showing the results of echocardiography. The vertical axis shows ejection fraction (EF) or fractional shortening (FS), and the horizontal axis shows the sample name.

[0015] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0016] <<Method for maturing cardiomyocytes>> The method for maturing cardiomyocytes according to this embodiment includes a preparation step of preparing a sample containing immature cardiomyocytes, and a pressure application step of continuously or intermittently applying hydrostatic pressure to the sample.

[0017] <Preparation Step> In this step, a sample containing immature cardiomyocytes is prepared. In this embodiment, "cardiomyocytes" refers to muscle cells that constitute cardiac muscle and express at least cardiac troponin (cTnT) or αMHC. Examples of cTnT include NCBI accession number NM_000364 for humans and NM_001130174 for mice. Examples of αMHC include NCBI accession number NM_002471 for humans and NM_001164171 for mice. "Immature cardiomyocytes" refer to TNNI1-positive cells that have just differentiated from pluripotent stem cells into cardiomyocytes. Other examples of markers for immature cardiomyocytes include MYH6.

[0018] In this embodiment, the sample is not particularly limited as long as it contains immature cardiomyocytes, but may be, for example, in the form of a cell suspension, a cell aggregate, or a three-dimensional structure. Examples of three-dimensional structures include artificial cardiac tissues, which will be described later. The shape of the three-dimensional structure is not particularly limited, and examples include dumbbell-like, band-like, string-like, sheet-like, doughnut-like, spherical, columnar, and cylindrical shapes.

[0019] (Cardiomyocytes) Immature cardiomyocytes can be produced, for example, by inducing differentiation from pluripotent stem cells. Examples of the pluripotent stem cells include the following cells.

[0020] (A) Embryonic stem cells Embryonic stem cells (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.

[0021] ES cells are embryonic stem cells derived from the inner cell mass of a blastocyst, an embryo at the eight-cell stage of a fertilized egg, or after the morula stage. They have the ability to differentiate into any cell that makes up an adult, known as pluripotency, and the ability to proliferate through self-replication. 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).

[0022] 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. Furthermore, maintenance of the cells by subculture can be carried out using a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). 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; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222:273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485, etc.

[0023] As a culture medium for producing ES cells, for example, DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 20% KSR, and 4 ng / ml bFGF is used, and the medium is cultured at 37°C, 5% CO ２ Human ES cells can be maintained in a humid atmosphere (H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932). ES cells must be passaged every 3 to 4 days, and passage is performed in a medium containing, for example, 1 mM CaCl. ２and 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 20% ​​KSR.

[0024] ES cells can generally be selected by real-time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog, etc. In particular, the expression of gene markers such as OCT-3 / 4, NANOG, and ECAD can be used as an index for the selection of human ES cells (E. Kroon et al. (2008), Nat. Biotechnol., 26:443-452).

[0025] Human ES cell lines, such as WA01 (H1) and WA09 (H9), are available from the WiCell Research Institute, and KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

[0026] (B) Spermatogonial stem cells Spermatogonial stem cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Like ES cells, these cells can be induced to differentiate into cells of various lineages. For example, when transplanted into mouse blastocysts, they can produce chimeric mice (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012). They are capable of self-renewal in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and spermatogonial stem cells can be obtained by repeated passage under the same culture conditions as ES cells (Takebayashi, Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 41-46, Yodosha Publishing, Tokyo, Japan).

[0027] (C) Embryonic Germ Cells Embryonic germ cells are cells established from primordial germ cells during the fetal stage and have pluripotency similar to that of ES cells. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Y. Matsui et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551).

[0028] (D) Induced Pluripotent Stem Cells Induced pluripotent stem (iPS) cells are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA, RNA, or protein. They have properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol.26:101-106 (2008); International Publication WO 2007 / 069666). The reprogramming factor may be composed of a gene that is specifically expressed in ES cells, its gene product or non-coding RNA, or a gene that plays an important role in maintaining the undifferentiated state of ES cells, its gene product or non-coding RNA, or a low-molecular-weight compound. Examples of genes contained in the reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1, and these reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, W O2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO 2010 / 111409, WO 2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al.(2008), Stem Cells. 26:2467-2474、HuangfuD, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell StemCell, 3, 568-574、Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479、Marson A, (2008), Cell Stem Cell, 3, 132-135、Feng B, et al. (2009), Nat Cell Biol. 11:197-203、RL Judson et al., (2009), Nat. Biotech., 27:459-461、Lyssiotis CA, et al.(2009), Proc Natl Acad Sci U S A. 106:8912-8917、Kim JB, et al. (2009), Nature. 461:649-643、Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503、Heng JC, et al. (2010), Cell Stem Cell. 6:167-74、Han J, et al. (2010), Nature. 463:1096-100、Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9.

[0029] Examples of the reprogramming factors include histone deacetylase (HDAC) inhibitors [e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, and M344, and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool® (Millipore), HuSH 29mershRNA Constructs against HDAC1 (OriGene))], MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), and glycogen inhibitors. synthase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., small molecule inhibitors such as BIX-01294, nucleic acid expression inhibitors such as siRNA and shRNA against Suv39hl, Suv39h2, SetDBl, and G9a), L-channel calcium agonists (e.g., Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (e.g., LY364947, SB431542, 616453, and A-83-01), p53 inhibitors (e.g., siRNA and shRNA against p53), ARID3A inhibitors (e.g., siRNA and shRNA against ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295, and miR-302, Wnt Signaling inhibitors (e.g., soluble These factors also include factors used for the purpose of improving establishment efficiency, such as Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISI, PITX2, and DMRTB1, and in this specification, factors used for the purpose of improving establishment efficiency will not be distinguished from reprogramming factors.

[0030] When the reprogramming factor is in the form of a protein, it may be introduced into somatic cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (for example, HIV-derived TAT and polyarginine), or microinjection.

[0031] On the other hand, when the reprogramming factor is in the form of DNA, for example, it can be introduced into somatic cells by techniques such as vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, microinjection, etc. Viral vectors include retroviral vectors, lentiviral vectors (Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007), adenoviral vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010 / 008054), etc. Artificial chromosome vectors include, for example, human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC), etc. The plasmid may be a mammalian cell plasmid (Science, 322:949-953, 2008). The vector may contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, and polyadenylation site to enable expression of the nuclear reprogramming substance. Furthermore, the vector may contain, as needed, a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a selection marker sequence such as a thymidine kinase gene or a diphtheria toxin gene, or a reporter gene sequence such as green fluorescent protein (GFP), β-glucuronidase (GUS), or FLAG. Furthermore, the above vector may have LoxP sequences before and after the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to it, in order to excise both the promoter and the gene after introduction into somatic cells.

[0032] Furthermore, when the reprogramming factor is in the form of RNA, it may be introduced into somatic cells by techniques such as lipofection or microinjection. To suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may also be used (Warren L, (2010) Cell Stem Cell. 7:618-630).

[0033] Examples of culture media for inducing iPS cells include DMEM, DMEM / F12, or DME culture media containing 10 to 15% FBS (these culture media may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc., as appropriate), or commercially available culture media [for example, a culture medium for culturing mouse ES cells (TX-WES culture medium, Thrombo-X), a culture medium for culturing primate ES cells (culture medium for primate ES / iPS cells, ReproCell), a serum-free medium (mTeSR, Stemcell Technology)], and the like.

[0034] Examples of the culture method include the following: First, the cells are cultured at 37°C and 5% CO ２ In the presence of bFGF, somatic cells are contacted with the reprogramming factors in a 10% FBS-containing DMEM or DMEM / F12 culture medium and cultured for about 4 to 7 days. The cultured somatic cells are then plated on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.), and starting about 10 days after contacting the somatic cells with the reprogramming factors, they are cultured in a bFGF-containing culture medium for primate ES cell culture. iPS-like colonies can be generated about 30 to about 45 days or more after the contact.

[0035] Alternatively, 37°C, 5% CO ２In the presence of ES cells, the cells are cultured on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) in a 10% FBS-containing DMEM culture medium (which may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) to produce ES-like colonies after about 25 to about 30 days or more. Desirably, somatic cells to be reprogrammed themselves are used instead of feeder cells (Takahashi K, et al. (2009), PLoS One. 4:e8067 or WO2010 / 137746), or extracellular matrix (e.g., Laminin-5 (WO2009 / 123349) and Matrigel (BD)) is used.

[0036] Other examples include culturing iPS cells using serum-free media (Sun N, et al. (2009), Proc Natl Acad Sci U S A. 106:15720-15725). Furthermore, to increase establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5:237-241 or WO2010 / 013845).

[0037] During the culture, the culture medium is replaced with fresh medium once a day from the second day onward. The number of somatic cells used for nuclear reprogramming is not limited, but may be increased to 100 cells per 100 cm culture dish. ２ Approximately 5 x 10 ３ ~Approx. 5×10 ６ It is the range of cells.

[0038] iPS cells can be selected based on the shape of the colonies they form. On the other hand, if a drug resistance gene that is expressed in conjunction with a gene (e.g., Oct3 / 4, Nanog) that is expressed when somatic cells are reprogrammed is introduced as a marker gene, the established iPS cells can be selected by culturing them in a culture medium (selective culture medium) containing the corresponding drug. Furthermore, if the marker gene is a fluorescent protein gene, iPS cells can be selected by observing them under a fluorescent microscope; if the marker gene is a luciferase gene, by adding a luminescent substrate; or if the marker gene is a chromogenic enzyme gene, by adding a chromogenic substrate.

[0039] The term "somatic cells" as used herein refers to any animal cell (preferably a mammalian cell, including a human cell) excluding germline cells such as eggs, oocytes, and ES 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, as well as primary culture cells, passaged cells, and established cell lines. In one aspect of this embodiment, the somatic cells are preferably mature, healthy somatic cells (somatic cells derived from a healthy individual). Specifically, examples of somatic cells include (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 (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0040] Furthermore, when iPS cells are used as a source of transplantation cells, it is desirable to use 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 matches the transplanted cells to an extent that an immune response can be suppressed with an immunosuppressant, for example, somatic cells with an HLA type that matches the three gene loci of HLA-A, HLA-B, and HLA-DR, or four gene loci including HLA-C.

[0041] (E) ES Cells Derived from Cloned Embryos Obtained by Nuclear Transfer ES cells derived from cloned embryos obtained by nuclear transfer (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 (T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502). That is, ES cells 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 are nt ES (nuclear transfer ES) cells. To generate nt ES cells, nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16:642-646) is combined with ES cell generation technology (Syoka Wakayama 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.

[0042] (F) Multilineage-differentiating Stress Enduring cells Multilineage-differentiating Stress Enduring cells (Muse cells) are pluripotent stem cells produced by the method described in WO2011 / 007900. Specifically, Muse cells are pluripotent cells obtained by trypsinizing fibroblasts or bone marrow stromal cells for a long period of time, preferably 8 or 16 hours, followed by suspension culture, and are SSEA-3 and CD105 positive cells.

[0043] In this embodiment, the preferred pluripotent stem cells are human iPS cells. In one aspect of this embodiment, the human iPS cells may be human iPS cells derived from mature, healthy somatic cells (human iPS cells derived from a healthy individual), or may be human iPS cells derived from diseased somatic cells (disease-specific iPS cells). In another aspect of this embodiment, the human iPS cells are preferably human iPS cells derived from a healthy individual.

[0044] The method for inducing cardiomyocytes from pluripotent stem cells is not particularly limited as long as it is a known method, and examples thereof include (1) a method performed in the absence of feeder cells and (2) a method performed in the presence of feeder cells. In this embodiment, (1) the method for inducing cardiomyocytes from pluripotent stem cells in the absence of feeder cells is exemplified by a method comprising the steps of (i) culturing induced pluripotent stem cells in a medium containing Activin A, and (ii) further culturing the induced pluripotent stem cells in a medium containing BMP4 and bFGF after step (i).

[0045] (1) Method for inducing cardiomyocytes from pluripotent stem cells in the absence of feeder cells (i) Step of culturing in a medium containing Activin A In this step, pluripotent stem cells are isolated by any method and may be cultured in suspension culture or in adherent culture using a coated culture dish. Adherent culture is preferred. Here, the isolation method may be a mechanical isolation method or a isolation solution having protease activity and collagenase activity (e.g., Accutase ＴＭ and Accumax ＴＭ Alternatively, the method may involve dissociating the pluripotent stem cells using a separation solution containing only collagenase activity, and then mechanically separating the pluripotent stem cells into small pieces. The pluripotent stem cells used herein are preferably colonies cultured to about 80% confluence in the dish used.

[0046] Suspension culture is the cultivation of cells in a non-adherent state to a culture dish. The suspension culture can be performed using, but is not limited to, a culture dish that has not been artificially treated to improve adhesion to the cells (e.g., coated with an extracellular matrix, etc.), or a culture dish that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA)).

[0047] Adherent culture is a culture method performed in a coated culture dish in a medium of choice. Examples of coating agents include Matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, or entactin, and combinations thereof. Preferably, the coating agent is Matrigel. More preferably, adherent culture is performed using the Matrigel sandwich method, in which induced pluripotent stem cells are attached to a Matrigel-coated culture dish, and Matrigel is then added to the medium to coat the entire pluripotent stem cells with Matrigel.

[0048] The medium in step (i) can be prepared by adding Activin A to a medium used for culturing animal cells as a basal medium.

[0049] Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM, Doulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. RPMI 1640 medium is preferred. The basal medium may contain serum or may be serum-free. If necessary, the basal medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS used in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The basal medium may also contain one or more substances, such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. An example of a preferred basal medium for step (i) is RPMI medium containing L-glutamine and B27 supplement.

[0050] In addition to Activin A, the medium used in step (i) may further contain one or more growth factors selected from the group consisting of Wnt1, Wnt3, Wnt3a, Wnt4, Wnt7a, TGF-β, Nodal, BMP2, BMP4, BMP6, BMP7, GDF, bFGF, and VEGF. A preferred growth factor is Wnt3a.

[0051] The concentration of Activin A added to the medium may be, for example, but not limited to, 10 ng / mL, 25 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 175 ng / mL, or 200 ng / mL. Preferably, the concentration of Activin A added to the medium is 100 ng / mL. In one aspect of this embodiment, the concentration of Activin A added to the medium may be 10 ng / mL or more and 200 ng / mL or less.

[0052] The concentration of Wnt3a added to the medium may be, but is not limited to, 10 ng / mL, 25 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 175 ng / mL, or 200 ng / mL. Preferably, the concentration of Wnt3a added to the medium is 100 ng / mL. In one aspect of this embodiment, the concentration of Wnt3a added to the medium may be 10 ng / mL or more and 200 ng / mL or less.

[0053] The culture temperature is, but is not limited to, about 30 to 40°C, preferably about 37°C. ２ The culture is carried out under an atmosphere containing CO ２ The concentration is preferably about 2 to 5%. The culture time is, for example, 1 to 5 days, preferably 1 day.

[0054] (ii) Culturing in a medium containing BMP and bFGF: In this step (ii), if the previous step was performed using suspension culture, the resulting cell population may be cultured directly on a coated culture dish in any medium. Examples of coating agents include Matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof. Matrigel is preferred. Alternatively, if the previous step was performed using adherent culture, the culture may be continued by replacing the medium.

[0055] The medium used in step (ii) can be prepared by adding BMP and bFGF to a medium used for culturing animal cells as a basal medium. The basal medium can be the same as that used in step (i) above.

[0056] The BMP used in step (ii) is preferably a BMP belonging to the TGFβ superfamily, such as BMP 2, BMP 4, and BMP 7. A preferred BMP is BMP 4.

[0057] The concentration of BMP4 added to the culture medium may be, but is not limited to, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 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, 17.5 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, or 50 ng / mL. Preferably, the concentration of BMP4 added to the culture medium is 10 ng / mL. In one aspect of this embodiment, the concentration of BMP4 added to the culture medium may be 0.1 ng / mL or more and 50 ng / mL or less.

[0058] The concentration of bFGF added to the medium may be, but is not limited to, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 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, 17.5 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, or 50 ng / mL. Preferably, the concentration of bFGF added to the medium is 10 ng / mL. In one aspect of this embodiment, the concentration of bFGF added to the medium may be 0.1 ng / mL or more and 50 ng / mL or less.

[0059] The culture temperature is, but is not limited to, about 30 to 40°C, preferably about 37°C. ２ The culture is carried out under an atmosphere containing CO ２ The concentration is preferably about 2 to 5%. The culture time is, for example, 1 to 10 days, preferably 4 days.

[0060] (2) Method in the Presence of Feeder Cells In this embodiment, 2) the method of inducing cardiomyocytes from pluripotent stem cells in the presence of feeder cells is exemplified by a method of co-culturing OP9 cells (Nishikawa, S. I. et al., Development 125, 1747-1757 (1998)) or END-2 cells (Mummery C. et al., Circulation. 107:2733-40 (2003)) with pluripotent stem cells or Flk1-positive cells derived from pluripotent stem cells.

[0061] The medium for co-culture with feeder cells can be prepared by adding appropriate additives to a medium used for culturing animal cells as a basal medium.

[0062] Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM, Doulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. RPMI 1640 medium is preferred. The basal medium may contain serum or may be serum-free. If necessary, the basal medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. The basal medium may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. Examples of preferred basal media include αMEM medium containing 10% FBS, αMEM medium containing 10% FBS, and DMEM medium containing 10% FBS.

[0063] Examples of additives to the basal medium in co-culture with feeder cells include 1 to 3 μg / mL of cyclosporin A, activin A, and BMP4.

[0064] The culture temperature is, but is not limited to, about 30 to 40°C, preferably about 37°C. ２ The culture is carried out under an atmosphere containing CO ２ The concentration is preferably about 2 to 5%. The culture time is the number of days required for cardiac troponin and / or αMHC to be expressed, for example, 10 to 20 days.

[0065] Preferred conditions include culturing Flk1-positive cells in αMEM medium containing 10% FBS for 4 days, isolating the cells, and co-culturing them with OP9 cells for 6 days using αMEM medium containing 3 μg / mL cyclosporine A and 10% FBS, or co-culturing them with END-2 cells for 16 days using DMEM medium containing 10% FBS.

[0066] For use in producing the artificial cardiac tissue of this embodiment, the obtained cardiomyocytes may be cultured in a basal medium further supplemented with VEGF, and if necessary, a Wnt signaling pathway inhibitor (e.g., XAV939, IWP4, etc.) may be added to the basal medium.

[0067] Examples of basal media to which VEGF is added include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM, Doulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. RPMI 1640 medium is preferred. The basal medium may contain serum or may be serum-free. If necessary, the basal medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS used in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. The basal medium may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. An example of a preferred basal medium for this step is RPMI medium containing L-glutamine and B27 supplement.

[0068] The concentration of VEGF added to the medium can be, for example, within the range of 10 ng / mL to 500 ng / mL, 25 ng / mL to 300 ng / mL, 40 ng / mL to 200 ng / mL, 50 ng / mL to 100 ng / mL, 60 ng / mL to 90 ng / mL, or 65 ng / mL to 85 ng / mL. Preferably, the concentration of VEGF added to the medium is 25 ng / mL to 75 ng / mL. The concentration of VEGF added to the medium may be, but is not limited to, 10 ng / mL, 25 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, or 200 ng / mL. Preferably, the concentration of VEGF added to the medium is 50 ng / mL.

[0069] The culture temperature is, but is not limited to, about 30 to 40°C, preferably about 37°C. ２ The culture is carried out under an atmosphere containing CO ２ The concentration is preferably about 2 to 5%. The culture time is, for example, 4 to 20 days, preferably 10 days.

[0070] In this embodiment, the content of the immature cardiomyocytes may be 10% to 80% or 20% to 70% of the total cells contained in the sample. The number of the immature cardiomyocytes can be counted by flow cytometry using TNNI1 as a marker molecule, for example.

[0071] (Vascular Wall Cells) In this embodiment, it is preferable that the sample further contains vascular wall cells. "Vascular wall cells" (hereinafter sometimes simply referred to as "wall cells") refer to cells expressing smooth muscle actin (SMA) and / or PDGFRB. Examples of SMA include NCBI accession number NM_001141945 for humans and NM_007392 for mice. Examples of PDGFRB include NCBI accession number NM_002609 for humans and NM_001146268 for mice.

[0072] The method for inducing mural cells from pluripotent stem cells is not particularly limited as long as it is a known method, and examples thereof include a method comprising the steps of: (I) culturing pluripotent stem cells in a medium containing Activin A; (II) culturing the cells obtained in step (I) in a medium containing BMP and bFGF; and (III) culturing the cells obtained in step (II) in a medium not containing VEGF.

[0073] (I) Step of culturing in a medium containing Activin A In this step, pluripotent stem cells may be isolated by any method and cultured in suspension culture, or in adhesion culture using a coated culture dish. Adhesion culture is preferred. Here, the isolation method may be a mechanical isolation method or a isolation solution having protease activity and collagenase activity (e.g., Accutase ＴＭ and Accumax ＴＭ Alternatively, the method may involve dissociating the pluripotent stem cells using a separation solution containing only collagenase activity, or may involve separating the pluripotent stem cells using a separation solution containing only collagenase activity, followed by mechanically separating the pluripotent stem cells into finer particles. The pluripotent stem cells used herein are preferably colonies cultured to 80% confluence in the dish used.

[0074] Suspension culture is the cultivation of cells in a non-adherent state to a culture dish. The suspension culture can be performed using, but is not limited to, a culture dish that has not been artificially treated to improve adhesion to the cells (e.g., coated with an extracellular matrix, etc.), or a culture dish that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA)).

[0075] Adherent culture is a culture method performed in a coated culture dish in a medium of choice. Examples of coating agents include Matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, or entactin, and combinations thereof. Preferably, the coating agent is Matrigel. More preferably, the adherent culture is performed using the Matrigel sandwich method, in which induced pluripotent stem cells are adhered to a Matrigel-coated culture dish, and Matrigel is then added to the medium to coat the entire pluripotent stem cells with Matrigel.

[0076] The medium in step (I) can be prepared by adding Activin A to a basal medium used for culturing animal cells.

[0077] Examples of basal media include IMDM medium, 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. RPMI 1640 medium is preferred. The basal medium may contain serum or may be serum-free. If necessary, the basal medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS used in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The basal medium may also contain one or more substances, such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. An example of a preferred basal medium for step (I) is RPMI medium containing L-glutamine and B27 supplement.

[0078] In addition to Activin A, the medium used in step (I) may further contain one or more growth factors selected from the group consisting of Wnt1, Wnt3, Wnt3a, Wnt4, Wnt7a, TGF-β, Nodal, BMP2, BMP4, BMP6, BMP7, GDF, bFGF, and VEGF. A preferred growth factor is Wnt3a.

[0079] The concentration of Activin A added to the medium may be, for example, but not limited to, 10 ng / mL, 25 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 175 ng / mL, or 200 ng / mL. Preferably, the concentration of Activin A added to the medium is 100 ng / mL. In one aspect of this embodiment, the concentration of Activin A added to the medium may be 10 ng / mL or more and 200 ng / mL or less.

[0080] The concentration of Wnt3a added to the medium may be, but is not limited to, 10 ng / mL, 25 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 175 ng / mL, or 200 ng / mL. Preferably, the concentration of Wnt3a added to the medium is 100 ng / mL. In one aspect of this embodiment, the concentration of Wnt3a added to the medium may be 10 ng / mL or more and 200 ng / mL or less.

[0081] The culture temperature is, but is not limited to, about 30 to 40°C, preferably about 37°C. ２ The culture is carried out under an atmosphere containing CO ２ The concentration is preferably about 2 to 5%. The culture time is, for example, 1 to 5 days, preferably 1 day.

[0082] (II) Step of culturing in a medium containing BMP and bFGF: In this step (II), if the previous step was performed using suspension culture, the resulting cell population may be cultured directly on a coated culture dish in any medium. Examples of coating agents include Matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof. Preferably, the coating agent is Matrigel. Alternatively, if the previous step was performed using adherent culture, the culture may be continued by changing the medium.

[0083] The medium used in step (II) can be prepared by adding BMP and bFGF to a basal medium used for culturing animal cells. The basal medium can be the same as that used in step (I) above.

[0084] The BMP used in this step (II) is preferably a BMP belonging to the TGFβ superfamily, such as BMP 2, BMP 4, and BMP 7. A preferred BMP is BMP 4.

[0085] The concentration of BMP4 added to the culture medium may be, but is not limited to, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 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, 17.5 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, or 50 ng / mL. Preferably, the concentration of BMP4 added to the culture medium is 10 ng / mL. In one aspect of this embodiment, the concentration of BMP4 added to the culture medium may be 0.1 ng / mL or more and 50 ng / mL or less.

[0086] The concentration of bFGF added to the medium may be, but is not limited to, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 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, 17.5 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, or 50 ng / mL. Preferably, the concentration of bFGF added to the medium is 10 ng / mL. In one aspect of this embodiment, the concentration of bFGF added to the medium may be 0.1 ng / mL or more and 50 ng / mL or less.

[0087] The culture temperature is, but is not limited to, about 30 to 40°C, preferably about 37°C. ２ The culture is carried out under an atmosphere containing CO ２ The concentration is preferably about 2 to 5%. The culture time is, for example, 1 to 10 days, preferably 2 days.

[0088] (III) Step of culturing in a VEGF-free medium: The cells obtained in step (II) are cultured in a basal medium that is a medium used for culturing animal cells that does not contain VEGF. The VEGF-free medium may be a medium that is substantially free of VEGF, for example, a medium having a VEGF concentration of less than 1 ng / mL, preferably less than 0.1 ng / mL, and more preferably 0.

[0089] The basal medium used in step (III) includes, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. RPMI 1640 medium is preferred. The basal medium may contain serum or may be serum-free. If necessary, the basal medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS used in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The basal medium may also contain one or more substances, such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. An example of a preferred basal medium used in step (III) is RPMI medium containing 10% FBS.

[0090] In this embodiment, the content of the vascular wall cells may be 5% to 80% or 10% to 70% of the total cells contained in the sample. The number of the vascular wall cells can be counted by flow cytometry using, for example, SMA or PDGFRB as a marker molecule.

[0091] (Vascular Endothelial Cells) In this embodiment, the sample preferably further contains vascular endothelial cells. The inclusion of vascular endothelial cells promotes the formation of a vascular network when the sample (e.g., ECT) is subjected to hydrostatic pressure. "Vascular endothelial cells" (hereinafter, may be simply referred to as "endothelial cells") refer to cells expressing any one of PE-CAM, VE-cadherin, and von Willebrand factor (vWF). PE-CAM is exemplified by NCBI accession number NM_000442 in the case of humans and NM_001032378 in the case of mice. VE-cadherin is exemplified by NCBI accession number NM_001795 in the case of humans and NM_009868 in the case of mice. Examples of vWF include NCBI accession number NM_000552 for humans and NM_011708 for mice.

[0092] The method for inducing endothelial cells from pluripotent stem cells is not particularly limited as long as it is a known method, and an example thereof includes the steps of: (a) culturing pluripotent stem cells in a medium containing Activin A and Wnt3a; (b) culturing the cells obtained in step (a) in a medium containing BMP and bFGF; and (c) culturing the cells obtained in step (b) in a medium containing VEGF.

[0093] Steps (a), (b), and (c) may be performed using a method similar to the method for inducing cardiomyocytes described above. Therefore, in this embodiment, a method for simultaneously inducing cardiomyocytes and endothelial cells, including steps (a), (b), and (c), can be used. Specific procedures include, for example, the method described in the Examples below (CM+EC protocol).

[0094] In this embodiment, cAMP, which induces endothelial cells, may be further added in step (c). The concentration of cAMP is, for example, greater than 0.5 mM and less than 2 mM, such as, but not limited to, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, and 1.9 mM. Preferably, it is 1 mM. The period for adding cAMP is not particularly limited, but is preferably 1 to 5 days, and particularly preferably 3 days.

[0095] In this embodiment, the content of the vascular endothelial cells may be 5% to 80% or 10% to 70% of the total cells contained in the sample. The number of the vascular endothelial cells can be counted by flow cytometry using, for example, PE-CAM, VE-cadherin, or vWF as a marker molecule.

[0096] In this embodiment, the sample is preferably an iPS cell-derived artificial cardiac tissue (ECT). "Artificial cardiac tissue" refers to an artificially produced three-dimensional structure, such as cardiac tissue or a structure similar thereto. "iPS cell-derived artificial cardiac tissue" refers to an artificial cardiac tissue containing at least cardiomyocytes differentiated from iPS cells. In one aspect of this embodiment, "iPS cell-derived artificial cardiac tissue" can also be understood as an artificial cardiac tissue having a genome of the same origin as the iPS cells prior to differentiation induction. Preferably, the artificial cardiac tissue further contains vascular endothelial cells, vascular wall cells, or both. Preferably, the artificial cardiac tissue is a structure that spontaneously repeats contraction and expansion.

[0097] The method for producing the artificial cardiac tissue is not particularly limited, and known methods can be used. For example, the artificial cardiac tissue can be obtained by mixing the cardiomyocytes, endothelial cells, and mural cells described above, culturing them in the presence of an extracellular matrix, and forming a three-dimensional structure. The mural cells are preferably mixed at a content of less than 30%, more preferably in the range of 5% to 25%, even more preferably in the range of 5% to 20%, and most preferably in the range of 10% to 20%.

[0098] In this embodiment, the extracellular matrix refers to a protein secreted outside the cell, and examples of the extracellular matrix include collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, fragments thereof, and combinations thereof. The extracellular matrix used in forming the artificial cardiac tissue is not particularly limited, but is an extracellular matrix containing at least type I collagen, and more preferably Matrigel (available from BD) containing type I collagen.

[0099] Culture media used for forming artificial cardiac tissue include, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. DMEM medium is preferred. The basal medium may contain serum or may be serum-free. If necessary, the basal medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS used in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. The basal medium may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. An example of a preferred medium for use in forming artificial cardiac tissue is DMEM medium containing 20% ​​FBS.

[0100] The artificial cardiac tissue can be formed by mixing the medium in which the above-mentioned mixed cells are suspended with an extracellular matrix, placing the mixture in a container of a desired shape, and allowing it to stand for a certain period of time. To facilitate easy handling of the artificial cardiac tissue, it is desirable that the portion of the container that comes into contact with the cell mixture be covered with a silicone membrane coated with type I collagen. Such containers are available commercially from Flexcell International. The shape of the container is preferably columnar, and more preferably cylindrical, to facilitate easy handling of the formed artificial cardiac tissue.

[0101] The formed artificial cardiac tissue can be stored in any medium, including, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM, Doulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. DMEM medium is preferred. The basal medium may contain serum or may be serum-free. If necessary, the basal medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS used in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. The basal medium may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. An example of a preferred medium for culturing artificial cardiac tissue is αMEM medium containing 10% FBS, 2-mercaptoethanol, and antibiotics.

[0102] <Pressure application step> In this step, hydrostatic pressure is applied to the sample continuously or intermittently. In this embodiment, "hydrostatic pressure" refers to the pressure exerted by still water or an aqueous solution (e.g., a culture medium, etc.). In this embodiment, "continuous application of hydrostatic pressure" refers to a state in which hydrostatic pressure is applied in a cycle described below for a predetermined period of time. "Intermittent application of hydrostatic pressure" refers to a state in which hydrostatic pressure is applied in a cycle described below and a state in which the cycle is stopped are alternately repeated.

[0103] In this embodiment, the method for applying hydrostatic pressure to the sample is not particularly limited as long as the environment is one in which the cells contained in the sample can survive. For example, a method for applying hydrostatic pressure using a MechanoCulture TR stimulator (MCTR, manufactured by CellScale) described in the Examples can be used. Here, the "survivable environment" can be, for example, an environment in which the cells are in contact with a medium used for culturing the cells.

[0104] In this embodiment, the hydrostatic pressure applied in the pressure application step (the hydrostatic pressure applied during the compression period described below) is preferably 10 kPa or more and 100 kPa or less, and more preferably 25 kPa or more and 75 kPa or less.

[0105] In this embodiment, a "cycle" when applying hydrostatic pressure refers to a repetitive unit of compression duration → hold duration → recovery duration → rest duration (see FIG. 2). During the compression duration, the pressure rises from the "pre-load pressure" to the load pressure, and the load pressure is maintained during the hold period. Thereafter, during the recovery period, the load pressure falls from the "pre-load pressure," and the "pre-load pressure" is maintained during the rest period.

[0106] In this embodiment, the manner in which the hydrostatic pressure increases during the compression period is not particularly limited as long as it increases to the load pressure, and may increase linearly or nonlinearly. The compression period is preferably 0.05 seconds or more and 0.5 seconds or less, and more preferably 0.1 seconds or more and 0.3 seconds or less.

[0107] In this embodiment, the hold period is preferably 0.025 seconds or more and 0.25 seconds or less, and more preferably 0.05 seconds or more and 0.15 seconds or less.

[0108] In this embodiment, the manner in which the hydrostatic pressure is reduced during the recovery period is not particularly limited as long as it is reduced to the pressure before loading, and may be reduced linearly or nonlinearly. The recovery period is preferably from 0.05 seconds to 0.5 seconds, and more preferably from 0.1 seconds to 0.3 seconds.

[0109] In this embodiment, the rest period is preferably 0.25 seconds or more and 0.75 seconds or less, and more preferably 0.4 seconds or more and 0.6 seconds or less.

[0110] In this embodiment, the cycle period is preferably 0.25 seconds to 4 seconds, and more preferably 0.5 seconds to 2 seconds. The cycle period can also be understood as the total period of the compression period, hold period, recovery period, and rest period in one cycle.

[0111] In the present embodiment, in the pressure application step, the hydrostatic pressure is preferably applied intermittently for 1 to 12 hours per day, and more preferably for 1 to 6 hours per day. In one aspect of the present embodiment, "applying hydrostatic pressure intermittently for 1 hour per day" can also be understood as maintaining a state in which hydrostatic pressure is applied for 1 hour in the above cycle, and then maintaining a state in which no hydrostatic pressure is applied for 23 hours.

[0112] In this embodiment, the pressure application step is preferably carried out for a period of 3 days or more and 8 days or less, and more preferably for a period of 3 days or more and 5 days or less.

[0113] Previously, hydrostatic pressure loading has been used in studies of tendons and bones, but not cardiac tissue. Furthermore, while electrical stimulation and stretching loading have been used as physical stimuli for cardiac tissue, compressive loading such as hydrostatic pressure has not. The present inventors discovered that applying a predetermined hydrostatic pressure to a sample containing immature cardiac muscle cells leads to efficient maturation of cardiac muscle cells, leading to the completion of this invention. The living heart beats continuously, even during development. During this process, cardiac muscle cells mature while receiving the loading stimuli associated with the pulsation. For this reason, it was speculated that continuous application of artificial hydrostatic pressure would be preferable. However, surprisingly, the present inventors have now discovered for the first time that intermittent application of artificial hydrostatic pressure results in more effective maturation. Furthermore, the present inventors have also discovered for the first time that hydrostatic pressure loading higher than normal blood pressure (10 kPa) promotes maturation.

[0114] In this embodiment, the term "mature cardiomyocytes" refers to TNNI3-positive cells that are formed by maturation of the immature cardiomyocytes after exposure to a hydrostatic pressure load. Other examples of markers for mature cardiomyocytes include MYH7 and NPPA.

[0115] <Method for producing mature cardiomyocytes> In this embodiment, the method for maturing cardiomyocytes can also be understood as a method for producing mature cardiomyocytes. That is, the method for producing mature cardiomyocytes according to this embodiment includes: a preparation step of preparing a sample containing immature cardiomyocytes; and a pressure application step of continuously or intermittently applying hydrostatic pressure to the sample. Details of each step are the same as those described in the section on the method for maturing cardiomyocytes.

[0116] <Artificial Heart Tissue> The artificial heart tissue according to this embodiment is an artificial heart tissue containing cardiomyocytes, and the cardiomyocytes contain TNNI3-positive cells.

[0117] In one aspect of this embodiment, the above-mentioned artificial cardiac tissue can also be understood as an artificial cardiac tissue in which immature cardiomyocytes contained in the precursor of the artificial cardiac tissue are matured by the method for maturing cardiomyocytes according to this embodiment.

[0118] In this embodiment, the content of the cardiomyocytes may be 10% to 80% or 20% to 70% of the total cells contained in the artificial cardiac tissue. The number of the cardiomyocytes can be counted by flow cytometry using cTnT as a marker molecule, for example.

[0119] In one aspect of this embodiment, the content of TNNI3-positive cells may be 5% to 70% or 10% to 50% of the total cells contained in the artificial cardiac tissue. The number of TNNI3-positive cells can be counted, for example, by flow cytometry using TNNI3 as a marker molecule.

[0120] In one aspect of this embodiment, the expression level of TNNI3 in the TNNI3-positive cells is preferably 100% to 1000%, and more preferably 200% to 800%, of the expression level of TNNI3 in immature cardiomyocytes. The expression level of TNNI3 can be estimated by quantitative PCR.

[0121] The artificial cardiac tissue may further contain vascular endothelial cells. By further containing vascular endothelial cells, the artificial cardiac tissue will have excellent mechanical contractility. In this embodiment, the content of the vascular endothelial cells may be 5% or more and 80% or less, or 10% or more and 70% or less, based on the total number of cells contained in the artificial cardiac tissue. The number of the vascular endothelial cells can be counted by flow cytometry using, for example, PE-CAM, VE-cadherin, or vWF as a marker molecule.

[0122] The artificial cardiac tissue may further contain vascular wall cells. By further containing vascular wall cells, the artificial cardiac tissue has excellent tissue strength. In this embodiment, the content of the vascular wall cells may be 5% or more and 80% or less, or 10% or more and 70% or less, based on the total number of cells contained in the artificial cardiac tissue. The number of the vascular wall cells can be counted by flow cytometry, for example, using SMA or PDGFRB as a marker molecule.

[0123] In one aspect of this embodiment, the artificial cardiac tissue may further include an extracellular matrix, specific examples of which are those described above.

[0124] <Therapeutic Agent for Cardiac Disease> This embodiment provides a therapeutic agent for cardiac disease comprising the above-described artificial cardiac tissue. The cardiac disease to which this embodiment is applicable is not particularly limited as long as it is a pathological condition resulting in a deficiency of cardiomyocytes, etc., and examples thereof include heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated phase hypertrophic cardiomyopathy, and dilated cardiomyopathy.

[0125] The artificial cardiac tissue of this embodiment may be used by directly suturing it in the formed shape to the site of myocardial cell deficiency, infarction, injury or disorder, or it may be transplanted using a biological glue such as fibrin glue used to adhere organs.

[0126] The amount of myocardial tissue used in the treatment of heart disease may be adjusted to suit the size of the affected area and the size of the body.

[0127] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0128] <Experimental Procedure> <Culturing and Differentiation Induction of Human iPS Cells> Maintenance culture of human iPS cells and subsequent differentiation induction were performed according to the following procedure. The following two types of human iPS cell lines were used: Four-factor (Oct3 / 4, Sox2, Klf4, and c-Myc) cell line (human iPS cell line derived from a healthy individual): 201B6 Disease-specific iPS cell line: HPS1799

[0129] (Maintenance culture of human iPS cells) First, expansion and maintenance culture of each iPS cell was carried out in StemFit AK02N medium (manufactured by Ajinomoto Co., Inc.; hereinafter, sometimes referred to as "AK02N medium"). After the cultured iPS cells reached a confluent state, the iPS cells were dissociated by adding TrypLE Select (manufactured by Thermo Fisher Scientific), and the iPS cells were suspended by further adding an amount (volume) of PBS (0.5 mM ethylenediaminetetraacetic acid) equal to the amount of TrypLE Select added. The iPS cells were then dissociated into single cells (5,000 to 8,000 cells / cm). ２ At the time of subculture, the cells were subcultured every 7 days in AK02N medium supplemented with iMatrix-511 silk (Fujifilm Wako Pure Chemical Industries, Ltd.) (final concentration: 0.125 μg / cm ２ ) (uncoated laminin fragment) and a ROCK inhibitor (Y-27632, final concentration 10 μM) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were used.

[0130] (Common Pre-Process in Differentiation Induction of Cardiovascular Cells) First, the pre-process common to the CM+EC protocol, CM protocol, and MC protocol described below will be explained. Note that cardiomyocytes (CM), vascular endothelial cells (EC), and vascular wall cells (MC) may be collectively referred to as "cardiovascular cells" (CV cells). First, iPS cells in a single cell state are cultured on a culture plate coated with Matrigel (dilution ratio 1:60) at a density of 360,000 to 400,000 cells / cm. ２The iPS cells were seeded in AK02N medium (containing a final concentration of 10 μM Y-27632) so that the final concentration was 1:1. After the cultured iPS cells reached confluence, the iPS cells were covered with Matrigel (diluted 1:60 with AK02N medium) one day before differentiation induction. After one day had passed, the medium was changed from AK02N medium to RPMI+B27 medium (day 0 of differentiation induction; d0), and the cells were cultured for 24 hours. The composition of RPMI+B27 medium is shown below. (Composition of RPMI+B27 medium) RPMI1640 medium (manufactured by Thermo Fisher) L-glutamine (final concentration 2 mM) (manufactured by Thermo Fisher) 1x B27 supplement without insulin (manufactured by Thermo Fisher) Activin A (final concentration 100 ng / mL) (manufactured by R&D) CHIR99021 (final concentration 5 μM, as needed) (manufactured by Tocris Bioscience)

[0131] Then, on day 1 (d1) of differentiation induction, bone morphogenetic protein 4 (BMP4, final concentration 10 ng / mL) (manufactured by R&D) and basic fibroblast growth factor (bFGF, final concentration 10 ng / mL) were added to the culture medium and cultured for 4 days (2 days for the MC protocol). The medium was not changed during this period. The subsequent procedures (post-processing) will be explained separately for the CM+EC protocol, CM protocol, and MC protocol.

[0132] (Post-processing of the CM+EC protocol) For differentiation into CM and EC, the medium was replaced with RPMI 1640 medium on day 5 (d5) of differentiation induction. The RPMI 1640 medium used at this time contained vascular endothelial growth factor (VEGF) 165 (final concentration 50 ng / ml) (Fujifilm Wako Pure Chemical Industries, Ltd.), and, as needed, IWP4 (final concentration 2.5 μM) (Stemgent) and XAV939 (final concentration 5 μM) (Merck). The medium was then replaced every other day with RPMI 1640 medium (insulin-free) supplemented with VEGF 165 (final concentration 50 ng / ml). Between day 11 (d11) and day 15 (d15) of differentiation induction, beating cells appeared.

[0133] (Post-stage steps of the CM protocol) For differentiation into CM, the medium was replaced with RPMI+B27 medium (containing insulin) on day 5 (d5) of differentiation induction. The RPMI+B27 medium used at this time contained IWP4 (final concentration 2.5 μM) (Stemgent) and XAV939 (final concentration 5 μM) (Merck) as needed. Thereafter, the medium was replaced with RPMI1640 medium (containing insulin) every other day. Between day 11 (d11) and day 15 (d15) of differentiation induction, beating cells appeared.

[0134] (Post-process of MC protocol) To control the proportion of vascular wall cells (MC) sufficient to form artificial cardiac tissue (ECT), a portion of the MC differentiation culture was used to induce MC differentiation as needed. That is, on day 3 (d3) of differentiation induction, the medium was replaced with RPMI+FBS medium and was replaced every other day. The composition of RPMI+FBS medium is shown below. (RPMI+FBS medium) RPMI1640 medium (manufactured by Thermo Fisher) L-glutamine (final concentration 2 mM) (manufactured by Thermo Fisher) Fetal bovine serum (FBS) (final concentration 10% by volume)

[0135] <<Flow Cytometry>> Flow cytometry was performed according to the following procedure. Human iPS cell-derived CV cells (CM, EC, MC) were dissociated by incubating with Accumax. The CV cells were then stained using the following surface marker-specific antibodies (one type alone or a combination of two types, at a dilution of 1:100). (Surface marker-specific antibodies) Phycoerythrin (PE)-conjugated anti-PDGFRβ antibody, clone 28d4 (BD) Allophycocyanin (APC)-conjugated anti-VE-cadherin antibody, clone 55-7h1 (BD)

[0136] To exclude dead cells from the analysis, cells were stained with the LIVE / DEAD fixable Aqua Dead Cell Staining Kit (Thermo Fisher). Cell surface markers were stained in PBS containing 5% FBS. Intracellular proteins were stained using cells fixed in PBS containing 4% paraformaldehyde (PFA). Cells were stained with APC-labeled anti-cardiac troponin T (cTnT) antibody (clone 13-11) (Thermo Fisher) using Zenon technology (Thermo Fisher) (1:50). Staining was performed in PBS containing 5% FBS and 0.75% saponin (Sigma). Stained cells were analyzed using a BD FACS Aria II (BD) or CytoFLEX S (Beckman Coulter). Data were collected from at least 10,000 events and analyzed using DIVA software (BD) or CytExpert software (Beckman Coulter).

[0137] <Preparation of Tissue Mold> Tissue molds were prepared using polydimethylsiloxane (PDMS) (Strex). The tissue molds were autoclaved and then coated with 1% Pluronic F127 for 1 hour. Before molding the tissue, the Pluronic F127 was removed and the tissue molds were thoroughly rinsed with PBS.

[0138] <<Preparation of Artificial Cardiac Tissue (ECT)>> To prepare ECT with or without ECs, cells differentiated from the CM+EC protocol (or cells differentiated from the CM protocol) and cells differentiated from the MC protocol were combined to a final MC concentration (cell density) of 10-20%. The combined cells were mixed with acid-soluble rat tail collagen type I (Sigma) and matrix factors (Matrigel, BD Biosciences).

[0139] The cells and matrix were mixed as follows: (1) First, 6 million cells were suspended in a culture medium (high-glucose modified Dulbecco's essential medium, Life Technologies) containing 20% ​​fetal bovine serum (Life Technologies). (2) Next, an acid-soluble type I collagen solution (2 mg / ml) (pH 3) was added to an alkaline buffer (0.2 M NaHCO ) on ice. ３ (3) Matrigel (15% of the total volume) was added to the neutralized collagen solution to obtain a matrix solution. (4) The cell suspension and the matrix solution were mixed.

[0140] The final concentration of type I collagen in the cell / matrix mixture was 0.67 mg / mL (total volume: 200 μL). The cell / matrix mixture was poured into a Pluronic F127-coated PDMS tissue mold and placed in a standard 12-well culture plate. The mixture was then incubated at room temperature for 1 hour at room temperature for 2 hours at room temperature. ２ Incubator (37°C, 5% CO ２ ) for 60 minutes to form an ECT. Once the ECT was formed, the tissue mold was immersed in a pre-culture medium. The composition of the pre-culture medium is as follows. Before hydrostatic pressure stimulation, the formed ECT was cultured for 14 days (or 21 days). During this time, the medium was changed every other day. (Pre-culture medium composition) Alpha minimum essential medium (αMEM, Life Technologies) 10% FBS 5x10 －５ M 2-mercaptoethanol (Sigma) 100 U / mL penicillin-streptomycin (Life Technologies)

[0141] Through the above steps, an ECT was prepared. The ECT corresponds to a sample containing cardiomyocytes.

[0142] <Hydrostatic Stimulation (Pressure Loading Step)> Hydrostatic stimulation was performed using a MechanoCulture TR stimulator (MCTR, manufactured by CellScale) installed in a tissue culture incubator. The tissue mold containing the ECT was transferred to the MCTR chamber, filled with medium, and the following hydrostatic stimulation protocol was set (see Figure 2). After hydrostatic stimulation, the ECT was returned to the 12-well culture plate. (Hydrostatic Stimulation Protocol) Loading pressure: 10 kPa, 50 kPa, or 100 kPa Hydrostatic stimulation cycle: compress duration: 0.2 s, hold duration: 0.1 s, recovery duration: 0.2 s, rest duration: 0.5 s Hydrostatic stimulation mode: continuous pressure loading (24 hours per day) or intermittent pressure loading (1 hour per day) Number of days performed: 3 or 8 days

[0143] <Histological and Fluorescence Analysis> ECTs were fixed in 4% PFA and embedded in paraffin. Tissue sections (thickness: 6 μm) were prepared from the fixed ECTs. The tissue sections were stained with hematoxylin-eosin, Sirius Red (SR), cTnT, and TUNEL.

[0144] For fluorescence microscopy, ECTs were stained with anti-cTnT antibody (Abcam, ab45932) (dilution: 1:500) and anti-CD31 antibody (monoclonal mouse IgG1, clone name 9G11) (R&D) (dilution: 1:500) together with DAPI (4',6-diamidino-2-phenylindole) (Thermo Fisher). Single cells were stained with anti-cTnT antibody (mouse monoclonal antibody, MS-295-P1) (dilution: 1:500) and anti-TOMM20 antibody (Abcam, ab78547) (dilution: 1:500) together with DAPI. Anti-mouse IgG antibody-Alexa 546 (Thermo Fisher) and anti-rabbit IgG antibody-Alexa 488 (Thermo Fisher) were used as secondary antibodies. ECTs or single cells were photographed using an all-in-one fluorescence microscope system, Zeiss LSM880 (mean fluorescence intensity of TOMM20 by ImageJ).

[0145] Electrical Stimulation Test: For electrical stimulation tests of ECTs, a custom-made electrical stimulation system (Strex) equipped with an electrical pulse generator was used. Two platinum electrodes were attached in parallel to ECTs cultured in a 12-well multiwell plate, and electric field stimulation was performed via the medium. Electrical stimulation was performed with a pulse width of 4 ms, an interval of 1000 ms (1 Hz), and a voltage of 30 V (peak-to-peak), gradually decreasing to 500 ms (2 Hz). Capture of electrical stimulation on ECTs was confirmed by simultaneous microscopic observation.

[0146] Analysis with MUSCLEMATION: MUSCLEMATION is a versatile open-source software with a video-based system used to assess contractile function. The software was used according to the manufacturer's instructions. ImageJ software was used with MUSCLEMATION installed as a plug-in. Movement amplitude was used for analysis.

[0147] Quantitative Analysis of Artificial Heart Tissue Quantitative analysis of the ECT, including cell number, cTnT-positive area, cell size, cell perimeter, TOMM20 mean fluorescence intensity (MFI), and thickness change of the ECT (with or without EC) over time, was analyzed using ImageJ software.

[0148] <Calcium Transient> Calibryte containing 0.04% PF-127 ＴＭ 520 AM (AAT Bioquest, final concentration 5 μM) loading solution was added to the tissue mold. ECT was performed at 37°C in 5% CO ２ The cells were incubated with dye loading buffer (dye loading solution) in an incubator for 60 minutes. ＴＭ The cells were then replaced with DMEM and analyzed. Videos of signal intensity were acquired through a FITC filter using a fluorescence microscope (CKX53) equipped with a camera system (DP27) and software (cellSens). Data analysis was performed using ImageJ software52 with manually selected ROIs and the "Plot Z-axis Profile" function.

[0149] Transmission Electron Microscopy Observation: Samples were fixed overnight in 2% glutaraldehyde + 4% PFA / 0.1 M phosphate buffer (pH 7.2) at 4° C. Fixed samples were sent to the Electron Microscopy and Histology Facility at Kyoto University for subsequent preparation, imaging, and data interpretation in a blind fashion.

[0150] <ECT Dissociation and Debris Removal Experiment> After washing the ECT twice with PBS, the ECT was incubated in a dissociation buffer. The composition of the dissociation buffer used here is as follows: (Composition of dissociation buffer) 120 mM NaCl, 5.4 mM KCl, 5 mM MgSO ４ 5 mM Na-pyruvate, 20 mM glucose, 20 mM taurine, 10 mM HEPES (pH 6.9), 30 μM CaCl

[0151] Next, the dissociated ECT-derived cells were subjected to debris removal experiments using debris removal solution (Miltenyi Biotec) according to the manufacturer's instructions. (1) The cell suspension was centrifuged at 300 × g for 10 minutes at 4 °C. (2) The supernatant was completely aspirated, and the cell suspension was carefully resuspended in 500 μl of cold PBS. An equal volume of cold debris removal solution was then added and gently layered on top of the cold PBS. (3) The tube was centrifuged at 3,000 × g for 10 minutes at 4 °C with full throttle and full brake. Three phases formed, and the upper two phases were completely aspirated and discarded. The tube was then filled with cold PBS and gently inverted three times. (4) The tube was centrifuged at 1,000 × g for 10 minutes at 4 °C with full throttle and full brake, and the supernatant was completely aspirated.

[0152] Sepharose Mitochondrial Stress Test: After dissociation of ECTs and removal of debris, purified single CMs were seeded onto Matrigel-coated Seahorse XFp assay plates (Agilent) at a density of 100,000 cells / well and grown in standard culture medium (αMEM) for 3 days. One hour before the test, the plates were replaced with Seahorse assay medium (XF RPMI supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine) and incubated in the Seahorse assay medium for 3 days under CO . ２ The cells were incubated at 37°C in a pH-free incubator. The XF Cell Mitostress Test Kit was used according to the manufacturer's instructions, with each compound at the following final concentrations: (Final concentrations of compounds used) Oligomycin 3 μM 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) 2 μM Rotenone / Antimycin A 2 μM

[0153] RNA Extraction, cDNA Synthesis, and Quantitative RT-PCR Total RNA was isolated using a Qiashredder (Qiagen) and purified using an RNeasy Mini Kit (Qiagen). These procedures were performed according to the manufacturer's instructions. The above purification process also included DNase treatment using an RNase-free DNase Set (Qiagen). RNA yield and purity were measured using a NanoDrop One (ThermoFisher Scientific). First-strand cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix (FSQ-201, TOYOBO) according to the manufacturer's instructions.

[0154] For each sample, 200 ng of total RNA was used for reverse transcription to cDNA. The RNA was purified using PowerUp™ according to the manufacturer's instructions. ＴＭThe samples were analyzed by quantitative real-time polymerase chain reaction (RT-PCR) using SYBR Green Master Mix (ThermoFisher Scientific) in Applied Biosystems StepOne Plus (ThermoFisher Scientific). Data analysis was performed using fold changes normalized to the gene expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0155] Mitochondrial DNA (MtDNA) was extracted from ECTs using DNA digestion buffer (Qiagen Cell Lysis solution containing 1:100 proteinase K). MtDNA was extracted using PowerUp™ according to the manufacturer's instructions. ＴＭ Analysis was performed by quantitative RT-PCR using Applied Biosystems StepOne Plus (ThermoFisher Scientific) with SYBR Green Master Mix (ThermoFisher Scientific). Primers for MtDNA were for the mitochondrial gene NADH dehydrogenase 1 (ND1). MtDNA expression levels were normalized to the expression of the RNA18S5 gene.

[0156] Single-Cell RNA Sequencing: After dissociation of ECTs and debris removal, purified single CMs were used for single-cell RNA sequencing (scRNA-seq) using the Chromium Single-Cell platform (10x Genomics). Cell barcoding, cDNA synthesis, and library construction were performed using the Single Cell Gene Expression 3' Kit Version 2 (10x Genomics, PN120237) or Single Cell Gene Expression 3' Kit Version 3 (10x Genomics, PN1000075) according to the manufacturer's instructions. scRNA-seq data were analyzed using Loope Browser 6, and pathway analysis was performed using iDEP 0.96 (ShinyGO 0.76.3).

[0157] <<Preparation of Model Animals and Transplantation>> Male athymic nude rats (F344 / N Jcl-rnu / rnu, CLEA Japan, Inc.) aged 10 to 13 weeks were used for transplantation. Myocardial infarction (MI) model rats were prepared as follows: First, the rat's left thoracotomy was performed to expose the heart. In the exposed heart, the left anterior descending coronary artery was ligated to create an MI model rat.

[0158] Rats whose hearts showed a left ventricular fractional shortening (FS) of less than 40% in echocardiograms taken immediately before MI induction were used in the following experiments. One week after MI induction, cell sheet transplantation was performed. The rats were randomly divided into three groups: rats transplanted with ECTs that had undergone MCTR training (MCTR group), rats transplanted with ECTs that had not undergone MCTR training (Control group), and rats that underwent sham surgery (Sham group). Three layers of ECTs were transplanted into the rat hearts. Echocardiography was performed before the MI model was created (PreMI), one week after the MI model was created (just before transplantation, PreTx), and two weeks (Tx_2w) and four weeks (Tx_4w) after transplantation.

[0159] Statistical Analysis: Data were expressed as mean ± SD. Differences between experimental groups were assessed by one-way repeated measures analysis of variance and t-test (nonparametric test) using GraphPad Prism (9.0). A p value of <0.05 was considered significant for all statistical tests.

[0160] <Experimental Results> <Preparation of Human iPS Cell-Derived Artificial Cardiac Tissue> Following the experimental procedure described above, artificial cardiac tissue (ECT) was prepared from human iPS cells by co-culturing cardiovascular cells differentiated from human iPS cells (201B6) with extracellular matrix proteins on a tissue mold (Fig. 1B). The cellular composition of the ECT immediately after preparation is shown in Fig. 1A. In Fig. 1A, the horizontal axis indicates the type of cells constituting the ECT, and the vertical axis indicates the proportion of each cell type present within the ECT. From the results of Fig. 1A, it was found that within the ECT, CM accounted for approximately 60%, EC for approximately 20%, and MC for approximately 20%. Note that all ECTs used in subsequent experiments were derived from the human iPS cell line (201B6).

[0161] <Hydrostatic Stimulation of ECT> First, we attempted to optimize the pressure load pattern on the ECT. The heart is continuously exposed to afterload pressure without rest. On the other hand, it has been reported that a sudden increase in afterload pressure in the ventricle via ascending aortic constriction (AAC), followed by the relief of the increased afterload and return to a normal state by removing the aortic constriction, further stimulates the re-entry of cardiomyocytes into the cell cycle (or increases cardiac myocytes).

[0162] To clarify the effects of continuous pressure overload, continuous pressure stimulation was applied under physiological (10 kPa, equivalent to approximately 75 mmHg) and non-physiological (50 kPa, equivalent to approximately 375 mmHg) conditions (Fig. 3, top panel). Histological evaluation revealed almost no cTnT staining, suggesting that continuous pressure overload may lead to tissue damage without an increase in cardiomyocytes in ECT (Fig. 4).

[0163] Based on the results shown in Figure 4, we performed intermittent pressure loading at 10 kPa, 50 kPa, and 100 kPa (1 hour per day for 3 days) (Figure 3, middle panel). ECTs loaded at 50 kPa showed higher cTnT-positive cardiomyocyte components, higher viable cell density, and higher expression of TNNT2, TNNI3, and CACNA1C genes, which are associated with higher collagen alignment (Figures 5 and 6). This indicates that hydrostatic pressure stimulation leads to the maturation of cardiomyocytes in the ECTs. On the other hand, no positive effect was observed in ECTs loaded at 10 kPa or 100 kPa (Figures 5 and 6). Note that the label "MCTR_2W_3D_10 kPa" in Figure 6 refers to a sample in which the formed ECTs were cultured for 2 weeks and then intermittently loaded at 10 kPa for 3 days. Furthermore, the description "Control_2W_3D" refers to a sample in which the formed ECT was cultured for two weeks. Other descriptions are also given in accordance with this description.

[0164] To assess the contractile function (pulsatile function) of the ECT, we used MUSCLEMATION, a versatile video-based system for quantifying ECT movement amplitude. In the control group without hydrostatic pressure stimulation, movement amplitude throughout the entire pulsatile cycle decreased with increasing frequency of external electrical stimulation, both before and after the training-equivalent period (observation period), demonstrating a strong negative correlation. However, in the MCTR group with hydrostatic pressure stimulation, movement amplitude throughout the entire pulsatile cycle clearly increased after training as the electrical stimulation frequency increased from 1000 ms (1 Hz) to 800 ms (1.25 Hz), despite being negative before training. These results indicate that intermittent hydrostatic pressure stimulation of the ECT at a nonphysiological pressure of 50 kPa induces a positive force frequency relationship (FFR).

[0165] To further optimize the hydrostatic stimulation method, we investigated the number of days of intermittent stimulation. We compared a 3-day (1 h / day) group with an 8-day (1 h / day) group, and also established corresponding control groups (control 3-day group and control 8-day group) (Figure 3, middle and bottom panels). For the four groups, we evaluated structural function by histological staining (Figures 7A and 7B-C), contractile force (Figure 7D-DE), and gene expression by RT-qPCR (Figure 7F-G). These results indicated that 3-day intermittent hydrostatic stimulation was more effective in promoting ECT maturation than 8-day intermittent hydrostatic stimulation.

[0166] Previously, hydrostatic pressure stimulation was performed after two weeks of culture of ECTs. Early-stage cardiomyocytes, newly differentiated from human iPS cells, are known to be more sensitive to electrical training than later-stage cardiomyocytes. This suggests that the timing of initiation of physical training is important for tissue maturation. Therefore, we compared the results of two-week culture before hydrostatic pressure stimulation with those of three-week culture before hydrostatic pressure stimulation using histological staining, MUSCLEMATION analysis, and RT-qPCR (Figures 7A, 7BC-C, 7DE-E, and 7FG-G). Histological staining results showed increased cTnT positivity and cell viability in both groups compared to the control group, with significant increases in the group cultured for two weeks before hydrostatic pressure stimulation (Figures 7A, 7BC-C). FFR was achieved in both the 2-week and 3-week pre-hydrostatic culture groups (Fig. 7D-E). The expression levels of TNNT, TNNI3, TNNI3 / TNNI1, and CACNA1C were increased in both groups. These results suggest that hydrostatic pressure stimulation of ECT cells has a positive effect in both the 2-week and 3-week pre-hydrostatic culture groups, with a particularly significant effect in the 2-week pre-hydrostatic culture group (Fig. 7F-G).

[0167] The arrangement of cardiomyocytes during ECT was compared between the control group and the MCTR group. The results are shown in Figure 7H. Figure 7H is a photograph showing the results of ECT fluorescence analysis. The results in Figure 7H indicate that cardiomyocytes were aligned in the same direction during ECT in the MCTR group compared to the control group (Control). It is known that in mature, in vivo cardiac tissue, cardiomyocytes are aligned in the same direction, allowing them to contract in unison in the same direction and exert contractile force. MCTR training showed that cardiomyocytes exhibited an arrangement (unidirectional alignment) similar to that of myocardium in mature cardiac tissue, and thus could exert contractile function similar to that of in vivo cardiac tissue.

[0168] These results indicate that hydrostatic pressure stimulation promotes the maturation of cardiomyocytes during ECT. Furthermore, it was found that hydrostatic pressure stimulation is optimally performed under conditions of 50 kPa, 1 hour / day, for 3 days after 2 weeks of culture before hydrostatic pressure stimulation.

[0169] Evaluation of FFR in ECTs: FFR was evaluated using ECTs trained with hydrostatic pressure stimulation under the optimal conditions described above. The ECTs showed a positive FFR, indicating tissue maturation (Figure 8A-B). The proportion of cardiomyocytes was higher in the positive FFR group than in the negative FFR group. The expression levels of TNNT, TNNI3, TNNI3 / TNNI1, and CACNA1C were increased in the positive FFR group. However, no significant differences were observed compared to the negative FFR group. As shown by PCR experimental data (Figure 7F-G), one mechanism of tissue maturation is expected to be improved calcium handling. Calcium transients could be visualized using Calibryte 520 (AAT Bioquest (ABD)). Representative spontaneous calcium transients in the control and MCTR groups are shown in Figure 8C-E-F. Measurement of numerous different parameters revealed a significant increase in calcium transients in the MCTR group. Peak calcium flux was increased by over 100%. Expression levels of PLN and SERCA2 were significantly increased in the MCTR group (Fig. 8C-E-F), indicating a high calcium handling capacity in the MCTR-trained ECT. This indicates that hydrostatic pressure stimulation induces cardiomyocyte maturation during ECT.

[0170] Mitochondrial function was also predicted as another mechanism of tissue maturation induced by MCTR training. Mitochondrial function was assessed using the Seahorse Mito Stress Test, and a significant increase in maximal oxygen consumption rate (OCR) was observed in MCTR-trained ECTs compared with the control group (Figure 9A-B). TEM observation of ECTs revealed a greater number of mitochondria in the MCTR group compared with the control group (Figure 9C-D-E, Figure 9C-D-E). These mitochondria were found to be located between sarcomeres (Figure 9C-D-E). Furthermore, confocal imaging revealed that the area positive for TOMM20 (a mitochondrial marker) was larger in cardiomyocytes in MCTR-trained ECTs (Figure 9C-D-E, Figure 9C-D-E, Figure 9C-D-E). Mitochondrial DNA was quantified by qPCR (Figure 9C-D-E, Figure 9C-D-E). These results demonstrate that MCTR training increases the amount of mitochondria in cardiomyocytes, enabling greater energy (ATP) production. Figure 9FG-G is a graph showing cell size (left), perimeter (center), and perimeter / area ratio (right). As cardiomyocytes mature, their size increases, and their perimeter also increases. Furthermore, as cardiomyocytes mature, their structure becomes more complex, resulting in more complex cell morphology. This increased complexity in cell morphology is supported by an increased perimeter / area ratio. In other words, the results of Figure 9FG-G demonstrate that MCTR training also leads to morphological maturation of individual cardiomyocytes.

[0171] To comprehensively understand the mechanism of tissue maturation mediated by MCTR training, we performed single-cell RNA-seq (Fig. 10AB, 10CD). The results demonstrated that MCTR training promoted functional maturation of cardiomyocytes contained in the artificial cardiac tissue, and revealed that the mechanism involved promotion of oxidative phosphorylation, which favors energy production.

[0172] The formation of vascular networks is one of the fundamental factors for establishing mature cardiac tissue. Endothelial cells are thought to be sensitive to hydrostatic pressure. Therefore, we further investigated the response of ECs to MCTR. ECTs with and without ECs were prepared (Fig. 11A, ABC). They differed in the time of ECT formation. The degree of contraction 1 hour after ECT compression and the subsequent beat were also different (Fig. 11B, ABC, B and C). In the ECT without EC group, FFR was negative (Fig. 11D, DEF, D). In the ECT without EC group, the expression levels of TNNT, TNNI3, TNNI3 / TNNI1, CACNA1C, PLN, SERCA2, MHY7, and MHY6 were decreased by MCTR training (Fig. 11E, DEF, E). Confocal imaging of tissue-removed ECTs confirmed the formation of a vascular network in the ECT group with ECs (Figure 12). On the other hand, no vascular network formation was observed in the ECT group without ECs (Figure 11 DEF-F). These results demonstrate that the inclusion of endothelial cells in ECTs promotes the formation of a vascular network.

[0173] In the experiment corresponding to Figure 12, when the rocking culture period after training was changed to one week, a vascular network composed of CD31-positive endothelial cells was observed inside ECTs that had undergone MCTR training and were "endothelial cell-containing" (EC-containing) (Figure 13). When electrical stimulation was applied to the ECTs in which this vascular network was observed, starting at 1000 ms and gradually increasing in frequency, a positive force-frequency relationship (FFR) was observed (Figure 14, bottom row) (suggesting that this is mature tissue). On the other hand, a control group with "endothelial cell-containing" (EC-containing) but not undergoing MCTR training showed a negative force-frequency relationship (FFR) (Figure 14, top row) (suggesting that this is immature tissue).

[0174] <<Transplantation Experiment Using MI Model Rats>> An ECT transplantation experiment was conducted using MI model rats. Rats whose hearts showed a left ventricular fractional shortening (FS) of less than 40% in an echocardiogram immediately before MI induction were used. One week after MI induction, cell sheets (ECT stacked in three layers) were transplanted. The rats were randomly divided into the following three groups. (Three groups) MCTR group: Rats transplanted with ECT that had undergone MCTR training Control group: Rats transplanted with ECT that had not undergone MCTR training Sham group: Rats that underwent a sham operation

[0175] Cardiac ultrasound examinations were performed before the creation of the MI model (PreMI), one week after the creation of the MI model (just before transplantation, PreTx), two weeks after transplantation (Tx_2w), and four weeks after transplantation (Tx_4w). The results are shown in Figure 15. The results in Figure 15 indicate that the MCTR group exhibited high cardiac function, especially two weeks after transplantation (Tx_2w).

[0176] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0177] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include meanings equivalent to the claims and all modifications within the scope of the claims.

Claims

1. A method for producing mature cardiomyocytes, comprising: a preparation step of preparing a sample containing immature cardiomyocytes; a pressure applying step of applying hydrostatic pressure to the sample continuously or intermittently; A method for producing mature cardiomyocytes, comprising:

2. The method for producing mature cardiomyocytes according to claim 1 , wherein the hydrostatic pressure applied in the pressure application step is 10 kPa or more and 100 kPa or less.

3. 3. The method for producing mature cardiomyocytes according to claim 1, wherein in the pressure application step, the hydrostatic pressure is applied intermittently at a frequency of 1 hour to 12 hours per day.

4. The method for producing mature cardiomyocytes according to claim 1 or 2, wherein the pressure application step is carried out for a period of 3 days or more and 8 days or less.

5. The method for producing mature cardiomyocytes according to claim 1 or 2, wherein the sample further contains vascular wall cells.

6. The method for producing mature cardiomyocytes according to claim 1 or claim 2, wherein the sample further contains vascular endothelial cells.

7. The method for producing mature cardiomyocytes according to claim 1 or 2, wherein the sample is an artificial cardiac tissue derived from iPS cells.

8. An artificial cardiac tissue comprising cardiomyocytes, The cardiomyocytes include TNNI3-positive cells.