Method for producing myocardial stem / progenitor cells and method for inhibiting myocardial fibrosis

Abstract

[Problem] To provide a method for efficiently reprogramming mature and immature cardiac muscle cells into cardiac muscle stem / precursor cells without involving genetic modification, and / or for inhibiting fibrosis. [Solution] According to the present invention, cardiac muscle stem / precursor cells can be maintained by culturing primary cardiac muscle cells in the presence of a ROCK inhibitor. Furthermore, fibrosis is inhibited by causing cardiac muscle cells cultured in the presence of these low-molecular-weight compounds, or exosomes or celectomes derived from the cardiac muscle cells, to act on cardiac fibroblasts.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international application claims priority based on Japanese Patent Application No. 2020-173581, filed with the Japan Patent Office on October 14, 2020, the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] The present invention relates to a method for producing cardiac stem / progenitor cells using a low molecular weight compound, a method for inhibiting fibrosis of cardiac cells, or a long-term culture method. [Background technology]

[0003] Remarkable advances in stem cell biology hold great promise for their application in myocardial regenerative medicine, but this has yet to be realized. Induced pluripotent stem cells (iPS cells), one of the most promising cell sources, still pose a risk of tumorigenesis. Although clinical studies have been conducted, practical application of iPS cells in clinical settings remains difficult (Non-Patent Documents 1-3). Meanwhile, recent research has demonstrated the possibility of direct reprogramming of cells of different lineages into cardiac progenitor-like cells. However, as with iPS cells, direct reprogramming involves genetic modification by introducing genes such as Gata4, Mef2c, and Tbx5, which poses unforeseen risks and has prevented their application in regenerative medicine (Non-Patent Document 4).

[0004] Recently, a series of reports have been published demonstrating the reprogramming of cardiac fibroblasts into cardiomyocytes (Non-Patent Document 5). These groundbreaking discoveries provide significant insights not only into myocardial stem cell theory but also into myocardial regeneration research. If such reprogramming can be reproduced, the resulting myocardial stem / progenitor cells are expected to become an innovative cell source for myocardial regenerative medicine. However, no method is known for reprogramming cardiomyocytes at various stages into myocardial stem / progenitor cells without genetic modification.

[0005] The present inventors and other groups have previously reported that combinations of certain small molecule inhibitors contribute to the induction and maintenance of stem cell pluripotency in the liver, stomach, etc. (Patent Document 1, Non-Patent Documents 6-9). However, there have been no reports to date on the relationship of small molecule inhibitors to the reprogramming of mature or immature cardiomyocytes into cardiac stem / progenitor cells. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Retable 2018-079714 [Non-patent literature]

[0007] [Non-Patent Document 1] Cell. 2014 Oct 9;159(2):428-39 [Non-patent document 2] Cell Metab. 2016 Apr 12; 23(4): 622-634 [Non-patent document 3] IntechOpen, 2019DOI: http: / / dx.doi.org / 10.5772 / intechopen.88878. [Non-patent document 4] Stem Cells International, Volume 2018, Article ID 1435746, https: / / doi.org / 10.1155 / 2018 / 1435746 [Non-Patent Document 5] Circulation Report. 2019, review, doi:10.1253 / circrep.CR-19-0104 [Non-patent document 6] Proc Natl Acad Sci US A. 2010 Aug 10;107(32):14223-8. [Non-Patent Document 7] Cell Stem Cell. 2017 Jan 5;20(1):41-55 [Non-patent document 8] Cell Stem Cell. 2016 Oct 6;19(4):449-461 [Non-Patent Document 9] eLIFE, 2019, https: / / doi.org / 10.7554 / eLife.47313.001 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for efficiently reprogramming mature or immature cardiomyocytes into cardiac stem / progenitor cells without genetic modification. In another aspect, the present invention is to provide a method for maintaining cardiac stem / progenitor cells in an undifferentiated state for a long period of time. In yet another aspect, the present invention is to provide a method for inhibiting fibrosis and / or defibrosis of fibroblasts. In yet another aspect, the present invention is to provide a method for inhibiting the onset and / or aggravation of cardiovascular disease and activating the development and / or function of the cardiovascular system. [Means for solving the problem]

[0009] To achieve the above-mentioned objectives, the present inventors have extensively investigated small molecular weight compounds that can contribute to cardiomyocyte reprogramming. As a result, they have found that culturing cardiomyocytes in the presence of a Rho kinase inhibitor maintains the expression of undifferentiated markers and suppresses the increased expression of mature cardiomyocyte markers, senescence markers, and / or endothelial cell markers. Thus, they have successfully reprogrammed cardiomyocytes using the small molecular weight compound Rho kinase inhibitor. Furthermore, they have found that cardiomyocytes cultured in the presence of a Rho kinase inhibitor can proliferate for long periods while maintaining their stem cell state, indicating that Rho kinase inhibitors maintain the undifferentiated state. Furthermore, they have found that cardiomyocytes treated with the ROCK inhibitor of the present invention suppress signaling pathways associated with cardiovascular disease and activate signaling pathways associated with cardiovascular development and / or function. Furthermore, they have found that co-culture of cardiomyocytes cultured in the presence of these small molecular weight compounds with fibroblasts and culture of fibroblasts in the presence of exosomes derived from these cardiomyocytes can suppress fibrosis. They have also found that these exosomes can defibrosate fibrotic cells. [Effects of the Invention]

[0010] According to the present invention, cardiac stem / progenitor cells with self-proliferation capacity can be safely and rapidly induced from various cardiac muscle cells without genetic modification. Furthermore, according to the present invention, cardiac muscle cells, including cardiac muscle stem / progenitor cells, can be stably cultured for a long period of time. Furthermore, treatment of cardiac muscle cells with the ROCK inhibitors of the present invention is expected to suppress the onset and / or exacerbation of cardiovascular disease and activate the development and / or function of the cardiovascular system. Furthermore, cardiac muscle cells treated with the ROCK inhibitors of the present invention, or secretomes or exosomes derived from such cardiac muscle cells, can safely and rapidly suppress fibrosis and / or defibrosing fibroblasts. The method of the present invention not only enables the supply of cardiac muscle cells for autologous / allogeneic transplantation, but can also be used for the treatment and prevention of cardiomyopathy, myocarditis, and other diseases associated with myocardial fibrosis. It can also be used for the production of model cells, the evaluation of therapeutic agents, and the evaluation of cardiotoxicity. Furthermore, since the method of the present invention can safely and rapidly induce and / or maintain cardiac muscle stem / progenitor cells from cardiac muscle cells without genetic modification, it can be applied to cardiac function regenerative medicine. [Brief explanation of the drawings]

[0011] [Figure 1] These figures show changes in cell morphology and proliferation of cardiomyocytes treated with a TGFβ receptor inhibitor or a ROCK inhibitor. A: Photographs showing that long-term culture under treatment with a TGFβ receptor inhibitor (A) alone or a ROCK inhibitor (Y) alone results in cardiomyocytes taking on the morphology of myocardial stem / progenitor cells. B: Graph showing proliferation of HCM cells under treatment with a TGFβ receptor inhibitor (A) alone or a ROCK inhibitor (Y) alone. C: Graph showing proliferation of primary human coronary artery smooth muscle cells PC-100-021 cells under treatment with a TGFβ receptor inhibitor (A) alone or a ROCK inhibitor (Y) alone. [Figure 2]This graph shows the mRNA expression of cardiac progenitor cell markers (GATA4, VCAM-1) and MYL2, a cardiomyocyte maturation marker, in cardiomyocytes treated with a TGFβ receptor inhibitor (A) or a ROCK inhibitor (Y). The vertical axis represents the expression level of each mRNA (relative mRNA level) when the expression level of cells before culture is set to 1. The letters on the horizontal axis represent the target mRNA, and the numbers represent the culture period (months). For each mRNA in each month, the left bar graph shows data for the drug-treated group, and the right bar graph shows data for the drug-untreated group (control). [Figure 3] A: Graphs showing the mRNA expression of cardiac progenitor cell markers (GATA4, VCAM-1), cardiomyocyte maturation marker (MYL2), and senescence markers (CDKN1A, CDKN2A) in cardiomyocytes treated with a TGFβ receptor inhibitor (A) or a ROCK inhibitor (Y). In each graph, the vertical axis represents the relative mRNA expression level (relative to the expression level of cells before culture), and the horizontal axis represents the culture period (days). B: Photographs showing the results of Western blot analysis of the expression of endothelial cell markers (CD31), myocyte markers (Troponin T), and actin in primary human cardiomyocytes (HCM) or primary human coronary artery smooth muscle cells (PC-100-021) treated with a TGFβ receptor inhibitor (A) or a ROCK inhibitor (Y). NT represents the untreated control, and HUVEC represents the results using human umbilical vein endothelial cells (positive for the endothelial cell marker CD31). [Figure 4]Graphs showing the results of inferring changes in signaling pathways by comparing the total RNA of cardiomyocytes treated with a ROCK inhibitor with the total RNA of untreated cardiomyocytes using Ingenuity® Pathway Analysis (IPA). A: Inferred results of changes in signaling pathways related to cardiovascular disease. B: Inferred results of changes in signaling pathways related to the development and function of the cardiovascular system. For activation z-scores in the graphs, see Bioinformatics. (2014); 30(4): 523-530. #Molecules indicates the number of molecules. [Figure 5] A: Graph showing the particle count of extracellular vesicles purified by ultracentrifugation from the culture supernatant of cardiomyocytes treated with a TGFβ receptor inhibitor or a ROCK inhibitor. B: Photographs showing the results of Western blotting to identify CD9, CD63, and CD81 molecules in extracellular vesicles purified by ultracentrifugation from the culture supernatant of cardiomyocytes treated with a TGFβ receptor inhibitor and a ROCK inhibitor. [Figure 6]Figures 1A and 1B show a schematic diagram and results of an experiment in which cardiomyocytes treated with a TGFβ receptor inhibitor or a ROCK inhibitor were co-cultured with fibroblasts activated by TGFβ treatment. A and C are graphs showing ACTA2 mRNA levels in fibroblasts co-cultured with HCM cells (A) or PC-100-021 cells (C). The vertical axis represents the ACTA2 mRNA expression level in each fibroblast (relative mRNA level), relative to the expression level in fibroblasts not treated with TGFβ, which is set to 1. The horizontal axis represents the presence or absence of TGFβ treatment and the drug treatment (from left to right: TGF-β(-): no TGFβ treatment, no drug treatment, no co-culture; -: TGFβ treatment, no drug treatment, no co-culture; NT: TGFβ treatment, no drug treatment, co-culture with cardiomyocytes; Y: TGFβ treatment, ROCK inhibitor treatment, co-culture with cardiomyocytes; A: TGFβ treatment, TGFβ receptor inhibitor treatment, co-culture with cardiomyocytes) (the same applies to the horizontal axis of the graphs in Figures 7 to 9). B and D: Photographs and graphs showing the results of Western blotting to determine the expression level of αSMA protein in fibroblasts co-cultured with HCM cells (B) or PC-100-021 cells (D). The photographs, from left to right, represent TGF-β(-): no TGFβ treatment, no drug treatment, no co-culture; -: TGFβ treatment, no drug treatment, no co-culture; NT: TGFβ treatment, no drug treatment, co-culture with cardiomyocytes; Y: TGFβ treatment, ROCK inhibitor treatment, co-culture with cardiomyocytes; A: TGFβ treatment, TGFβ receptor inhibitor treatment, co-culture with cardiomyocytes (the same applies to the photographs in Figures 7 to 9 below). The graphs represent the αSMA protein expression level of each fibroblast (relative protein level), where the expression level of αSMA protein in fibroblasts not treated with TGFβ is set to 1. [Figure 7]Figures 1 and 2 show a schematic diagram and results of an experiment in which cardiomyocytes treated with a TGFβ receptor inhibitor or a ROCK inhibitor were cocultured with fibroblasts activated by TGFβ treatment. A and C: Graphs showing ACTA2 mRNA levels in fibroblasts cocultured with HCM cells (A) or PC-100-021 cells (C). The vertical axis represents the ACTA2 mRNA expression level of each fibroblast (relative mRNA level) relative to the expression level of ACTA2 mRNA in fibroblasts treated with TGFβ, untreated, or not cocultured with cardiomyocytes, set at 1. B and D: Photographs and graphs showing the results of Western blotting of αSMA protein expression levels in fibroblasts cocultured with HCM cells (B) or PC-100-021 cells (D). The graphs represent the αSMA protein expression level of each fibroblast (relative protein level) relative to the expression level of αSMA protein in fibroblasts treated with TGFβ, untreated, or not cocultured with cardiomyocytes, set at 1. [Figure 8] Figures 1 and 2 show a schematic diagram and results of an experiment in which exosomes derived from cardiomyocytes treated with a TGFβ receptor inhibitor or a ROCK inhibitor were co-cultured with fibroblasts activated by TGFβ treatment. A and C: Graphs showing ACTA2 mRNA levels in fibroblasts cultured in the presence of exosomes derived from HCM cells (A) or PC-100-021 cells (C). The vertical axis represents the ACTA2 mRNA expression level of each fibroblast when the expression level of ACTA2 mRNA in fibroblasts not treated with TGFβ is set to 1 (relative mRNA level). B and D: Photographs and graphs showing the results of Western blotting of αSMA protein expression levels in fibroblasts cultured with exosomes derived from HCM cells (B) or PC-100-021 cells (D). The graphs represent the αSMA protein expression level of each fibroblast when the expression level of αSMA protein in fibroblasts not treated with TGFβ is set to 1 (relative protein level). [Figure 9]Figures 1 and 2 show a schematic diagram and results of an experiment in which exosomes derived from cardiomyocytes treated with a TGFβ receptor inhibitor or a ROCK inhibitor were co-cultured with fibroblasts activated by TGFβ treatment. A and C: Graphs showing ACTA2 mRNA levels in fibroblasts cultured in the presence of exosomes derived from HCM cells (A) or PC-100-021 cells (C). The vertical axis represents the ACTA2 mRNA expression level of each fibroblast (relative mRNA level), relative to the expression level in fibroblasts treated with TGFβ, untreated, and not treated with exosomes, set at 1. B and D: Photographs and graphs showing the results of Western blotting of αSMA protein expression levels in fibroblasts cultured with exosomes derived from HCM cells (B) or PC-100-021 cells (D). The graph shows the expression level of αSMA protein in each fibroblast (relative protein level), with the expression level of αSMA protein in fibroblasts treated with TGFβ, untreated with drugs, and not given exosomes set at 1. [Figure 10] Figure (A) and graph (B) show immunostaining results for HCM cells treated with a TGFβ receptor inhibitor or a ROCK inhibitor cocultured with fibroblasts activated by TGFβ treatment, using anti-αSMA, anti-fibronectin, and anti-collagen I antibodies as primary antibodies. From top to bottom, the figures show the following: TGFβ-untreated and no coculture (TGFβ(-)), TGFβ-treated and coculture (TGFβ(+)), TGFβ-treated and ROCK inhibitor-treated coculture (TGFβ(+)+Y), and TGFβ-treated and TGFβ receptor inhibitor-treated coculture (TGFβ(+)+A). The graph shows the expression levels of αSMA protein, fibronectin, and collagen I in fibroblasts untreated with TGFβ and not co-cultured (-), TGFβ-treated and co-cultured with cardiomyocytes treated with a ROCK inhibitor (Y), or TGFβ-treated and co-cultured with cardiomyocytes treated with a TGFβ receptor inhibitor (A), when the expression levels of αSMA protein, fibronectin, and collagen I in fibroblasts treated with TGFβ and not co-cultured (TGFB) are set to 1. [Figure 11]Exosomes derived from HCM cells treated with a TGFβ receptor inhibitor or a ROCK inhibitor were added to fibroblasts activated by TGFβ treatment and cultured. Immunostaining was performed using anti-αSMA, anti-fibronectin, and anti-collagen I antibodies as primary antibodies. From top to bottom, the figures show the following: TGFβ-untreated and no exosomes added (TGFβ(-)), TGFβ-treated and no exosomes added (TGFβ(+)), TGFβ-treated and with exosomes derived from ROCK inhibitor-treated cardiomyocytes added (TGFβ(+)+Y), and TGFβ-treated and with exosomes derived from TGFβ receptor inhibitor-treated cardiomyocytes added (TGFβ(+)+A). The graph shows the expression levels of αSMA protein, fibronectin, and collagen I in fibroblasts untreated with TGFβ and no co-culture (-), TGFβ-treated with exosomes derived from cardiomyocytes treated with a ROCK inhibitor (Y), or TGFβ-treated with exosomes derived from cardiomyocytes treated with a TGFβ receptor inhibitor (A), with the expression levels of αSMA protein, fibronectin, and collagen I in TGFβ-treated fibroblasts (without exosomes) (TGFB) set at 1. [Figure 12]Exosomes derived from HCM cells treated with a TGFβ receptor inhibitor or a ROCK inhibitor were added to fibroblasts activated by TGFβ treatment and cultured. Immunostaining was performed using anti-αSMA, anti-fibronectin, and anti-collagen I antibodies as primary antibodies. From top to bottom, the figures show the following: TGFβ-untreated and no exosomes (TGFβ(-)), TGFβ-treated and no exosomes (TGFβ(+)), TGFβ-treated and with exosomes derived from ROCK inhibitor-treated cardiomyocytes (TGFβ(+)+Y), and TGFβ-treated and with exosomes derived from TGFβ receptor inhibitor-treated cardiomyocytes (TGFβ(+)+A). Arrows in the figures indicate αSMA-positive cells. The graphs show the expression levels of αSMA protein, fibronectin, and collagen I in fibroblasts untreated with TGFβ and without co-culture, TGFβ-treated with exosomes derived from ROCK inhibitor-treated cardiomyocytes, or TGFβ-treated with exosomes derived from TGFβ-receptor inhibitor-treated cardiomyocytes, with the expression levels of αSMA protein, fibronectin, and collagen I in TGFβ-treated fibroblasts (without exosomes) set to 1. From left to right, the graphs show the results for fibroblasts untreated with TGFβ and without co-culture, TGFβ-treated with no exosomes, TGFβ-treated with exosomes derived from ROCK inhibitor-treated cardiomyocytes, and TGFβ-treated with exosomes derived from TGFβ-receptor inhibitor-treated cardiomyocytes. [Figure 13] Graphs showing the results of an IPA analysis of changes in existing signaling pathways in fibrotic cells. (A) Graph showing the top 10 signaling pathways that were altered when fibroblasts were activated, and (B) Graph showing the top 10 signaling pathways that were altered when activated fibroblasts were treated with exosomes obtained by treating cardiomyocytes with a ROCK inhibitor. [Figure 14]Graphs showing the results of IPA analysis of (A) the top 10 suppressed signaling pathways and (B) the top 10 activated signaling pathways in activated fibroblasts. Graphs also showing the results of IPA analysis of (C) the top 20 suppressed signaling pathways and (D) the top 20 activated signaling pathways when activated fibroblasts were treated with exosomes obtained by treating cardiomyocytes with a ROCK inhibitor. [Figure 15] (A) A graph showing the results of an analysis of exosomes in which selected microRNAs are highly expressed in Example 10. The horizontal axis represents expression levels. (B) A diagram showing that of the 513 genes targeted by the microRNAs contained in the selected exosomes, 18.5% are associated with cardiovascular disease. (C) A diagram showing the top five signaling pathways involved in genes targeted by the selected microRNAs. DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. Method for producing cardiac stem / progenitor cells from cardiac myocytes, and the produced cardiac stem / progenitor cells In one aspect, the present invention relates to a method for producing cardiac stem / progenitor cells, which comprises treating cardiomyocytes with a ROCK inhibitor (hereinafter also referred to as the "cardiomycologic reprogramming method of the present invention").

[0013] The "cardiomyocytes" used in the myocardial reprogramming method of the present invention may be either mature or immature cardiomyocytes. For example, cardiomyocytes may be cells expressing at least one cardiomyocyte marker gene (e.g., MYL2, cardiac troponin 1, GATA4, VCAM-1, Nkx2.5, etc.), preferably MYL2 and cardiac troponin 1.

[0014] The animal from which the cardiomyocytes used in the reprogramming method of the present invention are derived is preferably a mammal, such as a human, rat, mouse, guinea pig, rabbit, sheep, horse, pig, cow, monkey, etc., preferably a human, rat, or mouse, and most preferably a human.

[0015] Cardiac cells may be cardiomyocytes isolated from hearts removed from mammals (primary cardiomyocytes), immortalized cardiomyocytes (e.g., cardiomyocytes transfected with SV40 large T antigen), cardiomyocytes obtained from pluripotent stem cells such as embryonic stem cells (ES cells) or iPS cells, or mesenchymal stem cells by known differentiation induction methods (e.g., J. Clin. Inv., 1999; 103: 697-705.; Circ Res. 2009; 104(4): e30-41), and cardiomyocytes induced by direct reprogramming from fibroblasts (Circulation Report, Vol. 1 (2019), No. 12: pp. 564-567), but primary cardiomyocytes are preferred. Alternatively, cardiomyocytes may be cardiomyocytes present in the heart of a mammal.

[0016] When using primary cardiomyocytes, for example, in the case of rodents, it is preferable to use hearts excised from 10-20 week-old adults, although hearts from young animals up to 8 weeks old can also be used. In the case of humans, it is preferable to use a surgically excised adult heart tissue fragment, although hearts excised from dead fetuses can also be used. Alternatively, frozen cardiomyocytes isolated and purified from these excised hearts can also be used. Cardiomyocytes can be obtained from mammalian hearts or their tissue fragments by digesting the myocardial tissue with collagenase and removing non-parenchymal cells and cell debris by filtration, centrifugation, or other methods.

[0017] As used herein, "cardiomycotic stem / progenitor cells" (hereinafter also referred to as "CMSCs") refer to cells that are destined to differentiate into cardiac muscle and have unipotency and self-renewal capabilities. Preferably, the CMSCs herein have a high expression level of stem cell markers. For example, the expression level of stem cell markers in CMSCs herein may be higher than that of mature cardiomyocytes. As used herein, "stem cell marker" refers to one or more markers selected from Pdx1, Nkx6.1, Gata4, Vcam1, Hes1, Sox9, Foxa2, CK19, and CD133, preferably Gata4 and / or Vcam1. Preferably, the CMSCs herein have a low expression level of maturation markers. For example, the expression level of maturation markers in CMSCs herein may be lower than that of fully differentiated cardiomyocytes. As used herein, "maturation marker" refers to a cardiomyocyte maturation marker, and examples thereof include Myl2, Myl7, Myh7, Herg, Kcnq1, Tcap, Vcam1, and Sirpa. Furthermore, the CMSCs herein undergo less cell death upon treatment with a ROCK inhibitor. Preferably, the CMSCs herein have a low expression level of a senescence marker. For example, the expression level of a senescence marker in the CMSCs herein may be lower than that of mature cardiomyocytes or cardiomyocytes untreated with a ROCK inhibitor. As used herein, the term "senescence marker" refers to a senescence marker of cardiomyocytes, and examples include CDKN1A, CDKN2A, p53, and senescence-associated acid β-galactosidase (SA-β-Gal). The levels of the stem cell marker, maturation marker, senescence marker, and endothelial cell marker may be at the mRNA level or protein level.

[0018] As used herein, a ROCK inhibitor refers to a substance known to inhibit the function of Rho-associated kinase, such as N-[3-[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethylimidazo[5,4-d]pyridin-6-yl]oxyphenyl]-4-(2-morpholin-4-ylethoxy)benzamide (GSK269962A), fasudil hydrochloride, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y-27632), or 4-methyl-5-[[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline (H-1152), with Y-27632 being preferred. These ROCK inhibitors may be used alone or in combination of two or more compounds. The ROCK inhibitor may be in the free form, in the form of a salt such as a hydrochloride or sulfate, or in the form of a solvate or hydrate.

[0019] In the reprogramming method of the present invention, a small molecule signaling pathway inhibitor other than a ROCK inhibitor may be used in combination with a ROCK inhibitor, including, but not limited to, a GSK3 inhibitor and a MEK inhibitor.

[0020] When the reprogramming method of the present invention is performed in vitro, it can be performed by culturing cardiomyocytes in the presence of the inhibitors. Specifically, these inhibitors are added to the medium at effective concentrations and cultured. The medium used here may be any medium that can be used to culture cardiomyocytes, and commercially available media include CMC medium. Alternatively, a medium prepared by adding Supplement Pack Myocyte Cell GM (containing 5% FBS, 5 μg / mL insulin, 2 ng / mL FGF-β, and 0.5 ng / mL EGF) and 1% Antibiotic x Antimitotic to Myocyte Basal Medium, as described in the Examples, may also be used. The concentration of the ROCK inhibitor added to the medium can be appropriately selected from the ranges of, for example, 0.01-500 μM, 0.1-100 μM, or 1-50 μM, with 10 μM being more preferred.

[0021] Examples of culture vessels used for culture include dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, and culture bags. Culture vessels for cell suspension culture can be used. Alternatively, for adhesion fields, vessels whose inner surfaces are coated with a cell support substrate can be used to improve cell adhesion. Examples of such cell support substrates include collagen, atelocollagen, gelatin, Matrigel, poly-L-lysine, laminin, and fibronectin.

[0022] Cardiomyocytes are 10 2 -10 6 cells / cm 2 , preferably 10 3 -10 5 cells / cm 2The cardiomyocytes can be seeded onto a culture vessel at a cell density of 1000 kJ / cm. Cardiomyocytes can be cultured in a CO2 incubator (preferably with a CO2 concentration of about 5%) at 30-40°C (preferably about 37°C). The culture period can be, for example, 1-4 weeks, preferably 1-3 weeks. The medium may be replaced with fresh medium (which may contain the inhibitor) every 1-3 days. Induction into CMSCs can be confirmed by the expression of the stem cell markers in the cultured cardiomyocytes. The obtained CMSCs may also be isolated, if necessary, by a method utilizing the stem cell markers (e.g., FACS).

[0023] When the cultured cells reach 80% confluence, they are trypsinized to dissociate them and then transferred to a new culture vessel for 10 min. 3 -10 5 cells / cm 2 The cells are seeded at a density of 1000 x 1000 x 1000. Preferably, the medium is replaced with a medium containing a ROCK inhibitor. Stable CMSCs can be obtained after about 4 to 6 passages. After 10 or more passages, they may be cloned by a conventional method. As an example of a method for confirming that CMSCs are maintained after passage, the cells may be seeded at a low density (for example, 10 2 -10 3 cells / cm 2 ) into the above-mentioned culture vessel and observe or measure the morphology or number of the cells over time, or confirm the expression of CMSC markers.

[0024] Direct reprogramming of cardiac myocytes in vivo can be achieved by treating cardiac myocytes with a ROCK inhibitor in vivo. In this case, the treatment is performed by treating cardiac myocytes in a mammalian heart with a ROCK inhibitor. The ROCK inhibitor can be administered locally to the heart or to a desired site within the heart, or systemically.

[0025] The present invention also relates to CMSCs obtained by culturing cardiomyocytes in the presence of a ROCK inhibitor. These CMSCs can be used as fibroblast fibrosis inhibitors, as described below, or can be prepared as transplantable cardiomyocytes through proliferation and redifferentiation. For example, CMSCs can be redifferentiated into cardiomyocytes using known differentiation induction methods (e.g., J. Clin. Inv., 1999; 103:697-705; Circ Res. 2009; 104(4):e30-41). CMSCs or cardiomyocytes differentiated from CMSCs can be used, for example, to evaluate the cardiotoxicity of test substances, to prepare myocardium for transplantation, as a source of secretome release, and as inhibitors of cardiomyocyte fibrosis.

[0026] A method for evaluating the cardiotoxicity of a test substance includes culturing CMSCs or redifferentiated cardiomyocytes in the presence of the test substance. Treatment of CMSCs or cardiomyocytes with the test substance is typically performed by adding the test substance to a culture medium or solution in which the CMSCs or cardiomyocytes are cultured, but this method is not limited to this. For example, when the test substance is a protein, treatment may be performed by introducing a DNA vector expressing the protein into the cells. A method for evaluating the cardiotoxicity of a test substance may further include measuring or observing damage to CMSCs treated with the test substance, and determining that the test substance is myocardially toxic if damage to CMSCs is confirmed.

[0027] For example, a method for assessing the myocardial toxicity of a test substance may include treating cardiomyocytes with a ROCK inhibitor to obtain CMSCs, treating the obtained CMSCs with the test substance, measuring or observing damage to the CMSCs treated with the test substance, and determining that the test substance has myocardial toxicity if CMSC damage is confirmed. Alternatively, a method for assessing the myocardial toxicity of a test substance may include treating cardiomyocytes with a ROCK inhibitor to obtain CMSCs, redifferentiating the obtained CMSCs into cardiomyocytes, treating the redifferentiated cardiomyocytes with the test substance, measuring or observing damage to the cardiomyocytes treated with the test substance, and determining that the test substance has myocardial toxicity if cardiomyocyte damage is confirmed. The degree of damage may be measured, for example, by the viability, morphology, or apoptosis or necrosis markers of CMSCs or cardiomyocytes. Specifically, for example, if the addition of a test substance to the culture medium of CMSCs or cardiomyocytes reduces the viability of CMSCs or cardiomyocytes, the test substance is determined to be cardiotoxic; if there is no significant change in viability, the test substance is determined not to be cardiotoxic.

[0028] The CMSCs of the present invention can also be used to prepare myocardium for transplantation. The method for preparing myocardium for transplantation may include culturing and expanding CMSCs, redifferentiating the expanded CMSCs into cardiomyocytes, and preparing the redifferentiated cardiomyocytes as myocardium for transplantation. Thus, in one aspect, the present invention relates to myocardium for transplantation containing the CMSCs of the present invention or cardiomyocytes derived from the CMSCs of the present invention. Furthermore, the myocardium for transplantation can be used as a therapeutic or preventive agent for myocardial damage. Myocardial damage refers to a condition in which some abnormality occurs in the myocardium, causing abnormal cardiac function, and includes acute cardiovascular disease and chronic cardiovascular disease. Examples of chronic cardiovascular disease include cardiomyopathy (dilated cardiomyopathy, etc.), myocarditis, myocardial infarction, cardiac hypertrophy, and hypertension. CMSCs can be used by suspending them in an appropriate isotonic buffer (e.g., PBS). The size of the CMSC suspension varies depending on the type of heart disease and the severity of myocardial damage, but for example, in adults, 10 8 -10 11Transplantation can be performed by directly administering the cells into the myocardium or by directly administering them into the myocardium from the atrium using a catheter. Alternatively, the myocardium to be transplanted may be prepared as a myocardial sheet by co-culturing cardiomyocytes differentiated from the CMSCs of the present invention with vascular endothelial cells and vascular wall cells. The myocardial sheet is transplanted by attaching it to the treatment site in the mammalian heart.

[0029] 2. Methods for culturing cardiomyocytes and maintaining CMSCs In another aspect, the present invention relates to a method for culturing cardiomyocytes, comprising culturing cardiomyocytes in the presence of a ROCK inhibitor.In yet another aspect, the present invention relates to a method for maintaining and culturing CMSCs, comprising culturing CMSCs in the presence of a ROCK inhibitor.

[0030] Cardiomyocytes and CMSCs can be cultured by subculturing in accordance with the above-described culture method. In particular, the culture method of the present invention enables long-term culture while maintaining the stemness / progenitor properties of CMSCs. Herein, "maintenance of CMSCs" or "maintenance of stemness / progenitor properties" may refer to low levels of maturation markers and / or high levels of stem cell markers. For example, the levels of maturation markers expressed by cardiomyocytes or CMSCs after a long-term culture period may be lower than the levels of maturation markers expressed by cardiomyocytes cultured in the absence of a ROCK inhibitor. Furthermore, the levels of stem cell markers expressed by cardiomyocytes or CMSCs after a long-term culture period may be higher than the levels of stem cell markers expressed by cardiomyocytes cultured in the absence of a ROCK inhibitor. Herein, "long-term" refers to a culture period during which cardiomyocytes or induced CMSCs proliferate, compared to cardiomyocytes not treated with a ROCK inhibitor, and may refer to 20 days or more, 1 month or more, 40 days or more, or 2 months or more.

[0031] 3. Agents for inducing CMSCs from cardiomyocytes, agents for long-term culture of cardiomyocytes, or agents for maintaining CMSCs In one aspect, the present invention relates to a drug for inducing CMSCs from cardiomyocytes, a drug for long-term culture of cardiomyocytes, or a drug for maintaining CMSCs, which contains a ROCK inhibitor as an active ingredient. The drug containing a ROCK inhibitor may contain the ROCK inhibitor alone as an active ingredient, or may contain other drugs.

[0032] In another aspect, the present invention relates to the use of a ROCK inhibitor for the manufacture of a medicament for inducing CMSCs from cardiomyocytes. The present invention further relates to a method for inducing CMSCs from cardiomyocytes, comprising administering an effective amount of a ROCK inhibitor to a patient in need thereof. The present invention also relates to a ROCK inhibitor for use in the method for inducing CMSCs from cardiomyocytes.

[0033] 4. Secretome and exosomes In one aspect, the present invention relates to exosomes or secretomes derived from cardiomyocytes cultured in the presence of a ROCK inhibitor. As used herein, "secretome" is a collective term for useful components secreted into cell culture supernatant, including, for example, protein components such as various cytokines and chemokines, extracellular matrices such as ECM, and microparticles such as extracellular vesicles. Furthermore, as used herein, "exosomes" refer to endosomal membrane-derived vesicles formed during endocytosis and released from various cells, with a diameter of approximately 20 to 200 nm (preferably 50 to 150 nm). These vesicles are primarily composed of lipids, proteins, and nucleic acids (microRNA, messenger RNA, and DNA).

[0034] In another aspect, the present invention relates to a method for preparing secretomes or exosomes, comprising culturing cardiomyocytes in the presence of a ROCK inhibitor and recovering secretomes or exosomes from the cultured cardiomyocytes.

[0035] The secretome of the present invention can be obtained as a culture medium (e.g., culture supernatant) obtained by culturing cardiomyocytes or CMSCs in the presence of a ROCK inhibitor. The culture of cardiomyocytes and CMSCs can be carried out as described above.

[0036] The exosomes of the present invention can be recovered from the secretome. Exosomes can be recovered from culture medium or secretome using any known method or commercially available kit. Examples include ultracentrifugation (e.g., Thery C., Curr. Protoc. Cell Biol. (2006) Chapter 3: Unit 3.22.), polymer precipitation, immunoprecipitation, FACS, ultrafiltration, gel filtration, HPLC, and methods involving adsorption to a carrier such as beads using antibodies or lectins. Alternatively, exosomes may be recovered using a commercially available exosome isolation kit.

[0037] Among the above recovery methods, ultracentrifugation is the most commonly used standard method for isolating exosomes. The centrifugal force used in ultracentrifugation may be, for example, 50,000 × g or more, 100,000 × g or more, or 1,500,000 × g or more, or 300,000 × g or less, 250,000 × g or less, or 200,000 × g or less. The centrifugation time is not limited, but may be, for example, 30 to 120 minutes, 60 to 90 minutes, or 70 to 80 minutes. Furthermore, prior to centrifugation, if necessary, contaminants may be removed or reduced by filtration and / or centrifugation at a lower centrifugal force.

[0038] The presence of exosomes can be measured using a nanoparticle tracking system (e.g., an instrument such as NanoSight). Furthermore, molecules such as the tetraspanins CD9, CD63, and CD81 are present on the surface of exosome particles, and these molecules can serve as exosome markers. The presence of exosomes can also be confirmed by confirming the expression of these proteins and / or genes using immunoassays (e.g., Western blot).

[0039] 5. Fibrosis Inhibition Method, Fibrosis Inhibitor, Defibrillation Method, and Defibrillation Agent Secretomes or exosomes released by cardiomyocytes or CMSCs treated with a ROCK inhibitor can suppress cardiomyocyte fibrosis and defibrosify fibrotic cardiomyocytes. Therefore, in another aspect, the present invention relates to a method for suppressing fibrosis and a method for defibrosification using a ROCK inhibitor, cardiomyocytes cultured in the presence of a ROCK inhibitor, or a selectome or exosomes released by cardiomyocytes treated with a ROCK inhibitor. In this method, a ROCK inhibitor may be administered to directly act on cardiomyocytes in vivo to release secretomes or exosomes, cardiomyocytes treated with a ROCK inhibitor may be used, or the selectome or exosomes released by cardiomyocytes treated with a ROCK inhibitor may be isolated and / or purified and used. Furthermore, cardiomyocyte fibrosis or defibrosis of fibrotic cardiomyocytes may be achieved, for example, by the suppression of genes involved in signaling pathways involved in the activation or fibrosis of fibrotic cells by microRNAs contained in the selectome or exosomes released by cardiomyocytes treated with a ROCK inhibitor. Such signal transduction pathways include TGFB1, E2F1, EGF, HRAS, AGT, etc., and preferably TGFB1.

[0040] When a ROCK inhibitor is administered to directly treat cardiomyocytes in vivo, the ROCK inhibitor can act on cardiomyocytes already located in the vicinity of fibroblasts. Thus, the present invention includes a method for inhibiting fibrosis and a method for defibrosis of fibroblasts, which comprises treating cardiomyocytes located in a position where they can exert a paracrine effect on fibroblasts with a ROCK inhibitor.

[0041] When using cells, the method may be carried out by using cardiomyocytes that have been previously treated with a ROCK inhibitor and localizing the cardiomyocytes in the vicinity of fibroblasts or by co-culturing the two cells. Alternatively, cardiomyocytes that are already located in the vicinity of fibroblasts or co-cultured with fibroblasts may be treated with a ROCK inhibitor. More specifically, the present invention relates to a method for inhibiting fibroblast fibrosis, which comprises treating cardiomyocytes with a ROCK inhibitor and localizing the cardiomyocytes with fibroblasts at a position where the cardiomyocytes can exert their paracrine effect.

[0042] "Paracrine action" refers to the action of substances secreted from cardiomyocytes or CMSCs treated with a ROCK inhibitor on cells or tissues surrounding the cardiomyocytes or CMSCs. Therefore, a "location capable of exerting paracrine action" refers to a location where the secretome or exosomes produced from cardiomyocytes or CMSCs treated with a ROCK inhibitor can suppress fibrosis in target fibroblasts. Preferably, the cardiomyocytes or CMSCs are located near or adjacent to fibroblasts. When cardiomyocytes previously treated with a ROCK inhibitor are allowed to act on fibroblasts in vivo, the cardiomyocytes can be transplanted locally into the heart or a target site within the heart. Specifically, transplanting cardiomyocytes or CMSCs treated with the ROCK inhibitor of the present invention into the heart releases secretomes locally in the heart, suppressing fibrosis caused by surrounding fibroblasts through paracrine action.

[0043] When cardiomyocytes and fibroblasts are co-cultured, they may be cultured on the same surface, or may be cultured using a chamber or the like in a form in which selectomes or exosomes released from cardiomyocytes can act on fibroblasts. In the case of cultured cells, cardiomyocytes co-cultured with fibroblasts may be treated with a ROCK inhibitor to suppress fibroblast fibrosis.

[0044] Culture supernatants of cardiomyocytes or CMSCs treated with a ROCK inhibitor and exosomes isolated and purified from the culture supernatants suppress cardiomyocyte fibrosis. Therefore, the fibrosis suppression method of the present invention can also be performed using secretomes or exosomes released by cardiomyocytes or CMSCs treated with a ROCK inhibitor. Therefore, the present invention relates to a method for suppressing fibroblast fibrosis, which comprises treating fibroblasts with secretomes or exosomes extracted from cardiomyocytes cultured in the presence of a ROCK inhibitor. For example, the method of the present invention may be a method for suppressing fibroblast fibrosis, which comprises culturing cardiomyocytes in the presence of a ROCK inhibitor, recovering secretomes or exosomes from the cultured cardiomyocytes, and treating fibroblasts with the recovered secretomes or exosomes.

[0045] In this specification, the method for inhibiting fibroblast fibrosis may be a method for preventing or treating a disease accompanied by fibrosis of cardiomyocytes, and the agent for inhibiting fibroblast fibrosis may be a preventive or therapeutic agent for a disease accompanied by fibrosis of cardiomyocytes. Diseases accompanied by fibrosis of cardiomyocytes include diseases that develop due to fibrosis of cardiomyocytes and diseases that are exacerbated by fibrosis of cardiomyocytes, such as myocardial damage, myocardial fibrosis, myocardial infarction, heart failure, cardiac hypertrophy, and hypertension.

[0046] 6. Method for suppressing the onset and / or aggravation of cardiovascular disease, and suppressant MicroRNAs encapsulated in exosomes derived from cardiomyocytes treated with a ROCK inhibitor target genes associated with cardiovascular disease, specifically genes associated with cardiac necrosis and cell death, cardiac dilation, heart failure, and heart failure. Therefore, treating cardiomyocytes with a ROCK inhibitor can suppress the onset and / or worsening of cardiovascular disease. Furthermore, treating cardiomyocytes with a ROCK inhibitor can activate signaling pathways related to the development and / or function of the cardiovascular system. This can promote, for example, endothelial cell motility, angiogenesis, neovascularization, and vasculature development. Therefore, in another aspect, the present invention relates to an agent for suppressing the onset and / or worsening of cardiovascular disease, a therapeutic agent for cardiovascular disease, an agent for promoting endothelial cell motility, angiogenesis, angiogenesis, and vasculature development, which comprises treating cardiomyocytes with a ROCK inhibitor, a ROCK inhibitor, cardiomyocytes cultured in the presence of a ROCK inhibitor, or a selectome or exosomes released by cardiomyocytes treated with a ROCK inhibitor. In this method, a ROCK inhibitor may be administered to directly act on cardiomyocytes in vivo to release secretomes or exosomes, or cardiomyocytes treated with a ROCK inhibitor may be used, or the selectomes or exosomes released from cardiomyocytes treated with a ROCK inhibitor may be isolated and / or purified and used. Furthermore, this method may include suppressing signaling pathways related to the cardiovascular disease and / or activating signaling pathways related to the development and / or function of the cardiovascular system using microRNA encapsulated in exosomes derived from cardiomyocytes treated with a ROCK inhibitor. Examples of cardiovascular diseases include the above-mentioned acute cardiovascular diseases and chronic cardiovascular diseases (for example, cardiomyopathy (dilated cardiomyopathy, etc.), myocarditis, myocardial infarction, cardiac hypertrophy, hypertension, etc.), as well as ventricular dysfunction, left ventricular dysfunction, left heart disorder, cardiac dysfunction, familial cardiovascular disease, cerebrovascular dysfunction, left ventricular abnormality, ventricular abnormality, peripheral vascular disease, atherosclerosis, arteriosclerosis, vascular occlusion, vascular occlusion, arterial occlusion, congestive heart failure, and heart failure.

[0047] In another aspect, the present invention relates to a method for suppressing or treating the onset and / or aggravation of cardiovascular disease, comprising administering to a patient in need thereof an effective amount of a ROCK inhibitor, cardiomyocytes treated with a ROCK inhibitor, or a secretome or exosomes released by cardiomyocytes treated with a ROCK inhibitor. The present invention also relates to use of a ROCK inhibitor, cardiomyocytes treated with a ROCK inhibitor, or a secretome or exosomes released by cardiomyocytes treated with a ROCK inhibitor for the manufacture of an agent for suppressing or treating the onset and / or aggravation of cardiovascular disease. Alternatively, the present invention relates to a ROCK inhibitor, cardiomyocytes treated with a ROCK inhibitor, or a secretome or exosomes released by cardiomyocytes treated with a ROCK inhibitor for suppressing or treating the onset and / or aggravation of cardiovascular disease.

[0048] 7. Administration method and formulation Selectomes and exosomes can be obtained from cardiomyocytes treated with a ROCK inhibitor using the methods described above. Treatment with selectomes or exosomes can be performed in vitro by culturing cardiomyocytes or fibroblasts in the presence of selectomes or exosomes. In vivo treatment can involve administering selectomes or exosomes locally to the heart or to a desired site within the heart, or systemically.

[0049] The above-mentioned agents may contain, as an active ingredient, a ROCK inhibitor, cardiomyocytes treated with a ROCK inhibitor, or secretomes or exosomes released by cardiomyocytes treated with a ROCK inhibitor, or may contain other ingredients as needed. For example, when the agent is intended for administration to animals, the other ingredients may be pharmaceutically acceptable additives such as sterile water, physiological saline, buffers, excipients, binders, disintegrants, emulsifiers, surfactants, stabilizers, lubricants, diluents, flow enhancers, flavorings, colorants, and fragrances. For example, when the agents of the present invention are used for administration to animals, they can be administered orally or parenterally, such as in injections or infusions. These agents may also be administered orally as tablets, powders, granules, syrups, or parenterally as injections or infusions. The dosage may be an amount effective to achieve the intended purpose and can be determined depending on the symptoms, age, sex, weight, dosage form, etc.

[0050] All methods of the present invention may be performed in vivo, ex vivo, or in vitro, except where it is inconsistent to do so.

[0051] The present invention will be described in more detail below using examples, but these are not intended to limit the scope of the present invention. Note that all references cited throughout this specification are incorporated herein by reference in their entirety. [Example]

[0052] (Preparation of medium) Cardiomyocyte medium (CMM) was prepared as follows: Myocyte basal medium was supplemented with Supplement Pack Myocyte Cell GM (containing 5% FBS, 5 μg / mL insulin, 2 ng / mL FGF-β, and 0.5 ng / mL EGF) (Promocell, Cat# C-39270) and 1% Antibiotic x Antimitotic.

[0053] The culture medium for PC-100-021 was prepared as follows: Vascular Smooth Muscle Cell growth medium was supplemented with the Vascular Smooth Muscle Cell Growth Kit (containing 5% FBS, 5% L-glutamine, 50 μg / mL ascorbic acid, 5 ng / mL EGF, 5 μg / mL insulin, and 5 ng / mL FGF-β) (ATCC, Cat# PCS-100-042) and 1% Antibiotic x Antimitotic.

[0054] The complete medium used was Advanced DMEM (Gibco, cat. No. 12491).

[0055] Example 1: Cultivation of cardiomyocytes (1) Primary culture Primary human cardiomyocytes (HCM, Promocell) and primary human coronary artery smooth muscle cells (PC-100-021, ATCC) as a control were seeded onto culture dishes containing cardiomyocyte medium (CMM) and PC-100-021 culture medium, respectively, suitable for culturing cardiomyocytes, and plate cultured in an incubator (37°C, 5% CO2 / 95% air).

[0056] (2) Subculture The primary cultured human cardiomyocytes described above were collected by treatment with TrypLE Express (ThermoFisher) and then cultured in a collagen I-coated culture vessel (2 μg / cm 2 , 150mm dish) 9 × 10 3 cells / cm 2 The cells were seeded at 1000 x g for 1 hour and cultured in Vascular Smooth Muscle Cell growth medium or Myocyte basal medium. The cultured cells were frozen and stocked using CELLBANKER® 1 (Takara Bio).

[0057] Example 2: Effects of TGFβ receptor inhibitors and ROCK inhibitors in cardiomyocyte culture (1) Incubation in the presence of a test small molecule compound 0.5 × 10 human cardiomyocytes were subcultured onto 35 mm plates (IWAKI) containing 3 mL Vascular Smooth Muscle Cell growth medium or Myocyte basal medium with or without the small molecule compound 3-(6-methyl-2-pyridinyl)-n-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A-83-01:A) (final concentration 1 μM), a TGFβ receptor inhibitor, or the ROCK inhibitor (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide (Y-27632:Y) (final concentration 10 μM), or both compounds A-83-01 and Y-27632. 2 cells / cm 2 On day 3, the medium was replaced with Vascular Smooth Muscle Cell growth medium or Myocyte basal medium containing each low molecular weight compound. Thereafter, the medium was replaced every 3 days in the same manner.

[0058] (2) Time-lapse photography at low cell density After medium replacement, time-lapse photography was performed using a BZ9000 all-in-one fluorescence microscope (Keyence). Individual cells were tracked throughout the imaging period (110 days), and the final cell numbers derived from each cell type were counted.

[0059] Figure 1A shows changes in cell morphology of cardiomyocytes treated with A-83-01 or Y-27632. Treatment with A-83-01 or Y-27632 resulted in cardiac stem / progenitor cell-like morphology. In HCM, treatment with A-83-01 and Y-27632 induced long-term culture (Figure 1B). In PC-100-021, treatment with A-83-01 induced long-term culture (Figure 1C).

[0060] (3) Quantitative RT-PCR Total RNA was isolated from each cardiomyocyte after one and two months of culture in the presence of the test small molecule compound using the miRNeasy Mini Kit (QIAGEN). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer's guidelines. PCR was performed using the resulting cDNA as a template with Taqman Probe (ThermoFisher). Expression levels of the target genes were normalized to the endogenous control β-actin.

[0061] Figure 2 shows the expression levels of myocardial progenitor marker (GATA4, VCAM-1) mRNA and the mature cardiomyocyte marker MYL2 mRNA after culturing HCM cells and PC-100-021 cells in the presence of A-83-01 (A) or Y-27632 (Y). In both HCM and PC-100-021 cells, differentiation progressed with continued culture in the untreated (NT) group, with a decrease in the expression of progenitor markers (GATA4, VCAM-1) and an increase in the expression of the mature cardiomyocyte marker (MYL2). Treatment of HCM and PC-100-021 cells with Y-27632 suppressed the decrease in progenitor markers (GATA4, VCAM-1) and the increase in the mature cardiomyocyte marker (MYL2). Treatment with A-83-01, on the other hand, promoted the decrease in progenitor markers (GATA4, VCAM-1) and the increase in the mature cardiomyocyte marker (MYL2).

[0062] Figure 3A shows the expression levels of myocardial progenitor markers (GATA4, VCAM-1), the cardiomyocyte maturation marker MYL2, and the senescence markers (CDKN1A, CDKN2A) after culturing HCM and PC-100-021 cells in the presence of A-83-01 (A) or Y-27632 (Y). In both HCM and PC-100-021 cells, differentiation progressed with continued culture in the untreated (NT) group, with a decrease in the expression of progenitor markers (GATA4, VCAM-1) and an increase in the expression of the maturation marker (MYL2). Treatment of HCM and PC-100-021 cells with Y-27632 suppressed the decrease in progenitor markers (GATA4, VCAM-1) and the increase in the maturation marker (MYL2). On the other hand, treatment with A-83-01 promoted the upregulation of maturation markers (MYL2). Furthermore, senescence markers (CDKN1A, CDKN2A) were barely expressed in HCM cells and PC-100-021 cells treated with A-83-01(A) or Y-27632(Y). This indicates that treatment with A-83-01(A) or Y-27632(Y) resulted in minimal cell death. Furthermore, as shown in Figure 3B, HCM cells and PC-100-021 cells treated with A-83-01(A) or Y-27632(Y) did not express endothelial cell markers (CD31), but did express myocyte markers, confirming that these cells contained only myocytes and were not contaminated with endothelial cells in the primary culture.

[0063] (Example 3) Characteristics of signal transduction pathways in cardiomyocytes treated with a ROCK inhibitor The signaling pathways that were altered when cardiomyocytes were treated with Y-27632 (Y) compared to untreated cardiomyocytes were estimated using Ingenuity® Pathway Analysis (IPA) (QIAGEN) according to the manufacturer's instructions.

[0064] Cardiovascular disease-related pathways were inhibited in cardiomyocytes treated with the ROCK inhibitor Y-27632 (Y) compared with untreated cardiomyocytes. Specifically, signaling pathways related to ventricular dysfunction, left ventricular dysfunction, left heart disorder, cardiac dysfunction, familial cardiovascular disease, cerebrovascular dysfunction, left ventricular abnormalities, ventricular abnormalities, peripheral vascular disease, atherosclerosis, arteriosclerosis, vascular occlusion, vascular occlusion, arterial occlusion, congestive heart failure, and heart failure were suppressed (Figure 4A). These findings suggest that signaling pathways related to cardiovascular disease are reduced in cardiomyocytes treated with ROCK inhibitors. Furthermore, treatment of cardiomyocytes with Y-27632 (Y) suppressed signaling pathways related to cardiac contraction, blood pressure, and myocardial contraction, while activating signaling pathways related to endothelial cell motility, vasculogenesis, angiogenesis, and vasculature development (Figure 4B). Therefore, it was suggested that signaling pathways related to cardiovascular development and function were activated in cardiomyocytes treated with ROCK inhibitors. These changes in the signaling pathway also suggest that cardiomyocytes treated with ROCK inhibitors exhibit regenerative properties and are less susceptible to malignant transformation.

[0065] (Example 4) Purification and analysis of exosomes from cardiac stem / progenitor cells Cardiac stem / progenitor cells were cultured in complete medium until they reached 70% confluence. The complete medium was as described above. The complete medium was then replaced with Advanced DMEM (Gibco) and cultured for an additional 48 hours. The culture medium was then collected, centrifuged (2,000 × g, 10 minutes, 4°C), and filtered through a 0.22 μm filter. The cell pellet was discarded and the supernatant was collected. Exosomes were then isolated from the supernatant by subsequent ultracentrifugation. The exosomes were then ultracentrifuged (35,000 × g, 70 minutes, 4°C), dissolved in PBS, and stored.

[0066] Figure 5 shows the particle count (A) of exosomes (EVs), a type of secretome, purified by ultracentrifugation from the culture supernatant of cardiomyocytes treated with A-83-01 and Y-27632, measured using a nanoparticle capture system (Nanosight LM-10). Furthermore, the presence of CD9, CD63, and CD81 molecules contained in exosomes, as identified by Western blotting, was also shown. This demonstrates that exosomes were indeed recovered.

[0067] Example 5 Marker Expression in TGFβ-Stimulated Fibroblasts Co-Cultured with Cardiomyocytes Treated with TGFβ Receptor Inhibitors and ROCK Inhibitors Human cardiac fibroblasts were activated with TGFβ for 24 hours. Activated fibroblasts were co-cultured with cardiomyocytes treated with A-83-01 or Y-27632 for over 48 hours. Total RNA and protein were extracted for analysis of markers of fibrosis activation.

[0068] Figure 6 shows graphs showing the mRNA levels of ACTA2, a fibrosis-related gene (A, C), and the protein levels of αSMA, a fibrosis marker (B, D), in TGFβ-stimulated fibroblasts cocultured with cardiomyocytes treated with A-83-01 and Y-27632. The expression levels of ACTA2 and αSMA can be used as indicators of cellular fibrosis. While ACTA2 and αSMA expression levels increased with TGFβ-treated human cardiac fibroblasts undergoing fibrosis, they decreased upon coculture with cardiomyocytes, indicating that fibroblast activation was suppressed. These results demonstrate the inhibitory effect of secretome purified from cardiomyocytes treated with TGFβ receptor inhibitors and ROCK inhibitors on fibroblast activation.

[0069] Figure 7 shows graphs showing the mRNA levels of ACTA2, a fibrosis-related gene (A, C), and the protein levels of αSMA, a fibrosis marker (B, D), in TGFβ-stimulated fibroblasts cocultured with cardiomyocytes treated with A-83-01 and Y-27632. Graphs show mean ± standard deviation. The expression levels of ACTA2 and αSMA can be used as indicators of cellular fibrosis. While ACTA2 and αSMA expression levels increased with TGFβ-treated human cardiac fibroblasts undergoing fibrosis, they decreased upon coculture with cardiomyocytes, indicating that fibroblast activation was suppressed. These results demonstrate the inhibitory effect of secretome purified from cardiomyocytes treated with TGFβ receptor inhibitors and ROCK inhibitors on fibroblast activation.

[0070] Example 6: Marker expression in TGFβ-stimulated fibroblasts cultured in the presence of exosomes derived from cardiomyocytes treated with a TGFβ receptor inhibitor and a ROCK inhibitor Human cardiac fibroblasts were activated with TGFβ for 24 hours. Exosomes (EVs) derived from Y-27632-treated cardiomyocytes were then added, and the fibroblasts were cultured for an additional 48 hours. Total RNA and protein were extracted for analysis of fibrosis activation markers.

[0071] Figure 8 shows graphs showing the ACTA2 mRNA levels (A, C) and αSMA protein levels (B, D) expressed by fibroblasts activated by TGFβ treatment after incorporation of exosomes purified from cardiomyocytes treated with A-83-01 and Y-27632. ACTA2 and αSMA expression levels increased with TGFβ-treated human cardiac fibroblast fibrosis, but the levels were significantly reduced by the addition of exosomes, indicating that fibroblast activation was suppressed. Therefore, the effect of exosomes purified from cardiomyocytes treated with A-83-01 and Y-27632 on suppressing fibroblast activation was demonstrated.

[0072] Figure 9 shows graphs showing the ACTA2 mRNA levels (A, C) and αSMA protein levels (B, D) expressed by fibroblasts activated by TGFβ treatment after incorporation of exosomes purified from cardiomyocytes treated with A-83-01 and Y-27632. Graphs show mean ± standard deviation. ACTA2 and αSMA expression levels increased with TGFβ-treated human cardiac fibroblast fibrosis, but the levels were significantly reduced by the addition of exosomes, indicating that fibroblast activation was suppressed. Therefore, the effect of exosomes purified from cardiomyocytes treated with A-83-01 and Y-27632 on suppressing fibroblast activation was demonstrated.

[0073] (Example 7) Immunostaining As in Example 5 or 6, fibroblasts activated with TGFβ for 24 hours were co-cultured with HCM cells treated with A-83-01 or Y-27632, or cultured for 48 hours in the presence of exosomes (EVs) derived from the HCM cells. Next, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 minutes. The cell membrane was permeabilized with 0.1% Triton X dissolved in PBS. Blocking was performed with Blocking One (Nacalai Tesque) for 30 minutes, and the cells were incubated with primary antibodies (anti-αSMA, anti-fibronectin, anti-collagen I) for 1 hour at room temperature. Secondary antibodies conjugated to AlexaFluor 594 were then incubated for an additional hour. Nuclei were stained with DAPI (Vectashield).

[0074] The results of immunostaining when HCM treated with A-83-01 or Y-27632 were cocultured with fibroblasts activated by TGFβ treatment are shown in Figure 10. The expression of αSMA, fibronectin, and collagen I was significantly suppressed by coculture with HCM treated with a ROCK inhibitor, confirming the inhibitory effect on cardiac fibroblast fibrosis.

[0075] The results of immunostaining when HCM-derived exosomes treated with A-83-01 or Y-27632 were added to fibroblasts activated by TGFβ treatment and cultured are shown in Figure 11. The expression of αSMA, fibronectin, and collagen I was significantly suppressed by HCM-derived exosomes treated with a ROCK inhibitor, confirming the inhibitory effect on cardiac fibroblast fibrosis.

[0076] Figure 12 shows representative immunostaining results when exosomes derived from HCM treated with A-83-01 or Y-27632 were added to fibroblasts activated by TGFβ treatment and cultured. αSMA-positive cells are indicated by white arrows. Graphs show mean ± standard deviation. TGFβ treatment induces αSMA expression and increases the number of αSMA-positive cells. Furthermore, the addition of exosomes derived from HCM treated with Y-27632 suppressed TGFβ-induced expression and reduced the number of αSMA-positive cells. Similarly, the expression of fibronectin and collagen I was significantly suppressed by exosomes derived from HCM treated with Y-27632, confirming their inhibitory effect on cardiac fibroblast fibrosis.

[0077] Example 8: Changes in signal transduction pathways in fibroblasts cultured in the presence of exosomes derived from cardiomyocytes treated with a ROCK inhibitor We estimated the altered signaling pathways in activated fibroblasts and fibroblasts treated with exosomes derived from Y-27632 (Y)-treated cardiomyocytes compared to untreated cardiomyocytes using IPA according to the manufacturer's instructions.

[0078] In activated fibroblasts, the following signaling pathways were predicted to be promoted in the following order: liver fibrosis / activation of hepatocytes, CREB signaling in neurons, axon guidance signaling, increased cardiac hypertrophy signaling, Stathmin1-mediated breast cancer control, atherosclerosis signaling, liver fibrosis signaling pathway, STAT3 pathway, macrophage, fibroblast, endothelial role, and osteoarthritis pathway (Figure 13A).On the other hand, in fibroblasts treated with exosomes derived from Y-27632 (Y)-treated cardiomyocytes, the following signaling pathways were predicted to be promoted in the following order: CREB signaling in neurons, sperm motility, Stathmin1-mediated breast cancer control, cardiac hypertrophy signaling, STAT3 pathway, estrogen receptor signaling, macrophages, fibroblast, endothelial role, IL-15 production, liver fibrosis / activation of hepatocytes, and PTEN signaling (Figure 13B).

[0079] Example 9: Changes in activated signal transduction factors in fibroblasts cultured in the presence of exosomes derived from cardiomyocytes treated with a ROCK inhibitor To estimate more specific signaling factors for the signaling pathways estimated in Example 8, we estimated the signaling pathways that are suppressed or promoted in activated fibroblasts compared to untreated cardiomyocytes, as well as the signaling pathways that are suppressed or promoted when activated fibroblasts are treated with exosomes derived from HCM cells treated with a ROCK inhibitor compared to untreated cardiomyocytes. For this estimation, IPA was used according to the manufacturer's instructions.

[0080] In activated fibroblasts, the signaling pathways associated with NFκB (complex), HGF, Vegf, IL17A, IL1B, CSF2, CHUK, EGF, Tlr, and TLR4 were suppressed in this order, while the signaling pathways associated with TGFB1, Tgf beta, TGFB3, NUPR1, NORAD, TAZ, MRTFA, TGFBR1, TGFB2, and DRD2 were promoted in this order (Figure 14A, B).

[0081] On the other hand, when activated fibroblasts were treated with exosomes derived from HCM cells treated with a ROCK inhibitor, the signaling pathways involved were suppressed in the following order: ESR1, TCF7L2, IRGM, MRTFB, HGF, CD24, MRTFA, MAPK1, Vegf, NKX2-3, RC3H1, Irgm1, IL1RN, ACKR2, IL4, TRIM24, Tgf beta, SMAD4, ESR2, and PTGER4, while the signaling pathways involved were promoted in the following order: IFNL1, IRF7, IFNA2, MIR17HG, GRIN3A, Hbb-b1, Ifnar, IFNB1, IRF3, STAT1, STING1, SEL1L, RNY3, IRF1, Interferon alpha, PML, MAVS, IFNAR1, IFNG, and KLF3 (Figure 14C, D). Therefore, the signaling pathway activated in activated fibroblasts was found to be suppressed by exosome treatment, suggesting that specific fibroblast signaling pathways could be restored by treatment with exosomes derived from ROCK inhibitor-treated cardiomyocytes.

[0082] (Example 10) Functional analysis of microRNA We selected exosomes derived from ROCK inhibitor-treated HCM cells that expressed the highest levels of encapsulated microRNAs (see Figure 15A) and identified the target genes of those microRNAs. Specifically, the culture medium of ROCK inhibitor-treated HCM cells was replaced with serum-free medium. After 72 hours of culture, 500 ml of culture supernatant was collected. The culture supernatant was centrifuged at 10,000 × g for 30 minutes to remove cell debris and then ultracentrifuged at 100,000 × g at 4°C for 70 minutes in a Beckman Coulter ultracentrifuge (Optima-XE-90). The pellet was dissolved in PBS(-), further ultracentrifuged at 100,000 × g for 70 minutes at 4°C, and then dissolved in an appropriate amount of PBS(-). MicroRNA was extracted from this exosome fraction using the microRNAeasy kit (QIAGEN), and 8 ng of the extracted RNA was analyzed using a next-generation sequencer (DNAchip Research Institute).

[0083] The results showed that 18.5% of the 513 genes targeted by the selected microRNAs were associated with cardiovascular diseases such as cardiac necrosis and cell death, cardiac enlargement, and heart failure (Figure 15B). Furthermore, the target genes of the selected microRNAs were shown to be deeply involved in the TGFB1 signaling pathway (Figure 15C).

Claims

1. A method for producing cardiac stem / progenitor cells, comprising treating cardiac cells in vitro with a ROCK inhibitor, the treatment of the cardiomyocytes with the ROCK inhibitor is carried out by culturing the cardiomyocytes in the presence of the ROCK inhibitor at a final concentration of 10-500 μM, the culture period is one month or longer, and the level of a progenitor cell marker expressed by the cardiomyocytes after the culture period in the presence of the ROCK inhibitor is suppressed to decrease more than the level of the marker expressed by cardiomyocytes cultured in the absence of the ROCK inhibitor; the increase in the level of a cardiomyocyte maturation marker expressed by the cardiomyocytes after a culture period in the presence of the ROCK inhibitor is suppressed compared to the level of the marker expressed by cardiomyocytes cultured in the absence of the ROCK inhibitor; After a culture period in the presence of the ROCK inhibitor, the cardiomyocytes do not express a senescence marker or the expression level of the marker is lower than the expression level of the marker expressed by cardiomyocytes cultured in the absence of a ROCK inhibitor; After a culture period in the presence of the ROCK inhibitor, the cardiomyocytes do not express an endothelial cell marker; The method for producing cardiac stem / progenitor cells, wherein the ROCK inhibitor is Y27632.

2. The method of claim 1 , wherein the cardiomyocytes are human cardiomyocytes.

Images