Mature cardiomyocyte production method

Abstract

The present invention addresses the problem of providing a method for obtaining mature cardiomyocytes which can be used for screening in drug development and for cell transplantation, and said problem is solved by providing a mature cardiomyocyte production method including a step for performing partial reprogramming in immature cardiomyocytes.

Description

Method for producing mature cardiomyocytes

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

[0002] In recent years, attempts have been made to induce the differentiation of pluripotent stem cells, such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells, into various somatic cells for use in regenerative medicine, etc. Although several systems have been reported for inducing the differentiation of pluripotent stem cells into cardiomyocytes, most of the obtained cardiomyocytes are immature, similar to fetal cardiomyocytes, and require manipulation of maturation for practical use.

[0003] The following methods have been known for maturing cardiomyocytes: 1) Long-term culture (e.g., Non-Patent Documents 1 and 2) 2) Culture on an undiluted Matrigel layer (Matrigel mattress) (e.g., Non-Patent Document 3) 3) Combination of three-dimensional culture (biowire formation) and electrical stimulation (e.g., Non-Patent Documents 4 to 6) 4) Addition of a cardiomyocyte maturation promoter (e.g., Patent Document 1) 5) Transient expression of the Sall1 gene and the Mesp1 gene (e.g., Patent Document 2) 6) Transient expression of the CDKN1A gene (Patent Document 3)

[0004] As mentioned above, several techniques for maturing cardiomyocytes have been reported, but it is not known to obtain mature cardiomyocytes using reprogramming factors.

[0005] International Publication No. 2019 / 189554 Pamphlet Japanese Patent Publication No. 2017-60422 International Publication No. 2021 / 172542 Pamphlet

[0006] Circ J 2013; 77: 1307-1314Proc Natl Acad Sci US A. 2015 May 26;112(21):E2785-94Circ Res. 2015 December 4; 117(12): 995-1000.Nat Methods. 2013 August; 10(8): 781-787Nature. 2018 April; 556(7700): 239-243Circulation. 2016 November 15; 134(20): 1557-1567

[0007] An objective of the present invention is to provide a method for obtaining mature cardiomyocytes that can be used for drug discovery screening and cell transplantation.

[0008] As a result of intensive research to solve the above problems, the inventors have surprisingly found that partial reprogramming of immature cardiomyocytes can result in the maturation of cardiomyocytes and the production of mature cardiomyocytes, leading to the completion of the present invention.

[0009] The gist of the present invention is as follows: [1] A method for producing mature cardiomyocytes, comprising a step of performing partial reprogramming in immature cardiomyocytes. [2] A method for producing mature cardiomyocytes according to [1], wherein the partial reprogramming is performed by transiently increasing the expression level of reprogramming factors in immature cardiomyocytes. [3] A method for producing mature cardiomyocytes according to [2], wherein the reprogramming factors comprise at least Oct4, Sox2, Klf4, and cMyc. [4] A method for producing mature cardiomyocytes according to [2] or [3], wherein the reprogramming factors further comprise Nanog and Lin28. [4-a] A method for producing mature cardiomyocytes according to [2] or [3], wherein the reprogramming factors further comprise immune evasion factors E3, K3, and B18R. [5] A method for producing mature cardiomyocytes according to any of [2] to [4], wherein the transient increase in the expression level of the reprogramming factor is performed by introducing mRNA encoding the reprogramming factor into immature cardiomyocytes. [6] A method for producing mature cardiomyocytes according to any of [1] to [5], further comprising a step of culturing the immature cardiomyocytes that have been partially reprogrammed in the presence of an mTOR inhibitor. [7] The method for producing mature cardiomyocytes according to [6], wherein the mTOR inhibitor is Torin 1. [8] The method for producing mature cardiomyocytes according to any one of [2] to [7], wherein the increase in expression level is maintained for two days or more. [9] The method for producing mature cardiomyocytes according to any one of [1] to [8], wherein the cardiomyocytes are human cardiomyocytes.

[10] The method for producing mature cardiomyocytes according to any one of [1] to [9], wherein the immature cardiomyocytes are immature cardiomyocytes induced to differentiate from pluripotent stem cells.

[11] The method for producing mature cardiomyocytes according to

[10] , wherein the pluripotent stem cells are induced pluripotent stem cells.

[12] Mature cardiomyocytes produced by the method according to any one of [1] to

[11] .

[0010] According to the present invention, cardiomyocytes can be efficiently matured in a short period of time without using a special culture substrate, etc. In particular, by combining this with differentiation induction from pluripotent stem cells, it becomes possible to easily produce mature cardiomyocytes in large quantities.

[0011] (A) Schematic diagram showing one embodiment of partial reprogramming of iPS cell-derived immature cardiomyocytes by RNA introduction, and (B-F) epigenetic profiles of partially reprogrammed immature cardiomyocytes. Figures show in vitro analysis of RNA expression levels (A, B) and protein expression levels (C) of maturation-related genes in partially reprogrammed cardiomyocytes. (A) Mitochondrial stress assay of partially reprogrammed Torin1-treated hiPSC-derived cardiomyocytes (hiPSC-CMs) over 80 minutes (A), basal respiration (B), and maximal respiration (C). (D) ATP production based on oxygen consumption rate (OCR; pmol / min) in partially reprogrammed cardiomyocytes. (A) Phase-contrast microscopy image of engineered heart tissues (EHTs) composed of partially reprogrammed cells and maturation-treated with Torin1. Contractile force (μN / mm) during pacing of EHTs composed of partially reprogrammed hiPSC-derived cardiomyocytes (hiPSC-CMs) 2 ) (B).

[0012] The method for producing mature cardiomyocytes of the present invention includes a step of partially reprogramming immature cardiomyocytes.

[0013] In the present invention, cardiomyocytes refer to cells expressing cardiac troponin (cTNT). Examples of human cTNT include NCBI accession number NM_000364 and mouse cTNT include NM_001130176. Cardiomyocytes are not particularly limited, but are preferably derived from mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, pigs, monkeys, and humans), and more preferably from humans.

[0014] As cardiomyocytes mature, an isoform switch occurs, in which the expression of troponin I1 (TNNI1) decreases and the expression of troponin I3 (TNNI3) increases (Fikru B. Bedada, (2014) 3(4): 594-605.).

[0015] As used herein, immature cardiomyocytes are not particularly limited as long as they can be determined to be in an immature state based on myocardial marker expression levels, morphology and structure (e.g., sarcomeres, mitochondria), properties (e.g., pulsation state, potential physiological maturity, epigenome), etc.

[0016] For example, cardiac markers include markers expressed in mature cardiomyocytes, such as TNNI3 and βMHC (MYH7), while immature cardiomyocytes include cardiomyocytes that express these markers at low levels, such as cardiomyocytes that exhibit expression levels similar to those of fetal-like cardiomyocytes. An example of βMHC in humans is NCBI accession number NM_000257, and in mice is NM_080728. An example of TNNI3 in humans is NCBI accession number NM_000363, and in mice is NM_009406.

[0017] When the maturity of cardiomyocytes is evaluated using indicators such as the morphology, structure (e.g., sarcomeres, mitochondria), or properties (e.g., pulsation state, electrophysiological maturity) of cardiomyocytes, for example, the depth of the resting membrane potential measured by patch clamp or the like can be used as an indicator of electrophysiological maturity to determine whether the cardiomyocytes are mature or immature. Furthermore, when the ultrastructure of sarcomeres or mitochondria is used as an indicator, they can be observed using an electron microscope; analyzed using a microscope or flow cytometer with fluorescent labeling; or functionally analyzed using an extracellular flux analyzer or the like. These indicators can also be compared with control mature cardiomyocytes such as adult cardiomyocytes or control immature cardiomyocytes such as fetal cardiomyocytes to determine whether the cardiomyocytes being evaluated are mature or immature. Furthermore, the maturity of cardiomyocytes can also be evaluated using changes at the epigenome level as an indicator. Specifically, when changes at the epigenome level are used as an indicator, DNA methylation in a specific region can be used as an indicator. Examples of the specific region include the aforementioned cardiac marker genes, specifically, for example, regions of TNNI3 and βMHC (MYH7), etc. Preferably, the specific region may be the promoter region of the above genes.

[0018] Immature cardiomyocytes may be immature cardiomyocytes isolated from a living body (e.g., cardiomyocytes derived from fetal or neonatal mouse or rat fetuses), or may be cells induced to differentiate from pluripotent stem cells, as described below.

[0019] In the method of the present invention, partial reprogramming is performed on immature cardiomyocytes, thereby improving the maturity of the immature cardiomyocytes and obtaining mature cardiomyocytes.

[0020] As used herein, "mature cardiomyocytes" refers to cardiomyocytes that highly express TNNI3. Mature cardiomyocytes are also called adult-like cardiomyocytes. For example, when the expression level of TNNI3 is measured at the gene level or protein level and normalized using the expression level of a constitutive expression marker, cardiomyocytes with an expression level of TNNI3 that is at least two times, more preferably at least five times, even more preferably at least ten times, and particularly preferably at least twenty times that of fetal cardiomyocytes can be considered mature cardiomyocytes. That is, in the method of the present invention, partial reprogramming of immature cardiomyocytes can increase the expression level of TNNI3, which serves as an indicator of cardiomyocyte maturity, by at least two times, more preferably at least five times, even more preferably at least ten times, and particularly preferably at least twenty times that of immature cardiomyocytes.

[0021] The expression level of TNNI3 can be analyzed by, for example, measuring the amount of mRNA of these genes using PCR or the like; analyzing the amount of protein expression by Western blotting or the like; analyzing the amount of reporter molecule expression; or analyzing using a microscope or flow cytometer based on the fluorescence intensity of fluorescent labels or fluorescent reporters.

[0022] Reprogramming refers to the erasure or reconstitution of epigenetic modifications in the genome of somatic cells, resulting in the acquisition of pluripotency equivalent to that of cells in the early stages of development. Reprogramming is performed, for example, by introducing reprogramming factors so that somatic cells acquire pluripotency equivalent to that of cells in the early stages of development. Examples of cells that have been reprogrammed and acquired pluripotency equivalent to that of cells in the early stages of development include iPS cells, which will be described later.

[0023] In the present invention, "partial reprogramming" refers to a change in somatic cells that involves the erasure or rearrangement of epigenetic modifications on the genome, but does not result in the acquisition of pluripotency equivalent to that of cells in the early developmental stage, and thus retains the identity of the original cell. For example, "partial reprogramming" in immature cardiomyocytes can be confirmed by the occurrence of DNA demethylation of cardiomyocyte maturation-related genes (e.g., TNNI3, TNNI3K, MYH7, RYR2, and / or CAV3), while maintaining the cell morphology, cell function, and gene expression pattern of immature cardiomyocytes.

[0024] Partial reprogramming may be performed, for example, by transiently or temporarily performing a reprogramming operation on somatic cells. Examples of somatic cells to which partial reprogramming is performed in the present invention include immature cardiomyocytes. Specifically, the partial reprogramming of the present invention may be performed, for example, by transiently increasing the expression level of a reprogramming factor in immature cardiomyocytes. Transiently increasing the expression level of a reprogramming factor may be performed by introducing a reprogramming factor into immature cardiomyocytes. "Transiently increasing the expression level of a reprogramming factor" means that the expression level of a reprogramming factor is increased for only a certain period of time and is not increased after the period has elapsed. Specifically, this may mean that the expression level of a reprogramming factor is increased more than the expression level in cardiomyocytes for a certain period of time, and after the period has elapsed, the expression level is reduced to the same level as that in cardiomyocytes. The period for transiently increasing the expression level of a reprogramming factor will be described below.

[0025] In the present invention, the term "reprogramming factor" refers to a gene or its product that is used, alone or in combination with multiple factors, to induce the differentiation state of a cell to a more undifferentiated state, and includes, for example, a gene or its product that is used to induce dedifferentiation of a differentiated cell. In a narrower sense, the term can be used to refer to a gene or its product that can induce iPS cells from somatic cells.

[0026] Examples of preferred genes that can be used as reprogramming factors include F-box protein 15 (Fbx15, NM_152676, NM_015798), Nanog (NM_024865, AB093574), ERAS (ES cell expressed Ras; NM_181532, NM_181548), DPPA2 (NM_138815, NM_028615), Oct3 / 4 (also known as POU5F1; NM_002701, NM_203289, NM_013633, NM_001009178), Sox2 (NM_003106, NM_011443, XM_574919), and TCL1A (T-cell leukemia / lymphoma 1A; NM_021966, NM_009337), KLF4 (Kruppel-like factor 4; NM_004235, NM_010637), cateninβ1 (cadherin-associated protein beta 1; NM_001904, NM_007614; including the S33Y mutant), and c-Myc (NM_002467, NM_010849;These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include those described in 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, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO 2010 / 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, Huangfu D, et al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 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, R. L. Judson et.al. , (2009), Nat. Biotechnol. , 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci USA. 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.

[0027] A suitable example of a combination of genes that can be used as reprogramming factors is a combination containing at least four genes: Sox, KLF, Myc, and Oct (Takahashi, K. and Yamanaka S., Cell 126, 663-676, 2006; Lowry WE et al., Proc Natl Acad Sci USA, 105(8):2883-8, 2008; Masaki, H. et al., Stem Cell Res. 1:105-115, 2008; WO2007 / 69666). Here, Sox, KLF, Myc, and Oct refer to genes that are members of the Sox family, KLF family, Myc family, and Oct family, respectively. It is particularly preferred to combine genes so as to express one or more members of each of these four families. For example, Sox family genes can be any of Sox1, Sox2, Sox3, Sox15, and Sox17. Furthermore, KLF family genes can be used with KLF4 or KLF2. Myc family genes can be used with wild-type c-Myc, as well as T58A mutants, N-Myc, and L-Myc. Another preferred combination includes Sox, KLF, and Oct genes, or at least three genes: Sox, Myc, and Oct genes. Furthermore, combinations containing at least four or three genes, such as the NANOG gene (NM_024865, AB093574) and / or the LIN28 gene (NM_024674), are also highly beneficial. If one or more of the above genes are already expressed at sufficient levels, for example, endogenously, in the cells undergoing partial reprogramming, the use of these genes can be omitted.

[0028] Various cofactors can be used in combination with the reprogramming factors to increase the efficiency of reprogramming. Examples of cofactors include TERT (NM_198253, NM_009354), SV40 large T antigen (NC_001669.1, Fiers, W. (05-11-1978) Nature 273:(5658)113-120), HPV16 E6, HPV16 E7, and Bmil (NM_005180, NM_007552). Other suitable cofactors include p53 inhibitors (e.g., shRNA, siRNA, or dominant-negative mutants of p53), microRNAs derived from the miR-302 / 367 microRNA cluster, and immune evasion factors derived from vaccinia virus (E3, K3L, B18). These cofactors can be used in combination as appropriate.

[0029] Suitable examples of reprogramming factors and cofactors used for partial reprogramming include, for example, a combination of Oct4, Sox2, Klf4, cMyc, Nanog, Lin28, immune evasion factor E3, immune evasion factor K3, immune evasion factor B18R, and microRNAs derived from miR-302 / 367 microRNA.

[0030] Partial reprogramming may be achieved, for example, by increasing the expression levels of reprogramming factors. Partial reprogramming may be achieved by increasing the expression levels of at least Oct4, Sox2, Klf4, and cMyc. "Increasing the expression levels of Oct4, Sox2, Klf4, and cMyc" may refer to increasing the expression levels of Oct4, Sox2, Klf4, and cMyc mRNA and / or Oct4, Sox2, Klf4, and cMyc protein, or any combination thereof, where the expression levels of at least Oct4, Sox2, Klf4, and cMyc mRNA and / or protein are increased. Specifically, an increase in these expression levels may mean an increase of 5-fold or more, preferably 10-fold or more, more preferably 20-fold or more, even more preferably 50-fold or more, and most preferably 100-fold or more. Furthermore, the Oct4, Sox2, Klf4, and cMyc mRNAs or Oct4, Sox2, Klf4, and cMyc proteins may be derived from either endogenous or exogenous Oct4, Sox2, Klf4, and cMyc genes.

[0031] Partial reprogramming may also be achieved by increasing the expression levels of Nanog and Lin28.

[0032] "Increasing the expression levels of Nanog and Lin28" may refer to increasing the expression levels of Nanog and Lin28 mRNA and / or Nanog and Lin28 protein, or any combination thereof, where the expression level of at least one of Nanog and Lin28 mRNA and protein is increased. Specifically, an increase in these expression levels may mean an increase of 5-fold or more, preferably 10-fold or more, more preferably 20-fold or more, even more preferably 50-fold or more, and most preferably 100-fold or more, respectively. Furthermore, the Nanog and Lin28 mRNA or Nanog and Lin28 protein may be derived from either endogenous or exogenous Nanog and Lin28 genes.

[0033] Partial reprogramming may also be achieved by increasing the expression levels of immune evasion factors E3, K3, and B18R.

[0034] "Increasing the expression levels of immune evasion factors E3, K3, and B18R" may refer to increasing the expression levels of mRNA and / or protein of immune evasion factors E3, K3, and B18R (Hum Gene Ther. 2015 Nov;26(11):751-66.), or any combination thereof, where the expression level of at least one of the mRNA and protein of immune evasion factors E3, K3, and B18R is increased. Specifically, an increase in these expression levels may mean an increase of 5-fold or more, preferably 10-fold or more, more preferably 20-fold or more, even more preferably 50-fold or more, and most preferably 100-fold or more. Furthermore, immune evasion factors are typically factors used by viruses and the like to evade immunity, and the mRNA of the immune evasion factors E3, K3, and B18R or the proteins of the immune evasion factors E3, K3, and B18R may typically be derived from exogenous immune evasion factor E3, K3, and B18R genes, but this does not exclude cases where these genes are endogenously expressed or where the endogenous expression of these genes is increased.

[0035] Partial reprogramming may also be achieved by increasing the expression levels of microRNAs from the miR-302 / 367 microRNA cluster.

[0036] "Increasing the expression level of microRNAs derived from the miR-302 / 367 microRNA cluster" may refer to increasing the expression level of microRNAs known to be expressed from the miR-302 / 367 microRNA cluster (Hum Gene Ther. 2015 Nov;26(11):751-66.). The microRNAs whose expression levels are increased may be any of the microRNAs known to be expressed from the miR-302 / 367 microRNA cluster, or any microRNA. Specifically, the microRNA may include at least miR-302a. Increasing the expression level of microRNAs derived from the miR-302 / 367 microRNA cluster may refer to increasing the expression level of immature microRNAs such as pri-miRNAs and pre-miRNAs, increasing the expression level of mature microRNAs, or a combination thereof. Specifically, increasing the expression level may refer to increasing the expression level by 5-fold or more, preferably 10-fold or more, more preferably 20-fold or more, even more preferably 50-fold or more, and most preferably 100-fold or more. Additionally, the microRNAs may be mature or immature microRNAs and may be derived from either endogenous or exogenous miR-302 / 367 microRNA cluster genes.

[0037] When increasing the expression level of the above-exemplified reprogramming factors, the reprogramming factors present endogenously in immature cardiomyocytes may be activated, or the reprogramming factors may be exogenously introduced into immature cardiomyocytes and expressed. The activation of the reprogramming factors present endogenously in immature cardiomyocytes can be achieved, for example, by modifying the expression regulation mechanism of the endogenously present reprogramming factors.

[0038] The method for exogenously introducing reprogramming factors into immature cardiomyocytes is not particularly limited, but for example, the following method can be used.

[0039] When introducing genes in the form of DNA, vectors such as viruses, plasmids, and artificial chromosomes can be introduced into immature cardiomyocytes by techniques such as lipofection, liposomes, and microinjection. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and Sendai viral vectors. Examples of artificial chromosome vectors include human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs and PACs). Plasmids for mammalian cells can also be used. The vector can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, and polyadenylation sites to enable expression of the target gene. Furthermore, if necessary, it can contain selectable marker sequences such as drug resistance genes (e.g., kanamycin resistance genes, ampicillin resistance genes, puromycin resistance genes, etc.), thymidine kinase genes, and diphtheria toxin genes, as well as reporter gene sequences such as fluorescent proteins, β-glucuronidase (GUS), and FLAG. Examples of promoters include the SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF-α promoter, CAG promoter, and TRE promoter (a CMV minimal promoter containing a Tet-responsive element with seven consecutive tetO sequences). When the TRE promoter is used, it is desirable to simultaneously express a fusion protein of tetR and VP16AD or a fusion protein of reverse tetR (rtetR) and VP16AD in the same cell. Here, a vector containing the TRE promoter and capable of expressing a fusion protein of reverse tetR (rtetR) and VP16AD is an example of a drug-responsive inducible vector.Furthermore, the above vector may have transposon sequences before and after the expression cassette, in order to incorporate an expression cassette consisting of a promoter and a gene for a reprogramming factor that binds to the promoter into the chromosome of the cell and, if necessary, to excise the expression cassette. Examples of transposon sequences include, but are not limited to, piggyBac. In another embodiment, the vector may have LoxP sequences before and after the expression cassette for the purpose of removing the expression cassette.

[0040] When a drug-responsive induction vector is used, the timing of expression of the introduced reprogramming factor can be controlled. That is, the reprogramming factor can be introduced into cells in advance, and then expressed by adding a drug at the required time. For example, when obtaining immature cardiomyocytes from pluripotent stem cells as described below, the reprogramming factor can be introduced at the pluripotent stem cell stage, and when differentiation into immature cardiomyocytes is induced, a drug can be added to express the reprogramming factor. Any drug can be used that is appropriate in relation to the drug-responsive promoter, such as doxycycline. When using a vector containing a LoxP sequence, it is also possible to stop expression by introducing Cre into the cells after a desired period of time has elapsed.

[0041] When introduced in the form of RNA, it may be introduced into immature cardiomyocytes by techniques such as electroporation, lipofection, and microinjection. When introduced in the form of RNA, the RNA to be introduced includes mRNA encoding a reprogramming factor. When introducing a reprogramming factor in the form of RNA, it is preferable to co-introduce the aforementioned immune evasion factors (E3, K3L, B18). This is because immunogenic RNA activates the cellular defense mechanism, preventing the RNA encoding the reprogramming factor from differentiating.

[0042] When introduced in the form of a protein, it may be introduced into immature cardiomyocytes by techniques such as lipofection, fusion with a cell membrane-permeable peptide (for example, HIV-derived TAT and polyarginine), or microinjection.

[0043] The period for "increasing the expression level of reprogramming factors" in immature cardiomyocytes is not particularly limited as long as it is a period sufficient for immature cardiomyocytes to change into mature cardiomyocytes, and may be, for example, 12 hours or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 7 days or more, or 10 days or more, or 30 days or less, 20 days or less, 10 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less, or a compatible combination thereof. Specifically, the period for increasing the expression level of reprogramming factors may be, for example, 12 hours or more and 30 days or less, 1 day or more and 20 days or less, 2 days or more and 10 days or less, 5 days or more and 10 days or less, or 1 day or more and 3 days or less. When partial reprogramming is performed by transiently increasing the expression level of reprogramming factors in immature cardiomyocytes, there is an upper limit to the period for increasing the expression level of reprogramming factors. It is not necessary to continue the state in which the expression level of the reprogramming factor is increased after the immature cardiomyocytes have changed into mature cardiomyocytes, and it is preferable that the state in which the expression level of the reprogramming factor is increased is resolved after the immature cardiomyocytes have changed into mature cardiomyocytes. Furthermore, when the reprogramming factors include multiple genes, the period for increasing the expression level of each gene may be appropriately set for each gene.

[0044] The operation of "increasing the expression level of a reprogramming factor," such as the introduction of a reprogramming factor into cells, can be performed once or multiple times. For example, when the reprogramming factor is introduced in the form of a gene product, if the half-life of the gene product is short, the introduction may be performed multiple times. The number of times of introduction can be set appropriately, and examples include once, twice, three times, four times, five times, or more. Furthermore, when the reprogramming factor contains multiple genes, the number of times of introduction may be set appropriately for each gene.

[0045] The operation of "increasing the expression level of reprogramming factors" in immature cardiomyocytes can be initiated at an appropriate timing. For example, it may be initiated immediately after differentiation into immature cardiomyocytes has occurred. At this time, differentiation into immature cardiomyocytes can be confirmed by, for example, but not limited to, the expression of at least one cardiomyocyte marker gene selected from the group consisting of TNNI1, cardiac troponin (cTNT), and αMHC (α myosin heavy chain, MYH6), and can be confirmed by any appropriate means. For example, the operation of "increasing the expression level of reprogramming factors" may be performed on cells in which at least one of the cardiomyocyte marker genes is expressed.

[0046] By culturing immature cardiomyocytes after a partial reprogramming operation, immature cardiomyocytes can be converted (induced) into mature cardiomyocytes. As an example, immature cardiomyocytes can be converted (induced) into mature cardiomyocytes by culturing them in a state in which the expression level of reprogramming factors is increased, or after a transient state in which the expression level of reprogramming factors is increased. Culture conditions may be those used for culturing normal cardiomyocytes, for example, 30 to 40°C, preferably 36 to 38°C, and more preferably about 37°C. Furthermore, culture is preferably performed in an atmosphere of air containing oxygen and carbon dioxide, with the oxygen concentration preferably being about 5 to 20% and the carbon dioxide concentration preferably being about 2 to 5%.

[0047] The culture period is not particularly limited as long as it is sufficient for immature cardiomyocytes to transform into mature cardiomyocytes, and may be, for example, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, or 20 days or more. There is no particular upper limit, and culture may be continued as long as the properties of mature cardiomyocytes are maintained even after the immature cardiomyocytes have transformed into mature cardiomyocytes. Although this depends on the selection of reprogramming factors and / or their expression levels, as a guideline, mature cardiomyocytes can be obtained approximately 5 to 20 days after "increasing the expression level of the reprogramming factors."

[0048] The culture medium used for the partial reprogramming step and for culturing and maintaining immature cardiomyocytes after partial reprogramming can be any known medium, without particular limitation. Examples include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), StemPro34 (Invitrogen), StemFit AK02 medium (AJINOMOTO), Essential 6 medium (Thermo Fischer Scientific), and mixtures thereof. These media can be supplemented with known additives depending on the cells and culture conditions. For example, the medium may contain serum or may be serum-free. Furthermore, if necessary, the 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 1-thiolglycerol. It may also contain one or more substances, such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts. Furthermore, cytokines such as activin A, BMP4, bFGF, VEGF, and VEGF, as well as compounds such as mTOR inhibitors, GSK-3β inhibitors, and Wnt inhibitors, may be added as appropriate. Examples of mTOR inhibitors include Torin 1 (CAS: 1222998-36-8). The medium used to culture and maintain immature cardiomyocytes during the partial initialization step and after the partial initialization step may be the same medium or different media.

[0049] The medium used to culture immature cardiomyocytes after partial reprogramming preferably contains an mTOR inhibitor and further preferably contains Torin 1. In such cases, the content of Torin 1 contained in the medium may be, for example, 1 nM or more, 5 nM or more, 10 nM or more, 50 nM or more, 70 nM or more, 100 nM or more, 150 nM or more, 200 nM or more, or 500 nM or more, or 1000 nM or less, 500 nM or less, 300 nM or less, 250 nM or less, 200 nM or less, or 100 nM or less, or a compatible combination thereof. Specifically, the content of Torin 1 contained in the medium may be, for example, 1 nM or more to 1000 nM or less, 5 nM or more to 500 nM or less, 50 nM or more to 300 nM or less, 100 nM or more to 300 nM or less, 150 nM or more to 250 nM or less, or 200 nM.

[0050] As described above, immature cardiomyocytes that have been induced to differentiate from pluripotent stem cells can also be used.

[0051] Pluripotent stem cells are stem cells that have the pluripotency to differentiate into many cells present in the body and also have the ability to proliferate, and include any cells that can be induced into primitive endoderm. Pluripotent stem cells are not particularly limited, but include, for example, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, spermatogonial stem cells ("GS cells"), embryonic germ cells ("EG cells"), cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells). Preferred pluripotent stem cells are iPS cells and ES cells. Pluripotent stem cells are preferably derived from mammals, more preferably from primates, and even more preferably from humans.

[0052] Methods for producing iPS cells are known in the art, and iPS cells can be produced by, for example, introducing a reprogramming factor into any somatic cell. Examples of reprogramming factors include genes or gene products such as 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. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include those described in 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, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO 2010 / 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, Huangfu D, et al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 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, R. L. Judson et al. , (2009), Nat. Biotechnol. , 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci USA. 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.

[0053] Somatic cells used to generate iPS cells include 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. Specific 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 blood cells (peripheral blood cells, umbilical cord blood cells, etc.), 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.

[0054] Immature cardiomyocytes can be produced from pluripotent stem cells by known methods. Examples of methods for inducing differentiation of pluripotent stem cells into immature cardiomyocytes include those disclosed in the following documents: Laflamme MA & Murry CE, Nature 2011, May 19;473(7347):326-35; Review Funakoshi, S. et al. Sci Rep 8, 19111 (2016); Miki, K. et al. Cell Stem Cell. 2015 Jun 4;16(6):699-711. Other methods not specifically specified include, for example, a method for producing cardiomyocytes by forming cell masses (embryoid bodies) through suspension culture of induced pluripotent stem cells (WO2016 / 104614), a method for producing cardiomyocytes in the presence of a substance that inhibits bone morphogenic protein (BMP) signaling (WO2005 / 033298), and a method for producing cardiomyocytes in the presence of a substance that inhibits Activin I receptor signaling (WO2005 / 033298). Examples of such methods include a method for producing cardiomyocytes by sequentially adding cyclosporine A and BMP (WO2007 / 002136), a method for producing cardiomyocytes in the presence of a substance that promotes activation of the canonical Wnt signaling pathway (WO2007 / 126077), and a method for isolating FLk / KDR-positive cells from induced pluripotent stem cells and producing cardiomyocytes in the presence of cyclosporine A (WO2009 / 118928).Other methods include a method using cytokines to induce cardiomyocyte differentiation in embryoid body formation (Yang L, et al., Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature., 2008 May 22;453(7194):524-8), a method using adherent culture to induce cardiomyocyte differentiation without using cytokines (Lian X, et al., Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A., 2012 July 3;109(27):E1848-57), and a method using both adherent and suspension culture to induce cardiomyocyte differentiation without using cytokines (Minami I, et al., A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions. Cell Rep., 2012 Nov). 29;2(5):1448-60) have also been proposed.

[0055] When the immature cardiomyocytes are immature cardiomyocytes induced to differentiate from pluripotent stem cells, the "partial reprogramming" procedure can be initiated at an appropriate time. For example, in any of the above methods for producing cardiomyocytes or methods for inducing cardiomyocyte differentiation, the procedure may be initiated immediately after the immature cardiomyocytes have been induced to differentiate. In this case, the differentiation-induced immature cardiomyocytes may be cells in which the expression of at least one cardiomyocyte marker gene selected from the group consisting of TNNI1, cardiac troponin (cTNT), and αMHC (α myosin heavy chain, MYH6) has been confirmed. That is, for example, the "partial reprogramming" procedure may be performed on cells in which at least one of the cardiomyocyte marker genes is expressed.

[0056] Mature cardiomyocytes obtained by the production method of the present invention may be used as a cell preparation for transplantation into patients in need of cardiomyocyte transplantation. Examples of patients in need of cardiomyocyte transplantation include, but are not limited to, patients with diseases caused by cardiomyocyte deficiency, such as myocarditis, myocardial infarction, and myocardial injury. The amount of cells to be transplanted is selected appropriately depending on the type and severity of the disease, and the number of transplants may be one or multiple. The transplantation method is also not limited, and may be injection into the diseased area, or a cardiomyocyte cell sheet may be prepared and applied to the diseased area.

[0057] In one embodiment, mature cardiomyocytes obtained by the method of the present invention can be used in cardiac regenerative medicine. For example, a composition containing a cell mass of cardiomyocytes produced by the method of the present invention can be administered to the heart of a patient suffering from cardiac disease. Specifically, cardiomyocytes obtained by the method of the present invention can be transplanted into the heart of a patient with cardiac disease either as a cell suspension or in the form of a myocardial sheet (single or multilayer). For methods of producing myocardial sheets, see, for example, WO2012 / 133945, WO2013 / 137491, WO2014 / 192909, and WO2016 / 076368.

[0058] In another embodiment, the mature cardiomyocytes obtained by the method of the present invention are uniformly mature and can be used for drug screening for the treatment of heart disease or for evaluating the cardiotoxicity of drugs. For example, the efficacy and toxicity of a test drug can be evaluated by administering a test drug to the cardiomyocytes obtained by the method of the present invention and examining the response of the cardiomyocytes.

[0059] The present invention will be described in more detail below with reference to examples, but the aspects of the present invention are not limited to the following examples.

[0060] Example 1: Induction of differentiation of iPS cells into immature cardiomyocytes. 1390D4 cells were cultured in StemFit AK02N medium (Ajinomoto) on culture dishes coated with iMatrix511 (Nippi) as described by Nakagawa et al. (Sci. Rep. 2014;4:3594). Then, differentiation into cardiomyocytes was induced using a 6-well low-adhesion plate (Corning) using a modified protocol described by Dubois et al. (Nat Biotechnol. 2011 Oct 23;29(11):1011-1018).

[0061] Specifically, 1390D4 cells were dissociated into single cells using 0.5× TrypLE select (Thermo Fisher Scientific) (1× TrypLE select diluted with 0.5 mM EDTA), and then the cells were incubated in 2 mM L-glutamine (Invitrogen), 4×10 -4 2 × 10 cells were suspended in medium supplemented with M monothioglycerol (MTG), 50 μg / ml ascorbic acid (AA), 150 μg / ml transferrin, 10 μM ROCK inhibitor (Y-27632), 0.5% Matrigel (Corning), and 2 ng / ml BMP4 (R&D Systems). 6 Cells were seeded at 1.5 ml / well (Day 0: the day differentiation induction was initiated). Hereinafter, X days after the start of differentiation induction will be referred to as Day X.

[0062] On Day 1, 2 mM L-glutamine, 4 × 10 -4On day 3, 1.5 ml of StemPro-34 medium supplemented with MTG, 50 μg / ml AA, 150 μg / ml transferrin, 10 ng / ml bFGF (final 5 ng / ml), 12 ng / ml activin A (final 6 ng / ml), and 18 ng / ml BMP4 (final 10 ng / ml) was added to the wells. On day 3, the formed EBs were washed once with Iscove's modified Dulbecco's medium (IMDM; Invitrogen) and then resuspended in 2 mM L-glutamine, 4 × 10 -4 The cells were cultured in 3 ml of StemPro-34 medium supplemented with MTG, 50 μg / ml AA, 150 μg / ml transferrin, 10 ng / ml vascular endothelial growth factor (VEGF; R&D Systems), 1 μM IWP3 (Stemgent), 0.6 μM dorsomorphin, and 5.4 μM SB431542. On day 6, the medium was changed to 2 mM L-glutamine, 4 × 10 -4 The medium was replaced with 2 ml of StemPro-34 medium supplemented with MTG, 50 μg / ml AA, 150 μg / ml transferrin, and 5 ng / ml VEGF. The medium was then replaced with the same medium every 2–3 days. The plates were placed in a hypoxic environment (5% O2) from Day 0 to Day 10, after which they were transferred to a normoxic environment. Immature cardiomyocytes were obtained on Day 14.

[0063] Example 2 Partial Reprogramming of Immature Cardiomyocytes Day 14 immature cardiomyocytes obtained in Example 1 were dissociated and cultured at 1 × 10 6 The cells were plated at a cell density of 100 cells / well for 3 days to allow them to adhere (= Day 17 immature cardiomyocytes). rd The reprogramming factors were introduced using the Gen Reprogramming Kit (REPROCELL) (reference: Hum Gene Ther. 2015 Nov;26(11):751-66.) (d0: the day partial reprogramming started). Hereafter, X days after the start of partial reprogramming will be referred to as dX. StemRNA 3 rdThe Gen Reprogramming Kit includes three kits, each containing RNA for the expression of Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28, microRNAs from the 302 / 367 microRNA cluster, and RNA for the expression of immune evasion factors E3, K3, and B18R. After one day, the cells were again reprogrammed with StemRNA 3. rd Reprogramming factors were introduced using the Gen Reprogramming Kit (d1: Figure 1A). After partial reprogramming, iPS cell-derived immature cardiomyocytes were immunostained with cardiac troponin (cTnT) antibody and analyzed by flow cytometry. The results are shown in Figure 1B. Cells after partial reprogramming maintained cTnT expression and retained cardiomyocyte characteristics.

[0064] Example 3 Maturation of Immature Cardiomyocytes After Partial Reprogramming Cardiomyocytes obtained on day 2 after the initiation of partial reprogramming in Example 2 were cultured in the presence of 200 nM Torin 1 until day 9 to induce maturation of the cardiomyocytes.

[0065] After induction of maturation, DNA samples were collected and DNA methylation was evaluated using a DNA methylation array (Infinium Methylation EPIC array). The results are shown in Figures 1C to 1F. rd Cardiomyocytes (control) that had been cultured in the same manner but without partial reprogramming by introducing reprogramming factors using the Gen Reprogramming Kit were also evaluated as a control group.

[0066] The density plot (Figure 1C) showing the degree of methylation (β value) in the entire region revealed greater demethylation in the partial reprogramming group (RNA) compared to the control group. The density plot (Figure 1D) showing DNA methylation in pluripotency-related regions (OCT4, SOX2, KLF4, NANOG) showed no difference in methylation between the control and RNA groups. The density plot (Figure 1E) showing DNA methylation in cardiomyocyte maturation-related gene regions (TNNI3, TNNI3K, MYH7, RYR2, CAV3) showed greater DNA demethylation in the partial reprogramming group (RNA) compared to the control group, suggesting that partial reprogramming induces epigenetic changes in maturation-related genes. Furthermore, the promoter region of the cardiomyocyte maturation-related gene MYH7 (top panel of Figure 1F) showed greater DNA demethylation in the partial reprogramming group (RNA) compared to the control group. On the other hand, demethylation of the promoter region of the MYH6 gene (lower panel of Figure 1F) was not confirmed in the partial reprogramming group (RNA).

[0067] Cardiomyocytes (RNA) in which partial reprogramming was performed in Examples 1 and 2 and maturation was induced up to day 9, and stem RNA 3 rdRNA and protein samples were collected from cardiomyocytes (control) cultured in the same manner but without partial reprogramming by introducing reprogramming factors using the Gen Reprogramming Kit. RNA-seq analysis was performed using the collected RNA to analyze the expression of genes related to cardiomyocyte maturation (maturation-associated genes). The results are shown in Figures 2A and 2B. While Torin 1 administration slightly increased the expression of maturation-associated genes in the control group, the partial reprogramming group (RNA) showed a more pronounced increase in maturation-associated gene expression (Figure 2A). In the partial reprogramming group (RNA), the expression of maturation-associated genes MYH7 and TNNI3 was significantly increased, while the expression of MYH6 and TNNI1, which are expressed in immature cardiomyocytes, was not (Figure 2B). Western analysis was performed using the collected protein samples. The results are shown in Figure 2C. Increased expression of maturation markers TNNI3 and TCAP was confirmed in the RNA group. These changes would normally take approximately one to two months to occur without partial reprogramming. Therefore, partial reprogramming of immature cardiomyocytes accelerates their maturation, enabling them to become mature cardiomyocytes in a significantly shorter time than previously possible.

[0068] Example 4: Seahorse Mitochondrial Stress Test. The mitochondrial stress test was performed using a Seahorse Bioscience XFPro extracellular flux analyzer. The day before the assay, 40,000 day-26 cardiomyocytes (CMs) were seeded onto fibronectin-coated Seahorse culture microplates. CMs were prepared according to the manufacturer's instructions. Compound concentrations were optimized for the cells as follows: oligomycin 1.5 μM, FCCP 2.0 μM, and rotenone + antimycin A 0.5 μM. Data were analyzed using Wave Pro software. The results are shown in Figure 3. The results indicated that mature cardiomyocytes in the partial reprogramming group (RNA) exhibited improved respiratory function and ATP production.

[0069] Example 5: Generation of engineered heart tissues (EHTs) Single-cell suspensions of 201B7-luc-FUCCI-MYH6-iRFP670 were prepared by enzymatic digestion using Accumax at 37°C for 15 minutes. After dissociation, the single cells were resuspended in DMEM supplemented with 1% P / S (penicillin / streptomycin) and 10% fetal bovine serum (FBS). EHTs were generated in 24-well plates using an EHT silicone rack (DiNAQOR Deutschland GmbH) and a device (Sumitomo Bakelite Co., Ltd.). 1 x 10 6 Cells were mixed with EHT master mix (NCM medium, fibrinogen, Matrigel basement membrane matrix, 2x concentrated DMEM, and Y-27632). For one EHT, 97 μl of master mix and 3 μl of thrombin (100 U / ml) were mixed and pipetted into the device, which was then placed in an EHT silicone rack at 37°C. After 2 hours of incubation, the EHT silicone rack with the attached EHT was transferred to a 24-well plate containing EHT medium (DMEM, heat-treated bovine serum, penicillin / streptomycin, aprotinin, and insulin). Two days after EHT generation, a combination treatment was administered to promote ectopic maturation of EHT.

[0070] To measure contractile force, EHTs were paced using a C-Pace EM (1.0 Hz, 5 ms, 5-10 V). A 15-second video of the contraction was recorded using a BZ-X800 (KEYENCE). Contractile force was calculated using ImageJ according to the following formula: Contractile force (N) = (3 × 3.14 × E × R 4 × distance (mm)) / (64 × L 3 ) Contraction force (N / mm 2 ) = P / (3.14 × (tissue width / 2) 2 ) where E = 1.7 (MPa; Young's modulus), R = 1.0 (mm; pole diameter), and L = 12 (mm; pole length). Three contractions were selected to determine the length, and the maximum diastole, maximum systole, and tissue width were measured. Each parameter was entered into the equation, and the contractile force was calculated. The results are shown in Figure 4. As a result, it was found that mature cardiomyocytes in the partial reprogramming group (RNA) had improved contractile function.

Claims

1. A method for producing mature cardiomyocytes, comprising a step of partially reprogramming immature cardiomyocytes.

2. The method for producing mature cardiomyocytes described in claim 1, wherein the partial reprogramming is carried out by transiently increasing the expression level of a reprogramming factor in immature cardiomyocytes.

3. The method for producing mature cardiomyocytes according to claim 2, wherein the reprogramming factors include at least Oct4, Sox2, Klf4, and cMyc.

4. The method for producing mature cardiomyocytes according to claim 2 or 3, wherein the reprogramming factors further include Nanog and Lin28.

5. A method for producing mature cardiomyocytes described in claim 2 or 3, wherein transiently increasing the expression level of the reprogramming factor is carried out by introducing mRNA encoding the reprogramming factor into immature cardiomyocytes.

6. A method for producing mature cardiomyocytes according to claim 1 or 2, further comprising a step of culturing the immature cardiomyocytes that have been partially reprogrammed in the presence of an mTOR inhibitor.

7. The method for producing mature cardiomyocytes described in claim 6, wherein the mTOR inhibitor is Torin 1.

8. The method for producing mature cardiomyocytes described in claim 2 or 3, wherein the increase in expression level is maintained for two days or more.

9. The method for producing mature cardiomyocytes according to claim 1 or 2, wherein the cardiomyocytes are human cardiomyocytes.

10. A method for producing mature cardiomyocytes according to claim 1 or 2, wherein the immature cardiomyocytes are immature cardiomyocytes induced to differentiate from pluripotent stem cells.

11. The method for producing mature cardiomyocytes according to claim 10, wherein the pluripotent stem cells are induced pluripotent stem cells.

12. Mature cardiomyocytes produced by the method of claim 1 or 2.

Images