Method for producing mature cardiomyocytes

Abstract

Provided is a mature-cardiomyocyte production method including a step for increasing the expression level of cyclin-dependent kinase inhibitor 1 in immature cardiomyocytes.

Description

[Technical field]

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

[0002] In recent years, attempts have been made to induce 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. Several systems have been reported for inducing differentiation of pluripotent stem cells into cardiomyocytes, but most of the cardiomyocytes obtained are immature cardiomyocytes similar to fetal cardiomyocytes, and maturation manipulation is required for practical use.

[0003] The following methods have been known as methods 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 cardiomyocyte maturation promoters (e.g., Patent Document 1) 5) Transient expression of the Sall1 gene and the Mesp1 gene (for example, Patent Document 2) [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2019 / 189554 Brochure [Patent Document 2] JP 2017-60422 A [Non-patent literature]

[0005] [Non-Patent Document 1] Circ J 2013;77:1307-1314 [Non-Patent Document 2] Proc Natl Acad Sci US A. 2015 May 26;112(21):E2785-94 [Non-Patent Document 3] Circ Res. 2015 December 4; 117(12): 995-1000. [Non-Patent Document 4] Nat Methods. 2013 August; 10(8): 781-787 [Non-Patent Document 5] Nature. 2018 April; 556(7700): 239-243 [Non-Patent Document 6] Circulation. 2016 November 15; 134(20): 1557-1567 Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, several techniques for maturing cardiomyocytes have been reported, but there is room for improvement in terms of cost and technology. Therefore, an objective of the present invention is to provide a method for efficiently obtaining mature cardiomyocytes that can be used for drug discovery screening and cell transplantation in a short period of time. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems, and first found that expression of CDKN1A (cyclin-dependent kinase inhibitor 1, also known as p21) increases during the process of cardiomyocyte maturation using a cardiomyocyte maturation promoter (Patent Document 1). They then found that increasing the expression level of CDKN1A in immature cardiomyocytes allows for efficient production of mature cardiomyocytes in a short period of time, and have completed the present invention.

[0008] The gist of the present invention is as follows. [1] A method for producing mature cardiomyocytes, comprising the step of increasing the expression level of cyclin-dependent kinase inhibitor 1 in immature cardiomyocytes. [2] The method for producing mature cardiomyocytes described in [1], wherein the increase in the expression level is maintained for three days or more. [3] The method for producing mature cardiomyocytes described in [1] or [2], wherein the cardiomyocytes are human cardiomyocytes. [4] The method for producing mature cardiomyocytes according to any one of [1] to [3], wherein the immature cardiomyocytes are immature cardiomyocytes induced to differentiate from pluripotent stem cells. [5] The method for producing mature cardiomyocytes described in [4], wherein the pluripotent stem cells are induced pluripotent stem cells. [6] Mature cardiomyocytes proliferated by any of the methods described in [1] to [5]. Effect of the Invention

[0009] 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 with differentiation induction from pluripotent stem cells, mature cardiomyocytes can be easily and mass-produced. [Brief description of the drawings]

[0010] [Figure 1] 1 is a schematic diagram showing one embodiment of the differentiation induction method of the present invention. "Day" indicates the number of days from the start of differentiation induction. [Diagram 2] This is a schematic diagram of the changes detected during the differentiation and maturation of reporter iPS cells (1390D4 strain) used in the Examples into cardiomyocytes. Since EmGFP is inserted under the control of the TNNI1 gene promoter and mCherry is inserted under the control of the TNNI3 gene promoter, when the cells are induced to differentiate into cardiomyocytes, they first produce EmGFP-positive, mCherry-negative immature cardiomyocytes. Then, as maturation progresses, the expression level of EmGFP decreases and mCherry expression is induced, so they eventually become EmGFP-negative, mCherry-positive mature cardiomyocytes. [Diagram 3]Figure 3A shows the results of gene ontology (GO) enrichment analysis of mature cardiomyocytes (maturation promoter-treated cells) obtained from reporter iPS cells using a cardiomyocyte maturation promoter and immature cardiomyocytes (non-promoter-treated cells) obtained without treatment with the promoter. Figure 3B shows a list of genes included in "Kinase inhibitor activity" in Figure 3A. [Figure 4] Using the Gene Expression Database (GEO, Acc. No. GSE51483) of the National Center for Biotechnology Information (NCBI), the results of analysis of the time course of expression (mRNA amount) of CDKN1A gene and TNNI3 gene during mouse heart development (A) and the correlation of the expression of both genes (B). The analyzed samples at each stage in A are as follows: E8.5 heart tubes, left and right ventricle tissues at E9.5, E12.5, E14.5, E18.5, 3 days after birth (Postnatal 3d), adult heart (mCM). [Diagram 5] The results of analyzing the mRNA levels of CDKN1A and HOPX genes in human ES cells and mature cardiomyocytes obtained from the cells using the cardiomyocyte maturation promoter are shown (A, C). B is a graph analyzing the correlation between the expression levels of CDKN1A and HOPX genes in human cardiomyocytes using GEO:GSE46224. D shows the results of analyzing the mRNA levels of CDKN1A gene in human adult and human fetal cardiomyocytes using the above-mentioned GEO:GSE50704. HOPX is a gene that has been suggested to contribute to heart development by regulating serum response factor (SRF)-dependent cardiac-specific gene expression. [Figure 6] These graphs show the correlation between the expression levels of the CDKN1A gene and the TNNI3 gene (A), and the CDKN1A gene and the HIC1 gene (B) during human heart development, analyzed using the above GEO:GSE46224. [Figure 7]Mature cardiomyocytes (cells treated with promoters) and immature cardiomyocytes (cells not treated with promoters) obtained from reporter iPS cells were analyzed for the expression levels of cardiomyocyte-specific genes (Myh6: myosin heavy chain, α isoform (MHC-α), Myl7: myosin light chain 7, Ttn: titin, cTnT (=TNNT): troponin T2, cardiac type) and fibroblast-specific genes (Tgfbi: transforming growth factor, beta-induced, 68 kDa, Col11a1: collagen type XI alpha 1 chain, Fbln1: fibulin 1, Thy: thymidylate synthase) in promoter-treated cells compared to non-treated cells (A), and the results of principal component analysis (PCA, n=3) at 7, 14, and 21 days after differentiation induction (B). [Figure 8] During the process of differentiating reporter iPS cells into mature cardiomyocytes using a cardiomyocyte differentiation inducer, the results of an analysis of genes whose expression levels changed between Day 7 and Day 14, and between Day 7 and Day 21 (heat map: A), the results of an analysis of the number of genes whose expression increased or decreased between Day 7 and Day 21 (Venn diagram: B), and the change in mRNA levels of the TNNI3 or CDKN1A genes over time (C) are shown. [Figure 9] This is a graph showing an analysis of the mRNA levels of CDKN1A and TNNI3 at long time points (Day 40, Day 60) after the start of differentiation induction in mature cardiomyocytes obtained from reporter iPS cells by treatment with a maturation promoter. [Figure 10] The results of analyzing cardiomyocyte maturation-related genes and CDKN1A in mouse fetal hearts using GSE1479 data from GEO (Gene Expression Omnibus) are shown below. A shows the time course of CD36 gene expression (n=6), B shows the correlation analysis (Pearson correlation) between CD36 and CDKN1A, and C shows the correlation analysis (Pearson correlation) between HOPX and CDKN1A. [Figure 11]This graph shows the results of an analysis of differentially expressed genes (DEGs) in pathways related to CDKN1A during fetal myocardial development using GEO's GSE51483 data. * indicates p-value<0.05, ** indicates p-value<0.01, and *** indicates p-value<0.001 (Pearson r). [Figure 12] Heat maps showing the results of analysis of the atria and ventricles of mouse fetal hearts. (A) shows the time course of maturation-related gene expression in the atrial myocardium, and (B) shows the time course of maturation-related gene expression in the ventricle. [Figure 13] In the process of producing mature cardiomyocytes from reporter iPS cells by the method shown in Fig. 1 (a method of introducing and expressing an exogenous CDKN1A gene into immature cardiomyocytes to mature them), the results of analyzing cells 4 or 5 days (Day 20 or Day 21) after the introduction (start of expression) of the exogenous CDKN1A gene are shown. A is a graph showing the analysis results of the fluorescence intensity of mCherry and GFP. The image in B is a fluorescent micrograph detecting the fluorescence of mCherry and GFP, and the graph in B is a graph analyzing the amount of mRNA of a group of genes involved in the maturation of cardiomyocytes. "CDKN1A" in A and B represents cells infected with a lentivirus (virus particle) encoding the CDKN1A gene. In addition, "pLenti6.3" in A and "Control" in B both represent cells (negative control) infected with a lentivirus (virus particle) not encoding the CDKN1A gene. [Figure 14] The results are from an analysis of mature cardiomyocytes obtained from reporter iPS cells by the method described in Figure 1. A shows a heat map of the mRNA levels of a set of genes related to cardiomyocyte maturation compared to negative control cells. B shows phase contrast microscopy images and fluorescent observation images of TNN3 and / or TNN1 for mature cardiomyocytes on Day 60 (all images taken from the same field of view). [Figure 15]The results of evaluating the maturation-inducing effect of CDKN1A in more immature early cardiomyocytes are shown in (A). The workflow for the CDKN1A expression experiment in early immature cardiomyocytes is shown in (A). "Day" indicates the number of days from the start of differentiation induction. CDKN1A was expressed in cardiomyocytes on day 14. (B) is a graph showing the mRNA expression of CDKN1A and TNNI3 on ​​day 30 analyzed by quantitative PCR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The method for producing mature cardiomyocytes of the present invention comprises the step of increasing the expression level of cyclin-dependent kinase inhibitor 1 (CDKN1A) in immature cardiomyocytes.

[0012] In the present invention, cardiomyocytes refer to cells expressing at least one marker gene selected from the group consisting of cardiac troponin (cTNT), αMHC (α myosin heavy chain, MYH6), and βMHC (MYH7). Examples of cTNT include NCBI accession number NM_000364 for humans and NM_001130176 for mice. Examples of αMHC include NCBI accession number NM_002471 for humans and NM_001164171 for mice. Examples of βMHC include NCBI accession number NM_000257 for humans and NM_080728 for mice. Although the cardiomyocytes are not particularly limited, they are preferably derived from mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, pigs, monkeys, and humans), and more preferably derived from humans.

[0013] It is known that 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.).

[0014] In this specification, immature cardiomyocytes refer to cardiomyocytes in which the expression level of TNNI3 is very low and the expression level of TNNI1 is predominant. Immature cardiomyocytes are also called fetal-like cardiomyocytes.

[0015] In this specification, mature cardiomyocytes refer to cardiomyocytes in which the expression level of TNNI1 is very low and TNNI3 is predominantly expressed. Mature cardiomyocytes are also called adult-like cardiomyocytes. For example, when the expression levels of TNNI3 and TNNI1 are measured at the gene or protein level and compared after standardization using the expression levels of constitutive expression markers, cardiomyocytes in which the expression level of TNNI3 is 5 times or more, more preferably 10 times or more, even more preferably 25 times or more, and particularly preferably 100 times or more, of the expression level of TNNI3 in fetal cardiomyocytes can be considered as mature cardiomyocytes.

[0016] The expression levels of TNNI3 and TNNI1 can be analyzed, for example, by measuring the amount of mRNA of these genes using PCR, etc.; by analyzing the amount of protein expression by Western blotting, etc.; by analyzing the amount of reporter molecule expression; or by analyzing using a microscope or flow cytometer based on the fluorescence intensity of fluorescent labels or fluorescent reporters.

[0017] In addition, the maturity of cardiomyocytes may be determined by, in addition to the expression levels of TNNI3 and TNNI1, morphology, structure (e.g., sarcomere, mitochondria), properties (e.g., pulsation state, electrophysiological maturity), etc. For example, the depth of the resting membrane potential by patch clamp or the like can be used as an indicator of electrophysiological maturity. Indicators of the fine structure of sarcomeres and mitochondria can be observed by electron microscopy; analyzed by a microscope or flow cytometer using fluorescent labeling; or functionally analyzed by 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 to be evaluated are mature or immature.

[0018] As used herein, CDKN1A refers to cyclin-dependent kinase inhibitor 1 (also known as p21), and there are no particular limitations on its sequence or the biological species from which it is derived, so long as it is capable of exerting the function of maturing immature cardiomyocytes. For example, an example of the human CDKN1A gene is the gene registered as Gene ID 1026 in NCBI (https: / / www.ncbi.nlm.nih.gov / gene / 1026). The nucleotide sequence of this CDKN1A gene is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2. The CDKN1A gene may be a gene that hybridizes with a polynucleotide having a complementary nucleotide sequence of SEQ ID NO: 1 under stringent conditions (e.g., conditions of washing with 0.1×SSC, 0.1% SDS after hybridization at 65° C.), so long as it is capable of exerting the function of maturing immature cardiomyocytes. In addition, the CDKN1A protein may be a protein having an amino acid sequence that has 90% or more, 95% or more, or 98% or more identity with the amino acid sequence of SEQ ID NO: 2, so long as it is capable of exerting the function of maturing immature cardiomyocytes.

[0019] As used herein, "increasing the expression level of CDKN1A" refers to increasing the expression level of CDKN1A mRNA and / or CDKN1A protein, specifically meaning increasing 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. The increase may be based on, for example, the expression level of CDKN1A in immature cardiomyocytes or fetal cardiomyocytes in which the expression ratio of TNNI1 / TNNI3 is 10 or more. Furthermore, the CDKN1A mRNA or CDKN1A protein may be derived from either an endogenous or exogenous CDKN1A gene.

[0020] When increasing the expression level of CDKN1A mRNA and / or CDKN1A protein, the CDKN1A gene present endogenously in immature cardiomyocytes may be activated, or the CDKN1A gene may be exogenously introduced into immature cardiomyocytes to be expressed. The CDKN1A gene present endogenously in immature cardiomyocytes may be activated, for example, by modifying the expression regulation mechanism of the endogenous CDKN1A gene.

[0021] The method for exogenously introducing the CDKN1A gene into immature cardiomyocytes is not particularly limited, but for example, the following method can be used.

[0022] When the gene (DNA) is introduced, for example, vectors such as viruses, plasmids, and artificial chromosomes can be introduced into immature cardiomyocytes by lipofection, liposomes, microinjection, and other techniques. 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 (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). Plasmids for mammalian cells can be used. The vector can contain control sequences such as promoters, enhancers, ribosome binding sequences, terminators, and polyadenylation sites so that the target gene can be expressed, and can further contain, as necessary, selection marker sequences such as drug resistance genes (e.g., kanamycin resistance genes, ampicillin resistance genes, and puromycin resistance genes), thymidine kinase genes, and diphtheria toxin genes, and reporter gene sequences such as fluorescent proteins, β-glucuronidase (GUS), and FLAG. Examples of promoters include 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 (CMV minimal promoter having a Tet responsive sequence with 7 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 having a TRE promoter and capable of expressing a fusion protein of reverse tetR (rtetR) and VP16AD is an example of a drug-responsive induction vector.In addition, the above vector may have a transposon sequence before and after the expression cassette, in order to incorporate an expression cassette consisting of a promoter and a CDKN1A gene bound thereto into the chromosome of a pluripotent cell, and further excise the expression cassette as necessary. The transposon sequence is not particularly limited, but an example thereof is piggyBac. In another embodiment, the vector may have a LoxP sequence before and after the expression cassette for the purpose of removing the expression cassette.

[0023] When a drug-responsive induction vector is used, the expression time of the introduced CDKN1A gene can be controlled. That is, the CDKN1A gene can be expressed by introducing the CDKN1A gene into cells in advance and adding a drug at the required time. For example, when obtaining immature cardiomyocytes from pluripotent stem cells as described below, the CDKN1A gene can be introduced at the stage of pluripotent stem cells, and when the cells are induced to differentiate into immature cardiomyocytes, a drug can be added to express the CDKN1A gene. As the drug, a drug appropriate in relation to the drug-responsive promoter can be used, for example, doxycycline, etc. can be used. When a vector having a LoxP sequence is used, it is also possible to terminate expression by introducing Cre into the cells after a desired period of time has elapsed.

[0024] When it is introduced in the form of RNA, it may be introduced into pluripotent stem cells by techniques such as electroporation, lipofection, and microinjection.

[0025] When it is introduced in the form of a protein, it may be introduced into pluripotent stem cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (eg, HIV-derived TAT and polyarginine), and microinjection.

[0026] The period for "increasing the expression level of CDKN1A" in immature cardiomyocytes may be a period sufficient for immature cardiomyocytes to change into mature cardiomyocytes, and is not particularly limited, 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, or 10 days or more. There is no particular upper limit, and the state in which the expression level of CDKN1A is increased may be maintained even after immature cardiomyocytes have changed into mature cardiomyocytes, but it is not necessarily necessary to continue the state in which the expression level of CDKN1A gene is increased after immature cardiomyocytes have changed into mature cardiomyocytes.

[0027] The operation of "increasing the expression level of CDKN1A", such as introduction of the CDKN1A gene into cells, can be carried out once or multiple times. For example, when CDKN1A is introduced in the form of a gene product, if the half-life of the gene product is short, the introduction may be carried out multiple times. The number of introductions can be appropriately set, and examples include two, three, four, five or more times.

[0028] The operation of "increasing the expression level of CDKN1A" in immature cardiomyocytes can be started at an appropriate timing. For example, it may be started immediately after differentiation into immature cardiomyocytes occurs. At this time, differentiation into immature cardiomyocytes can be confirmed by, for example, the expression of at least one cardiomyocyte marker gene selected from the group consisting of TNNI1, cardiac troponin (cTNT), αMHC (α myosin heavy chain, MYH6) and βMHC (MYH7), but is not limited thereto, and can be confirmed by appropriate means. As an example, the operation of "increasing the expression level of CDKN1A" may be performed on cells in which at least one of the cardiomyocyte marker genes is expressed.

[0029] By culturing immature cardiomyocytes under conditions in which "CDKN1A expression levels are increased," it is possible to convert (induce) immature cardiomyocytes into mature cardiomyocytes. The culture conditions may be those used for normal cardiomyocyte culture, for example, 30 to 40° C., preferably 36 to 38° C., and more preferably about 37° C. In addition, the culture is preferably performed in an atmosphere of air containing oxygen and carbon dioxide, with the oxygen concentration being preferably about 5 to 20% and the carbon dioxide concentration being preferably about 2 to 5%.

[0030] The culture period is not particularly limited as long as it is sufficient for immature cardiomyocytes to change into mature cardiomyocytes, but similar to the period of "increasing the expression level of CDKN1A" described above, it is, 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, or 10 days or more. There is no particular upper limit, and the culture may be continued as long as the properties of mature cardiomyocytes can be maintained even after the immature cardiomyocytes have changed into mature cardiomyocytes. Although it depends on the expression level of CDKN1A, as a guideline, mature cardiomyocytes can be obtained about 12 to 13 days after "increasing the expression level of CDKN1A" (around day 30 in the method described in Figure 1). After that, the proportion of mature cardiomyocytes continues to increase, and after about 22 to 23 days (around day 40 in the method described in Figure 1), 70% or more, 80% or more, or 90% or more of the total cells become mature cardiomyocytes.

[0031] The medium used to culture and maintain immature cardiomyocytes in a state where the expression level of CDKN1A is increased can be any known medium without any particular limitation. For example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, 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 mixed media thereof can be used. These media can be supplemented with additives known per se depending on the cells and culture conditions. For example, the media may contain serum or may be serum-free. Furthermore, if necessary, the media 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, and 1-thiolglycerol, or may contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. Furthermore, cytokines such as activin A, BMP4, bFGF, VEGF, and VEGF, and compounds such as GSK-3β inhibitors and Wnt inhibitors may be added as appropriate.

[0032] The immature cardiomyocytes may be cardiomyocytes isolated from a living body, but are preferably immature cardiomyocytes induced to differentiate from pluripotent stem cells.

[0033] Pluripotent stem cells are stem cells that have the pluripotency to differentiate into many cells present in the living body and also have the ability to proliferate, and include any cell 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. The origin of pluripotent stem cells is preferably mammalian, more preferably primate, and even more preferably human.

[0034] Methods for producing iPS cells are known in the art, and iPS cells can be produced by introducing reprogramming factors into any somatic cells. 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, or 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, 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, 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, Huangfu D,et al. 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, RLJudson et al.,(2009), Nat.Biotechnol.,27:459-461, Lyssiotis CA,et al.(2009),Proc Natl Acad Sci US A.106:8912-8917, Kim JB,et al. (2009), Nature.461:649-643, Ichida JK, et al. (2009), Cell Stem Examples of the combinations include those described in 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, and Maekawa M, et al. (2011), Nature. 474: 225-9.

[0035] The somatic cells used to generate iPS cells include fetal (baby) somatic cells, neonatal (baby) 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 mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0036] Immature cardiomyocytes can be produced from pluripotent stem cells by a method known per se. Examples of methods for inducing differentiation of pluripotent stem cells into immature cardiomyocytes include the methods 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 examples include, but are not limited to, 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 suppresses bone morphogenic protein (BMP) signaling (WO2005 / 033298), a method for producing cardiomyocytes by sequentially adding Activin A and BMP (WO2007 / 002136), a method for producing cardiomyocytes in the presence of a substance that promotes activation of the canonical Wnt signal 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). In addition, we have reported a method of inducing cardiomyocyte differentiation using cytokines 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 of inducing cardiomyocyte differentiation without using cytokines in adhesion culture (Lian X, et al., Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling., Proc Natl Acad Sci US A., 2012 July 3;109(27):E1848-57), and a method of inducing cardiomyocyte differentiation without using cytokines by combining adhesion culture and suspension culture (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.

[0037] When the immature cardiomyocytes are immature cardiomyocytes induced to differentiate from pluripotent stem cells, the operation of "increasing the expression level of CDKN1A" can be started at an appropriate timing. For example, in any of the above-mentioned methods for producing cardiomyocytes or methods for inducing differentiation of cardiomyocytes, the operation may be started immediately after the immature cardiomyocytes are induced to differentiate. In this case, the differentiation-induced immature cardiomyocytes may be, for example, cells in which the expression of at least one cardiomyocyte marker gene selected from the group consisting of TNNI1, cardiac troponin (cTNT), αMHC (α myosin heavy chain, MYH6) and βMHC (MYH7) has been confirmed. That is, the operation of "increasing the expression level of CDKN1A" may be performed on cells in which at least one of the cardiomyocyte marker genes is expressed, for example.

[0038] In the present invention, mature cardiomyocytes obtained by increasing the expression of CDKN1A have excellent properties such as improved engraftment ability and the ability to function as myocardium when transplanted into myocardial tissue, and are therefore suitable for use as a cell preparation for transplantation to patients in need of cardiomyocyte transplantation. Patients in need of cardiomyocyte transplantation include, but are not limited to, patients with diseases caused by the loss of cardiomyocytes, such as myocarditis, myocardial infarction, and myocardial injury. The amount of cells to be transplanted is appropriately selected depending on the type and severity of the disease, and the number of transplants may be one or more. The method of transplantation is also not limited, and may be injection into the diseased area, or a cardiomyocyte sheet may be prepared and applied to the diseased area.

[0039] In one embodiment, the 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, the cardiomyocytes obtained by the method of the present invention may be transplanted into the heart of a patient with cardiac disease as a cell suspension or in the form of a myocardial sheet (single layer or multilayer). For the method of producing a myocardial sheet, see, for example, WO2012 / 133945, WO2013 / 137491, WO2014 / 192909, and WO2016 / 076368.

[0040] In another embodiment, the mature cardiomyocytes obtained by the method of the present invention are uniformly matured and can be used for drug screening for the treatment of cardiac diseases or for evaluating the cardiotoxicity of drugs. For example, the effect or 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. EXAMPLES

[0041] 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.

[0042] <Reporter cells> In order to easily detect the degree of maturation of cardiomyocytes, the reporter cells described in the examples of Patent Document 1 were used as cells for inducing differentiation into cardiomyocytes. The cells are double knock-in human iPS cell lines (1390D4 lines) in which the gene sequences of EmGFP and mCherry are inserted into the gene locus of TNNI1 and TNNI3, respectively, via the 2A sequence. In the 1390D4 line, TNNI1 and EmGFP, and TNNI3 and mCherry are each expressed polycistronically, so that the expression level of TNNI1 can be evaluated using the expression level of EmGFP as an indicator, and the expression level of TNNI3 can be evaluated using the expression level of mCherry as an indicator. Therefore, by using the 1390D4 strain, the change from immature cardiomyocytes to mature cardiomyocytes can be detected by a change in fluorescent color (Figure 2).

[0043] Method 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.) and differentiation into cardiomyocytes was performed using a 6-well low-attachment plate (Corning) according to a modified protocol described by Dubois et al. (Nat Biotechnol. 2011 Oct 23;29(11):1011-1018.). 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 with 2 mM L-glutamine (Invitrogen), 4×10 -4 The cells were suspended in medium supplemented with 1 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), and 2 × 10 6 Cells were seeded at 1.5 ml / well (Day 0: the day differentiation induction was started). Hereinafter, X days after the start of differentiation induction is referred to as Day X. On Day 1, 2 mM L-glutamine, 4 × 10 -4 1.5 ml of StemPro-34 medium supplemented with MTG, 50 μg / ml AA, 150 μg / ml transferrin, 10 ng / ml bFGF (5 ng / ml final), 12 ng / ml activin A (6 ng / ml final) and 18 ng / ml BMP4 (10 ng / ml final) 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 -4They 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 μ M IWP3 (Stemgent), 0.6 μ m dorsomorphin, and 5.4 μ m 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. Thereafter, the medium was replaced with the same medium every 2-3 days. The plate was placed in a hypoxic environment (5% O2) from day 0 to day 10, and thereafter was transferred to a normoxic environment. On day 14, immature cardiomyocytes were obtained.

[0044] Method 2: Maturation of immature cardiomyocytes using cardiomyocyte maturation promoters (positive control experiment) Using the following T112 and T623 as maturation promoters, mature cardiomyocytes were obtained from pluripotent stem cells by the method described in Test Example 2 of Patent Document 1. Specifically, T112 or T623 was added to the medium for 6 days from Day 10 to Day 16 (final concentration 40 nM). In the Examples, mature cardiomyocytes obtained by this method may be called "promoter-treated cells", and cells that were only induced to differentiate into cardiomyocytes without the promoter treatment (immature cardiomyocytes) may be called "promoter-untreated cells". For comparative analysis of both types of cells, cells from the same day after the start of differentiation induction were used. [ka]

[0045] Method 3: CDKN1A gene transfer using lentiviral vector (Example, Figure 1) Immature cardiomyocytes obtained by method 1 on day 14 were dissociated and cultured at 1 × 10 6 Cells were plated at a cell density of 100 cells / well and allowed to adhere for 3 days (= Day 17 immature cardiomyocytes). In parallel, HEK293FT cells were transfected with pLenti6.3-CDKN1A (pLenti6.3 / V5DEST, in which the human CDKN1A gene was cloned; obtained from Thermofisher Scientific) to package lentiviral particles containing the CDKN1A gene. The human CDKN1A cDNA clone was obtained from the DNA type derived from clone ID H04D013O09. The medium was replaced 24 hours after transfection, and the culture supernatant containing the viral particles was harvested 24 hours later. The immature cardiomyocytes on Day 17 were cultured in the HEK293FT-derived culture supernatant for 24 hours to be infected with the viral particles. After that, the culture was continued, and expression analysis at each time point and expression of reporter proteins were analyzed. A lentiviral vector encoding blue fluorescent protein (BFP) was used as a positive control for lentiviral infection.

[0046] Method 4: CDKN1A gene transfer to earlier immature cardiomyocytes using lentiviral vectors (Example) As in Method 3, culture supernatant containing lentiviral particles carrying the CDKN1A gene from pLenti6.3-CDKN1A was collected. Immature cardiomyocytes on Day 14 obtained by Method 1 were cultured in the culture supernatant for 24 hours to be infected with the viral particles. A lentiviral vector encoding blue fluorescent protein (BFP) was used as a positive control for lentiviral infection. After that, the culture was continued, and the expression of CDKN1A and TNNI3 was analyzed by quantitative RT-PCR at Day 30. GAPDH was also used as an internal control for quantitative RT-PCR.

[0047] result <Identification of new genes that contribute to cardiomyocyte maturation> A comparative analysis of gene expression profiles was performed between mature cardiomyocytes obtained from 1390D4 cells by method 1 and immature cardiomyocytes not treated with the promoter. Figure 3(A) shows the results of OG enrichment analysis of the gene group whose expression was enriched in the promoter-treated cells compared to the promoter-untreated cells. Several GO terms were found to be significantly enriched (p<0.05), and it was revealed that the expression of the gene group with kinase inhibitory activity as GO was significantly enriched. The gene group included the 15 types of genes shown in Figure 3 (Figure 3(B)).

[0048] Based on the results of various analyses, we focused on CDKN1A and analyzed the expression changes of the CDKN1A and TNNI3 genes during mouse cardiac development using the NCBI gene expression information database (GEO, Acc. No. GSE51483) (Figure 4). As cardiac development progressed, the expression levels of both genes increased, and CDKN1A expression reached a level similar to that of adult mice at the later stages of development (Fig. 4A). Furthermore, the transcription levels of both genes were significantly correlated (Fig. 4B).

[0049] Next, mature cardiomyocytes were produced from human ES cells using the maturation promoter, and the expression level of CDKN1A was analyzed (Figure 5). Expression of CDKN1A was barely detectable in human ES cells (Figure 5A), and expression of HOPX was also very low (Figure 5C). However, in mature cardiomyocytes derived from human ES cells, the expression levels of both CDKN1A and HOPX were significantly higher than in the ES cells (Figures 5A and 5C). Furthermore, the expression levels of both genes in human cardiomyocytes were significantly correlated (Figure 5B). Figure 5D shows the expression levels of CDKN1A in human adult and human fetal cardiomyocytes. The expression level of CDKN1A was significantly higher in human adult cardiomyocytes than in human fetuses. Therefore, it has been revealed that in human cardiomyocytes, expression of CDKN1A increases significantly with maturation, not only during the process of maturation of cardiomyocytes derived from pluripotent stem cells (including iPS cells and ES cells) by applying a maturation-promoting agent, but also during the process of maturation (naturally) in vivo.

[0050] Figure 6 shows the results of an analysis using GEO:GSE46224 of the correlation between the expression levels of the CDKN1A gene and the TNNI3 gene (A), and the CDKN1A gene and the HIC1 gene (tumor suppressor gene) (B) during human heart development. It can be seen that expression of the CDKN1A gene increases with heart development, and this increase correlates with increased expression of TNNI3 and HIC1. From these results, it is believed that CDKN1A controls the expression levels of HIC1 and TNNI3.

[0051] In mature cardiomyocytes obtained using the cardiomyocyte maturation promoter, the expression of cardiomyocyte-specific genes was significantly increased and the expression of fibroblast-specific genes was significantly decreased compared to untreated immature cardiomyocytes (Figure 7A). Furthermore, the results of principal component analysis 7, 14, and 21 days after differentiation induction (Figure 7B) revealed that the gene expression pattern changed dramatically between days 7 and 14.

[0052] Figure 8 shows a heat map analysis of genes whose expression levels changed between Day 7 and Day 14 or Day 7 and Day 21 in promoter-treated cells (A), and a Venn diagram of the number of genes whose expression increased or decreased between Day 7 and Day 21 (B). The expression of a large number of genes changed (Figure 8B), but it was shown that many of the genes whose expression increased or decreased on Day 21 had already increased or decreased at the time of Day 14 (Figure 8B). It was also revealed that the expression level of TNNI3 remained almost unchanged until Day 7, decreased on Day 14, and then increased, whereas CDKN1A continued to increase from Day 7 to Day 21 (Figure 8C). Furthermore, it was also revealed that the expression levels of CDKN1A and TNNI3 were significantly higher on Day 40 than on Day 20, and there was no significant difference between Day 40 and Day 60 (Figure 9).

[0053] Furthermore, using GEO:GSE1479, the correlation between the genes related to cardiomyocyte maturation and the expression of CDKN1A in mouse fetal hearts was examined. As a result, as shown in Figure 10, during the process of mouse heart development, a significant correlation was found between CDKN1A and CD36, a known mature marker gene, and HOPX1, a transcription factor related to maturation.

[0054] Also, using GEO:GSE51483, a group of genes (DEG; Differentially Expressed Genes) showing expression fluctuations during fetal myocardial development was extracted, and the results of pathway analysis of the signals related to CDKN1A are shown in Figure 11. For the analysis, R2: genomics analysis and visualization platform (http: / / r2.amc.nl / ) was used, and the high-expression group and the low-expression group were defined as those with a difference of 1 SD (standard deviation) or more from the average value. According to this result, significant correlations were found in pathways involving myocardial maturation, such as "cardiac muscle contraction and maturation".

[0055] Figure 12 shows the time-course changes in the expression of genes related to maturation, analyzed separately for the atria and ventricles of the mouse fetal heart using GEO:GSE51483. As a result, in both the atria and ventricles, similar to other genes related to maturation, it was found that the expression of CDKN1A increased over time.

[0056] From the above results, it was suggested that CDKN1A may contribute to the maturation of immature cardiomyocytes and is involved in the maturation of both the atria and ventricles, and may serve as a biomarker for maturation.

[0057] <Analysis of the effect of CDKN1A on promoting cardiomyocyte maturation> According to Method 3, a viral vector expressing the CDKN1A gene was introduced into immature cardiomyocytes obtained by inducing the differentiation of reporter iPS cells to examine the effect of CDKN1A on cardiomyocyte maturation. Four to five days after transfection (Day 20-21), the mCherry fluorescence intensity was significantly higher in the cardiomyocytes transfected with the CDKN1A expression vector than in the cardiomyocytes transfected with the negative control vector (Figure 13A, left graph), and the GFP fluorescence intensity was significantly lower (Figure 13A, right graph). In the cells transfected with the CDKN1A expression vector, many cardiomyocytes positive for both TNNI1 and TNNI3 were observed at Day 30, but many cardiomyocytes were TNNI1-negative and TNNI3-positive at Day 40 (Figure 13B, image panel). Furthermore, when the expression of genes related to cardiomyocyte maturation was analyzed, all of them were significantly increased in the cardiomyocytes transfected with the CDKN1A expression vector compared to the cardiomyocytes transfected with the negative control vector (Figure 13B, graph). Therefore, it was shown that increasing the expression level of CDKN1A in immature cardiomyocytes significantly promotes their maturation as cardiomyocytes. As a result, it was revealed that 30 days after the start of differentiation induction (Day 30), the expression level of TNNI1 decreased and the expression level of TNNI3 increased significantly, and after 40 days (Day 40), the expression level of TNNI1 was very low, and mature cardiomyocytes that predominantly expressed TNNI3 were obtained.

[0058] Next, the expression of a set of genes known to be involved in the maturation of cardiomyocytes was analyzed. As a result, the expression of all of the above genes, except for HCN4, was increased in cardiomyocytes transfected with the CDKN1A expression vector compared to cardiomyocytes transfected with the negative control vector (Figure 14A). Therefore, it was strongly suggested that when the expression level of CDKN1A is increased in immature cardiomyocytes, the expression of (original) genes involved in maturation is basically induced, accelerating maturation. It was also suggested that CDKN1A may directly or indirectly control the expression of known genes involved in the maturation of cardiomyocytes. Furthermore, when cardiomyocytes into which the CDKN1A expression vector had been introduced were observed under a microscope on day 60, elongated, deformed TNNI1-negative, TNNI3-positive mature cardiomyocytes were observed to be close to each other, similar to mature cardiomyocytes in vivo (Figure 14B).

[0059] Furthermore, the maturation-inducing effect of CDKN1A in more immature early cardiomyocytes was evaluated according to method 4. CDKN1A gene was introduced into immature cardiomyocytes on day 14 of differentiation induction using lentivirus, and gene expression was evaluated on day 30. A significant increase in TNNI3 expression was confirmed (Figure 15). These results confirmed that CDKN1A also has a mature cardiomyocyte-inducing effect in immature cardiomyocytes on day 14 of differentiation induction, which are cells that have just differentiated into earlier cardiomyocytes.

[0060] These results demonstrated that CDKN1A has the effect of promoting the maturation of immature cardiomyocytes by stimulating the expression of genes necessary for maturation.

Claims

1. A method for producing mature cardiomyocytes, comprising the step of increasing the expression level of cyclin-dependent kinase inhibitor 1 in immature cardiomyocytes by introducing the CDKN1A gene.

2. The method for producing mature cardiomyocytes according to claim 1 , wherein the increase in the expression level is maintained for 3 days or more.

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

4. The method for producing mature cardiomyocytes according to any one of claims 1 to 3, wherein the immature cardiomyocytes are immature cardiomyocytes induced to differentiate from pluripotent stem cells.

5. The method for producing mature cardiomyocytes according to claim 4 , wherein the pluripotent stem cells are induced pluripotent stem cells.

6. A method for producing mature cardiomyocytes described in any one of claims 1 to 5, wherein the CDKN1A gene is a gene encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 2 and that has the function of maturing immature cardiomyocytes.

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