Method for proliferating cardiomyocytes, method for producing cardiomyocytes, and cardiomyocyte proliferating agent

Abstract

Provided are a method for proliferating cardiomyocytes, a method for producing cardiomyocytes, and a cardiomyocyte proliferating agent capable of contributing to cardiac regenerative medicine, that make it possible to induce proliferation of human cardiomyocytes by a drug. The method for proliferating cardiomyocytes, method for producing cardiomyocytes, and cardiomyocyte proliferating agent, including a step for culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega hydroxylase inhibitors, and fatty acid synthesis inhibitors

Description

Method for growing cardiomyocytes, method for producing cardiomyocytes, and agent for growing cardiomyocytes

[0001] The present invention relates to a method for proliferating and producing cardiomyocytes and a cardiomyocyte proliferating agent for use therein. This application claims priority to U.S. Patent Application No. 63 / 157,834, provisionally filed in the U.S. on March 8, 2021, the contents of which are incorporated herein by reference.

[0002] Currently, there are just under 100,000 cases of myocardial infarction in Japan each year. If human cardiac tissue could be repaired after injury, it could dramatically reduce deaths from myocardial infarction. However, the human heart is generally considered to be an organ that does not regenerate. For example, the myocardium of zebrafish and one-week-old mice retains the ability to proliferate and can regenerate after cardiac injury. However, human cardiomyocytes have low proliferation capacity, with turnover of only a few percent per year, and are thought to have almost no cardiac regeneration capacity.

[0003] Based on the theory that the regenerative potential of the heart may be determined by the proliferation potential of cardiomyocytes, various studies have been conducted in the hope that cardiac regeneration may be possible by enabling proliferation of cardiomyocytes. However, no sufficiently effective method has been established to date. In particular, most reports have been presented on mouse cardiomyocytes and hearts, and there is little knowledge about human cells, which may have different mechanisms of proliferation arrest.

[0004] The present inventors have succeeded in inducing cardiomyocytes from mouse and human pluripotent stem cells with high efficiency, and have been searching for and identifying compounds that induce the proliferation of these cardiomyocytes. In Non-Patent Document 1, the present inventors have already identified and reported a compound cocktail that induces the proliferation of cardiomyocytes derived from mouse ES cells and human iPS cells.

[0005] Uosaki et al. , Circ Cardiovasc Genet. , 2013; 6:624-633.

[0006] As mentioned above, the present inventors have discovered a technique for inducing proliferation of cardiomyocytes derived from mouse ES cells and human iPS cells in Non-Patent Document 1. However, the circumstances under which proliferation ceases in mouse and human cardiomyocytes are different, and it is thought that there may be a mechanism specific to human cells. Therefore, it is expected that further elucidation of the human cell-specific mechanism and the discovery of methods and necessary factors for proliferation of human cardiomyocytes will make a significant contribution to cardiac regenerative medicine.

[0007] The present invention has been made in consideration of the above circumstances, and aims to provide a method for proliferating cardiomyocytes, a method for producing cardiomyocytes, and a cardiomyocyte proliferation agent that enable the induction of human cardiomyocyte proliferation using drugs and can contribute to cardiac regenerative medicine.

[0008] Embodiments of the present invention have the following aspects. [1] A method for proliferating cardiomyocytes, comprising a step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, and a fatty acid synthesis inhibitor. [2] The method for proliferating cardiomyocytes, wherein the compound is an ALK inhibitor. [3] The method for proliferating cardiomyocytes, wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor. [4] The method for proliferating cardiomyocytes, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox. [5] The method for proliferating cardiomyocytes, wherein the cardiomyocytes are cardiomyocytes derived from pluripotent stem cells. [6] The method for proliferating cardiomyocytes, wherein the pluripotent stem cells are derived from humans. [7] A method for producing cardiomyocytes, comprising a step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, and a fatty acid synthesis inhibitor. [8] The method for producing cardiomyocytes, wherein the compound is an ALK inhibitor. [9] The method for producing cardiomyocytes, wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.

[10] The method for producing cardiomyocytes, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.

[11] The method for producing cardiomyocytes, wherein the cardiomyocytes are cardiomyocytes derived from pluripotent stem cells.

[12] The method for producing cardiomyocytes, wherein the pluripotent stem cells are derived from humans.

[13] A cardiomyocyte proliferation agent, comprising one or more compounds selected from the group consisting of an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, and a fatty acid synthesis inhibitor as an active ingredient.

[14] The cardiomyocyte-proliferating agent as described above, wherein the compound is an ALK inhibitor.

[15] The cardiomyocyte-proliferating agent as described above, wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.

[16] The cardiomyocyte-proliferating agent as described above, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.

[17] The cardiomyocyte-proliferating agent as described above, wherein the cardiomyocytes are cardiomyocytes derived from pluripotent stem cells.

[18] The cardiomyocyte proliferation agent as described above, wherein the pluripotent stem cells are derived from a human.

[0009] According to the present invention, it is possible to provide a method for proliferating cardiomyocytes, a method for producing cardiomyocytes, and a cardiomyocyte proliferating agent that can induce proliferation of human cardiomyocytes using a drug and contribute to cardiac regenerative medicine.

[0010] 1 is a schematic diagram showing the procedure for inducing cardiomyocyte differentiation in this example. FIG. 2 is a graph showing the cTnT(+) % of cardiomyocytes in this example, and a schematic diagram of a cardiomyocyte culture plate. FIG. 3 is a schematic diagram showing the procedure for evaluating proliferation for selecting a positive control in this example. FIG. 4 is a photograph showing cell staining of each positive control compound in this example. FIG. 5 is a graph showing the amount of cells in S phase when each positive control compound in this example is added. FIG. 6 is a graph showing the amount of cells in M ​​phase when each positive control compound in this example is added. FIG. 7 is a schematic diagram showing the procedure for proliferation of cardiomyocytes in this example. FIG. 8 is a photograph showing immunostaining of proliferation of cardiomyocytes in this example. FIG. 9 is a graph showing cell number evaluation of proliferation of cardiomyocytes in this example. FIG. 10 is a graph showing the number of EdU+ / cTnT+ nuclei counted for a compound picked up in the first screening in this example. FIG. 11 is a graph showing the number of EdU+ / cTnT+ nuclei counted for another compound picked up in the first screening in this example. 1 is a graph showing the count of EdU+ / cTnT+ nuclei for another compound picked up in the first screening of this example. It is a photograph showing a portion of the immunostaining that was the basis for the count in this example. It is a schematic diagram showing the method of the cell number evaluation test in this example. It is a photograph showing a bright field after 1 day and 1+5 days of culture in the cell number evaluation test in this example. It is a graph showing the evaluation of the total cell number in the test using various ALK5 inhibitors in this example. It is a graph showing the evaluation of cTnT(+)% in the test using various ALK5 inhibitors in this example. It is a graph showing the evaluation of the cTnT(+) cell number in the test using various ALK5 inhibitors in this example. It is a schematic diagram showing one embodiment of the operation of inducing cardiomyocyte differentiation.

[0011] The method for proliferating cardiomyocytes, the method for producing cardiomyocytes, and the cardiomyocyte proliferating agent according to the present invention will be described below with reference to embodiments, although the present invention is not limited to the following embodiments.

[0012] (Method for Proliferating Cardiomyocytes) The method for proliferating cardiomyocytes of this embodiment includes a step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, and a fatty acid synthesis inhibitor.

[0013] An ALK inhibitor broadly refers to a component that inhibits the function of ALK (activin receptor-like kinase). ALK inhibitors are, for example, nucleic acids (such as siRNA, miRNA, and antisense oligonucleotides), proteins, or small molecule compounds.

[0014] The ALK inhibitor is preferably a TGF-β receptor type-1 (ALK5) inhibitor, and is preferably one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.

[0015] A cytochrome P450 omega-hydroxylase inhibitor broadly refers to a component that inhibits the function of cytochrome P450 omega-hydroxylase. The cytochrome P450 omega-hydroxylase inhibitor may be, for example, a nucleic acid (such as siRNA, miRNA, or antisense oligonucleotide), a protein, or a small molecule compound. A preferred cytochrome P450 omega-hydroxylase inhibitor is 12(S)-hydroxy-16-heptadecynoic acid.

[0016] Fatty acid synthesis inhibitor broadly refers to a component that inhibits fatty acid synthesis.Fatty acid synthesis inhibitor is, for example, nucleic acid (such as siRNA, miRNA, and antisense oligonucleotide), protein, or low molecular weight compound.Preferably, fatty acid synthesis inhibitor is C75 or cerulenin.

[0017] The amount of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors, i.e., the amount used in the method for growing cardiomyocytes described below, is preferably in the range of 0.5 to 40 μM, more preferably 2 to 10 μM, in terms of concentration relative to the medium, when used in culturing cardiomyocytes.

[0018] In this embodiment, cardiomyocytes broadly refer to myocardial cells, specifically cells with the ability to self-pulsate. Cardiomyocytes also include myocardial progenitor cells. Cardiomyocytes also broadly include myocardial cells that form pulsating muscles and electrically conductive tissue, and cells capable of generating vascular smooth muscle. Cardiomyocytes and myocardial progenitor cells may coexist or exist independently. Cardiomyocytes and myocardial progenitor cells broadly include cells that are positive for cardiac markers such as cardiac troponin (cTNT) or α-myosin heavy chain (αMHC). Cardiomyocytes also include cell populations that contain a higher proportion of cardiomyocytes than other cell types. Preferably, the cell population contains 50% or more cardiomyocytes, preferably 60% or 70% or more, more preferably 80% or more, and particularly preferably 90% or more.

[0019] The cardiomyocytes are preferably cardiomyocytes derived from pluripotent stem cells. Known pluripotent stem cells include ES cells, ntES cells, GS cells, EG cells, Muse cells, and iPS cells. In this embodiment, iPS cells are preferably used as the pluripotent stem cells. iPS cells are pluripotent stem cells induced from somatic cells, and have pluripotency and self-renewal capabilities. Pluripotency means the ability to differentiate into all three germ layer lineages. Furthermore, self-renewal capabilities means the ability to proliferate while maintaining an undifferentiated state.

[0020] Furthermore, the pluripotent stem cells are more preferably pluripotent stem cells derived from humans, and particularly preferably iPS cells derived from humans (human iPS cells, HiPS cells, human iPS cells).

[0021] iPS cells can be obtained by conventionally known methods. For example, the techniques described in K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676 or WO2007 / 069666 can be used. Specifically, iPS cells can be generated by introducing specific reprogramming factors in the form of DNA or protein into somatic cells. As the reprogramming factor, genes 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, or the proteins encoded thereby, can be used alone or in combination. When the reprogramming factor is DNA, it can be introduced into cells using vectors such as viruses, plasmids, or artificial chromosomes, lipofection, liposomes, or microinjection. When the reprogramming factor is a protein, it can be introduced into cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide, or microinjection.

[0022] Methods for obtaining cardiomyocytes derived from pluripotent stem cells, i.e., inducing differentiation of cardiomyocytes from pluripotent stem cells, can be performed by known methods. For example, the techniques described in Tohiyama, S., et al. (2013). Cell Stem Cell 12(1):127-137, Laflamme et al., Nat Biotech. 2007 Sep;25(9):1015-24, and Uosaki et al., Plos ONE. 2011;6(8):e23657 can be used. Alternatively, a pre-existing myocardial induction medium or an existing kit containing the medium can also be used.

[0023] Specifically, various cardiac differentiation inducers can be used to induce differentiation from pluripotent stem cells into cardiomyocytes. Examples of cardiac differentiation inducers include mesodermal induction factors (activin A, BMP4, bFGF, VEGF, SCF, etc.), cardiac determination factors (VEGF, DKK1, etc.), Wnt signal inhibitors (XAV939, IWR-1, IWP-2, IWP-4, etc.), BMP signal inhibitors (NOGGIN, etc.), TGFβ / activin / NODAL signal inhibitors (SB431542, etc.), retinoic acid signal inhibitors, and cardiac differentiation factors (e.g., VEGF, bFGF, DKK, etc.). These cardiac differentiation inducers can be used alone or in combination.

[0024] The procedure for inducing differentiation of pluripotent stem cells into cardiomyocytes involves first culturing the pluripotent stem cells and inducing their differentiation into mesoderm. Inducing differentiation into mesoderm can be achieved by culturing the pluripotent stem cells for 1 to 7 days in the presence of the mesoderm induction factor. For example, in this embodiment, culturing is preferably performed for 4 to 5 days in the presence of BMP4. Next, culturing is performed for 1 to 4 days in the presence of the cardiac differentiation induction factor. For example, in this embodiment, culturing is preferably performed for 2 to 3 days in the presence of the cardiac differentiation induction factor. It is more preferable to use the endogenous Wnt inhibitor Dkk-1 or the Wnt signal inhibitors XAV939 and IWP-4 as the cardiac differentiation induction factor. These cardiac differentiation induction factors may be used in combination, for example, XAV939 and IWP-4 may be used in combination. Furthermore, in this embodiment, it is also preferable to culture the pluripotent stem cells for 1 to 2 days in the presence of Matrigel as a pretreatment before inducing differentiation into mesoderm, and then further culture the cells in the presence of bFGF when inducing differentiation into mesoderm. This procedure induces differentiation into cardiomyocytes with higher efficiency, and pulsating cells appear around 4 to 10 days after the start of the culture to induce differentiation into mesoderm.

[0025] The method for growing cardiomyocytes of this embodiment includes a step of culturing the cardiomyocytes in the presence of one or more of the compounds. For example, the culture is preferably carried out in a medium to which one or more of the compounds have been added. The medium may contain other components in addition to the above components.

[0026] The culture for proliferation of cardiomyocytes may be carried out by isolating cardiomyocytes obtained by the differentiation induction or the like and transplanting them again onto a plate for culture. Alternatively, the culture for proliferation may be carried out again on the plate where the culture for differentiation induction was carried out. In this embodiment, it is preferable to seed the cardiomyocytes obtained by differentiation induction again at a low density and carry out the culture for proliferation of cardiomyocytes. The seeding density is approximately 0.1 to 5 × 10 ４ cells / cm ２ Preferably, it is 0.5 to 2.0 × 10 ４ cells / cm ２ is.

[0027] Culturing for proliferation of cardiomyocytes can be carried out by conventionally known means and conditions. For example, the culture temperature is 30 to 40°C, preferably about 37°C, and CO ２ The concentration is about 2% to 5%, O ２ The concentration is what is called a hypoxic condition, for example, about 5% to 20%.

[0028] The medium used for culturing cardiomyocytes is not particularly limited and can be selected appropriately depending on the purpose. For example, a basal medium can be used. Examples of basal media that can be used include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, α-MEM 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), and mixed media containing these. The basal medium may contain serum or may be serum-free. When serum is contained, a basal medium containing 5% to 20% by mass of serum can be used.

[0029] The medium may contain other components. Examples of other components include nutrients and other additives. Examples of nutrients include serum substitutes. Serum substitutes may include one or more of 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, or 1-thiolglycerol. Other components may include one or more of lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, or inorganic salts.

[0030] In this embodiment, for example, RPMI medium is used as the medium, and the cells are cultured in the presence of insulin as an antibiotic.

[0031] The cardiomyocytes obtained by the cardiomyocyte proliferation method of this embodiment can be used to treat human heart disease (cardiac disease). Here, cardiac disease broadly includes diseases of the heart or diseases requiring cardiac treatment. To use the cardiomyocytes of this embodiment for treatment, the obtained cardiomyocytes can be administered to a patient, or the obtained cardiomyocytes can be used as a therapeutic agent for heart disease. The cardiomyocytes can be administered by suspending the cells in a liquid and administering them to the myocardium, or by attaching the cells via a sheet, bandage, or the like. Cardiac disease broadly includes heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated phase hypertrophic cardiomyopathy, dilated cardiomyopathy, and other diseases, as well as defects due to damage, and is not limited as long as it affects the heart.

[0032] (Method for Producing Cardiomyocytes) The method for producing cardiomyocytes of this embodiment includes a step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, and a fatty acid synthesis inhibitor. The compound and the step of culturing cardiomyocytes can be selected from those described in the method for proliferating cardiomyocytes. That is, the method for producing cardiomyocytes of this embodiment preferably includes the steps in the method for proliferating cardiomyocytes. In other words, by adding the steps in the method for proliferating cardiomyocytes to the cardiomyocytes being cultured, the cardiomyocytes proliferate, and an amount of proliferated cardiomyocytes can be produced.

[0033] (Cardiomyocyte Proliferation Agent) The cardiomyocyte proliferation agent of this embodiment contains, as an active ingredient, one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. The compounds can be selected from those described in the cardiomyocyte proliferation method.

[0034] The cardiomyocyte proliferating agent of this embodiment may be used in the cardiomyocyte proliferating method and cardiomyocyte manufacturing method described above, i.e., may be added to proliferate cultured cardiomyocytes.

[0035] When the cardiomyocyte proliferation agent of this embodiment is used in a method for proliferation of cardiomyocytes and a method for producing cardiomyocytes, the cardiomyocyte proliferation agent of this embodiment can be used by adding it to the culture system of cardiomyocytes being cultured.

[0036] In another embodiment, the cardiomyocyte proliferation agent may be administered to a patient. In this case, the cardiomyocytes are preferably those within the patient's body. In particular, the cardiomyocytes are preferably those of a patient with a heart disease or in need of heart treatment. Examples of heart disease or conditions requiring heart treatment include those described in the cardiomyocyte proliferation method.

[0037] In this case, the cardiomyocyte proliferating agent may contain other components as appropriate. For example, it may contain components conventionally known to be used in the treatment of cardiac and other circulatory diseases. Furthermore, the cardiomyocyte proliferating agent containing an ALK inhibitor may also be used for its anti-cancer therapeutic effect.

[0038] (Effects of the Present Embodiment) The method for proliferating cardiomyocytes, the method for producing cardiomyocytes, and the cardiomyocyte proliferating agent of the present embodiment use an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, or a fatty acid synthesis inhibitor. These compounds have the effect of inducing the proliferation of human cardiomyocytes.

[0039] The present inventors conducted a compound screening using human iPS cell-derived cardiomyocytes to identify compounds capable of inducing human cardiomyocyte proliferation. As a result, in a compound library screening using human cardiomyocytes, the above-mentioned group of compounds with cardiomyocyte proliferation activity was identified. The compounds were also able to induce two or more divisions in human cardiomyocytes cultured for approximately three months. On the other hand, some cardiomyocytes showed no responsiveness to the compounds. We are currently working to clarify the differences between these two types of cardiomyocytes through comprehensive gene expression analysis. Furthermore, the compounds did not show any significant cardiomyocyte proliferation effects on mouse ES cell-derived cardiomyocytes, suggesting the existence of a proliferation control mechanism specific to human cardiomyocytes.

[0040] Previous reports on myocardial proliferation have shown that p38 inhibition mediates adult CM proliferation via cyclin A activation, neureglin1 mediates ERBB2-ERBB4-ERK signaling, the Hippo pathway mediates Yap-PI3K-Akt activation, followed by GSK-3β inactivation and IGF activation, miR-15 family inhibition mediates prolonged proliferation, and Meis1 deletion inhibits the activation of CDK inhibitors (p15, p16, p21). However, there have been no reports to date that the above-mentioned compound groups, ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, or fatty acid synthesis inhibitors, are involved in the proliferation of cardiomyocytes.

[0041] In particular, ALK inhibitors have been found to increase the number of human cardiomyocytes by approximately 1.5 to 2 times. Cardiomyocytes that respond well to ALK inhibitors have been confirmed to divide three times, which corresponds to an eightfold increase in cell number compared to before division. The effect of ALK inhibitors is specific to human cells, and their effect on mouse cardiomyocytes is weak. There have been no reports to date regarding the control of cardiomyocyte proliferation by ALK inhibitors. Furthermore, some ALK inhibitors have already undergone clinical trials as anticancer drugs, and their use as pharmaceuticals is possible.

[0042] In order to enable cardiomyocyte proliferation in humans and realize cardiac regenerative therapy, a clear effect on human cardiomyocytes is required, and the present invention has the potential to achieve this with any of the above-mentioned compound groups, i.e., a single compound, thereby solving the problems of the past. The cardiomyocyte proliferation method, cardiomyocyte production method, and cardiomyocyte proliferation agent of this embodiment are expected to contribute to the development of new cardiac regenerative medicine by elucidating the molecular mechanisms of myocardial proliferation and responsiveness to proliferation stimuli and enabling human cardiomyocytes to proliferate.

[0043] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following descriptions.

[0044] <Induction of Cardiomyocyte Differentiation> Due to the need to perform analyses using cardiomyocytes induced from human iPS cells, we attempted to establish a human iPS-cardiomyocyte proliferation screening system. The inventors have established a system (Uosaki et al., Plos ONE, 2011, etc.) that more efficiently differentiates into cardiomyocytes by adding Matrigel on day 1 and bFGF on days 1-5 to the 2D cardiomyocyte differentiation induction method DD-protocol (e.g., Laflamme et al., Nat Biotech, 2007, as mentioned above) (Figure 19). In this method, Dkk-1 is added in the presence of RPMI medium and B27 on days 5-7 to differentiate into cardiomyocytes. This differentiation system can also be further purified to isolate only cells expressing a certain marker (platelet-derived growth factor receptor α; PDGFRα) (Fukushima et al., PLoS One. 2020 Nov 2; 15(11): e0241287.), and this purification procedure was carried out in the following examples.

[0045] The procedure for inducing cardiomyocyte differentiation used in this example is outlined in chronological order in Figure 1. Human iPS cells (201B6) were provided by Professor Shinya Yamanaka of Kyoto University. bFGF (4 ng / mL, Final Concentration) was added to the iPS cells during culture in conditioned medium from day -2 or day -3, and Matrigel (1 / 60 of the medium volume) was further added from day -1 to day 0. The cells were cultured in RPMI medium + B27 insulin(-) from day 0 to day 5, with Activin A added from day 0 to day 1, and BMP4 (addition amount: 1 ng / mL, final conc.) and bFGF (addition amount: 10 ng / mL, final conc.) added from day 1 to day 5. On day 5, only cells expressing PDGFRα were purified and re-cultured in RPMI medium + B27 (addition amount: 2% of RPMI) insulin(+), and Wnt signal inhibitors (XAV939, IWP4) were further added from day 5 to day 7.

[0046] In this system, iPS cells at the start of culture became mesoderm cells from day 5, and pulsating cells appeared from around day 9, resulting in the production of cardiomyocytes. Figure 2 shows the cardiac troponin T expression rate (cTnT(+)%) of cells obtained using this system. This differentiation system was shown to produce cardiomyocytes with a cTnT(+)% of over 97% (graph on the left side of Figure 2). These cardiomyocytes were re-seeded on plates at a lower density and used for subsequent analysis (schematic diagram on the right side of Figure 2).

[0047] <Consideration of analytical conditions> First, the analysis conditions were examined by determining the point of differentiation of the myocardium to be used, the myocardial seeding conditions, the analytical conditions, etc. Figure 3 shows a schematic diagram of proliferation assessment (from Patent Document 1) for selecting a positive control. A positive control for proliferation assessment was established based on a compound discovered by the present inventors in a mouse cardiomyocyte proliferation screening system. Using mouse cardiomyocytes induced from ES cells and mouse cardiomyocytes isolated from embryo, neonate, and adult mice, myocardial proliferation analysis was performed using various kinase inhibitors (ZM336372, SU1498, KN62, KN93, BIO, CHIR, and SB203580 in the figure). Note that CHIR refers to CHIR99021.

[0048] Each compound was added to mouse ES-cardiomyocytes (mESCM) and cultured. Figure 4 shows cell staining in cultures treated with DMSO (control), BIO, SU1498, and KN93. In BIO, SU1498, and KN93, dark cardiomyocytes (cTnT) staining was observed, while light EdU and DAPI staining was observed. Figure 5 shows the cell mass in the S phase (EdU-positive CM), and Figure 6 shows the cell mass in the M phase (pH 3, i.e., phosphohistone positive). Figures 5 and 6 each show *P<0.05 compared to the DMSO group (Dunn test, n=3). These results indicate that BIO, SU1498, and KN93 exhibit myocardial proliferation potential. Similarly, the results of the investigations were summarized, and it was found that the GSK3β inhibitors BIO and CHIR99021, the p38 MAPK inhibitor (p38i) SB203580, and the ERK activator SU1498 had myocardial proliferation ability.

[0049] Next, when these compounds were added to human iPS-cardiomyocytes, only a slight myocardial proliferation effect was observed when each compound was added alone (not shown). On the other hand, as described below, the number of EdU+ / cTnT+ cell nuclei, the screening criteria, was relatively stable and exceeded 1.5 times. However, because BIO is cytotoxic to human iPS-cardiomyocytes, we decided to use Wnt3a, which is located upstream of BIO (Figure 3). Therefore, in subsequent screening systems, we used a combination of four drugs: Wnt3a, CHIR99021, p38i (SB203580), and SU1498, as positive controls.

[0050] <Proliferation of human cardiomyocytes> The stage of differentiation of the myocardium to be used, the myocardial seeding conditions, the analysis conditions, etc. were determined. The flow of cardiomyocyte proliferation is shown in Figure 7. Cardiomyocytes (d19) induced from human iPS cells were cultured under low density conditions (1.33 x 10 ４ cells / cm ２After 24 hours of culture in the presence of serum, cardiomyocytes were confirmed to have adhered to the plate, and each of the positive control compounds was added at the desired concentration. After two days of culture, 20 μM of EdU was added. After another day of culture, the cells were fixed and immunostained with cardiac troponin T (cTnT), a cardiomyocyte marker, and then photographed using an in Cell Analyzer 6000.

[0051] Representative photographs are shown in Figure 8. In cardiomyocytes expressing cTnT (dark in the photograph), EdU and DAPI (light in the photograph) were detected. The negative control showed cTNT and faint EdU signals, while the positive control showed cTNT and more abundant EdU and DAPI signals (respectively indicated by arrows). The number of EdU / cTnT nuclei (white arrows in the image) was counted and evaluated using analysis software (Developer Toolbox 1.9), and the results are shown in Figure 9. Addition of the positive control showed a more than 1.5-fold increase in the number of EdU / cTnT nuclei.

[0052] <Proliferation of human cardiomyocytes> After the conditions were met, Cayman Bio-active lipid screening library I & II were analyzed. Bio-active lipid screening library I contained over 785 compounds to be screened, stored in 1.0 mM DMSO in a 96-well matrix tube. Bio-active lipid screening library II contained over 185 compounds to be screened, stored in 0.1 mM DMSO in a 96-well matrix tube.

[0053] Other screening conditions were as follows: Cell seeding conditions: Cardiomyocytes induced to differentiate from hiPS cells were used. Cell seeding density: 1.33 x 10 ４ cells / cm ２The reagent was added the day after cell seeding (20 hours later). Evaluation conditions: cTnT(+) / EdU(+) nuclear numbers. 3 days (72 hours) after compound addition, cells were fixed and stained. EdU incorporation was evaluated at 24 hours. Compound conditions: Cayman Bio active lipid I & II screening library plate 1-11: 10 μM, 2 μM (stock: 1.0 mM) plate 12-14: 1 μM, 0.2 μM (stock: 0.1 mM)

[0054] 1st screening: - One well was set up for each condition. - Six visual fields were photographed per well. - Counts were performed in the six visual fields, and the values ​​were graphed with n = 6. Next, - Evaluation was performed using the EdU+ / cTnT+ nuclear numbers. - Those whose values ​​were 1.5 times higher than the negative control were selected. 2nd screening: - Three wells were set up for each condition. - Six visual fields were photographed per well. - Counts were performed in 18 (3 x 6) visual fields, and the values ​​were graphed with n = 18. Next, - Evaluation was performed using the EdU+ / cTnT+ nuclear numbers. - Those whose values ​​were significantly higher than the negative control were selected.

[0055] As a result of the screening, the number of hit compounds in the first screening was 103 out of 982 Cayman compounds. Next, in the second screening, three compounds showed a significant increase at both concentrations analyzed. These three compounds were: R2290-098: 12(S)-hydroxy-16-Heptadecynoic Acid, R2290-358: C75, and R2290-370: Cerulenin.

[0056] Compounds selected in the first screening were added at two concentrations: I; 10 μM, 2 μM; II; 1 μM, 0.2 μM, and analyzed in the same manner. EdU+ / cTnT+ nuclear numbers were counted and graphed. The results are shown in Figures 10, 11, and 12. As shown in Figure 10, R2290-098:12(S)-hydroxy-16-Heptadecynoic Acid and R2290-358:C75 showed significant increases when added at both concentrations. For R2290-370:Cerulenin, a significant increase was observed only at one concentration (2 μM). Furthermore, compounds that share the same target as these three compounds were found to significantly increase the number of proliferating cardiomyocyte nuclei at one of the added concentrations (Figures 11 and 12). These compounds were R2289-949: LY364947 and R2289-880: SB431542, respectively. Further analysis was conducted on the five compounds that were found to have myocardial proliferation effects, including these compounds.

[0057] Representative immunostaining photographs used to count the number of EdU / cTnT cell nuclei are shown in Figure 13. The photographs also show that the addition of each compound (C75, LY364947, 12(S)-hydroxy-16-hepatadecynoic acid, cerulenin, SB431542) tended to increase the number of EdU / cTnT nuclear numbers (bright signals) compared to the negative control.

[0058] <Evaluation by Cardiomyocyte Count> In the cardiomyocyte proliferation screening, EdU+ / cTnT+ nuclear numbers were used as an index of evaluation. EdU+ / cTnT+ indicates cardiomyocytes that underwent DNA synthesis during the period (24 hours) during which EdU was added. Unlike cardiomyocytes of rodents such as mice, most human cardiomyocytes are mononuclear cells, but some cells temporarily become binuclear during division (bi-nuclear cells), and an increase in EdU+ / cTnT+ nuclear numbers does not necessarily correspond to an increase in the number of cardiomyocytes. Therefore, conditions were set for a system in which evaluation was performed using the number of cardiomyocytes.

[0059] An outline of the evaluation method is shown in Figure 14. As in the case of screening, cardiomyocytes induced to differentiate from hiPS cells were seeded at low density (0.93 x 10 ４ cells / cm ２ After confirming cell adhesion, each of the above compounds was added. The number of cardiomyocytes was counted 1 day and 1+5 days later. cTNT FACS analysis was also performed.

[0060] Bright-field photographs of the cells after 1 day and 1+5 days of culture are shown in Figure 15. The cTnT(+) value in the negative control was 93.8%, and the cTnT(+) value in the positive control was 97.5%. It can be seen that the addition of the positive control tended to increase the number of cardiomyocytes.

[0061] Next, various ALK5 inhibitors were added, and analysis was performed to determine whether the number of cardiomyocytes increased. LY364947 and SB431542 are both ALK5 inhibitors, and as described above, an increase in the number of cardiomyocytes was observed. This increase in the number of cardiomyocytes may be due to inhibition of ALK5 signaling. Therefore, analysis was performed using other ALK5 inhibitors, A83-01 and RepSox. A83-01 is a potent inhibitor of ALK5 and is known to also inhibit ALK4 and ALK7. On the other hand, RepSox (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphththyridine) (shown as "TGFbR KI" in Figures 16 to 18) is a selective ALK5 inhibitor.

[0062] For each ALK5 inhibitor, the total cell count after addition is shown in Figure 16, the cTnT(+) % in Figure 17, and the number of cTnT(+) cells in Figure 18. n = 3 to 9. Of these, the addition of A83-01 in particular significantly increased the number of cardiomyocytes. RepSox (referred to as TGFbR KI) also increased the number of cardiomyocytes to a similar extent as SB431542. These results suggest that inhibiting ALK5 signaling leads to an increase in cardiomyocyte number, and that ALK5 inhibitors may be used as pharmaceutical compounds to increase cardiomyocyte number.

[0063] The method for proliferating cardiomyocytes, the method for producing cardiomyocytes, and the cardiomyocyte proliferating agent of the present invention enable the induction of human cardiomyocyte proliferation using drugs, and can contribute to cardiac regenerative medicine.

Claims

1. A method for proliferating cardiomyocytes, comprising culturing the cardiomyocytes in the presence of one or more compounds selected from the group consisting of an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, and a fatty acid synthesis inhibitor.

2. The method for proliferation of cardiomyocytes according to claim 1 , wherein the compound is an ALK inhibitor.

3. The method for proliferating cardiomyocytes according to claim 2 , wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.

4. The method for proliferating cardiomyocytes according to claim 3, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.

5. The method for proliferating cardiomyocytes according to any one of claims 1 to 4, wherein the cardiomyocytes are cardiomyocytes derived from pluripotent stem cells.

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

7. A method for producing cardiomyocytes, comprising culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, and a fatty acid synthesis inhibitor.

8. The method for producing cardiomyocytes according to claim 7 , wherein the compound is an ALK inhibitor.

9. The method for producing cardiomyocytes according to claim 8 , wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.

10. The method for producing cardiomyocytes according to claim 9, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.

11. The method for producing cardiomyocytes according to any one of claims 7 to 10, wherein the cardiomyocytes are cardiomyocytes derived from pluripotent stem cells.

12. The method for producing cardiomyocytes according to any one of claims 7 to 11, wherein the pluripotent stem cells are derived from a human.

13. An agent for proliferation of cardiomyocytes, comprising as an active ingredient one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.

14. The method of claim 13, wherein the compound is an ALK inhibitor.

15. The cardiomyocyte proliferation agent according to claim 14, wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.

16. The cardiomyocyte proliferation agent according to claim 15, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.

17. The cardiomyocyte proliferation agent according to any one of claims 13 to 16, wherein the cardiomyocytes are cardiomyocytes derived from pluripotent stem cells.

18. The cardiomyocyte proliferation agent according to any one of claims 13 to 17, wherein the pluripotent stem cells are derived from humans.

19. A method for proliferating cardiomyocytes described in any one of claims 1 to 6, wherein the compound comprises a fatty acid synthesis inhibitor.

20. A method for proliferating cardiomyocytes according to any one of claims 1 to 6, wherein the compound comprises C75 or cerulenin.

21. A method for producing cardiomyocytes described in any one of claims 7 to 12, wherein the compound comprises a fatty acid synthesis inhibitor.

22. A method for producing cardiomyocytes described in any one of claims 7 to 12, wherein the compound includes C75 or cerulenin.

23. A cardiomyocyte proliferation agent described in any one of claims 13 to 18, wherein the compound comprises a fatty acid synthesis inhibitor.

24. A cardiomyocyte proliferation agent described in any one of claims 13 to 18, wherein the compound comprises C75 or cerulenin.