Method for producing myocardial stem / progenitor cell and method for suppressing myocardial fibrosis

Abstract

To provide a method for efficiently reprogramming mature and juvenile cardiomyocytes into myocardial stem / progenitor cells without genetic modification, and / or for suppressing fibrosis.SOLUTION: Myocardial stem / progenitor cells can be maintained by culturing primary cardiomyocytes in the presence of a ROCK inhibitor. Furthermore, fibrosis is suppressed by causing cardiomyocytes cultured in the presence of these low molecular weight compounds or an exosome or secretome derived from the cardiomyocytes to act on cardiac fibroblasts.SELECTED DRAWING: None

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, and incorporates by reference the entire contents of Japanese Patent Application No. 2020 - 173581 into this international application.

Technical Field

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

Background Art

[0003] Remarkable progress in stem cell biology occupies an important position in its application in myocardial regenerative medicine, but it has not yet been realized. Induced pluripotent stem cells (iPS cells), one of the most promising cell sources, still have a risk of tumor formation. Regarding their application to actual clinical practice, although there are clinical studies, their practical use is considered difficult (Non - Patent Documents 1 to 3). On the other hand, recent research has shown that cells of different lineages can be directly converted (direct reprogramming) into myocardial progenitor - like cells. However, like the case of iPS cells, direct reprogramming involves genetic modification by introducing genes such as Gata4, Mef2c, and Tbx5, so unexpected risks still exist and it cannot be applied to regenerative medicine (Non - Patent Document 4).

[0004] Recently, the fact that cardiac fibroblasts are reprogrammed into cardiomyocytes has been successively reported (Non - Patent Document 5). These innovative discoveries provide great insights not only into the myocardial stem cell theory but also into myocardial regeneration research. That is, if such reprogramming can be reproduced, the resulting myocardial stem / progenitor cells are expected to be an innovative cell source in myocardial regenerative medicine. However, a method for reprogramming cardiomyocytes at various stages into myocardial stem / progenitor cells without genetic modification is not known at all.

[0005] The present inventors and other groups have previously reported that combinations of certain small molecule inhibitors contribute to the induction and maintenance of pluripotency of stem cells in the liver, stomach, etc. (Patent Document 1, Non-Patent Documents 6 to 9). However, the relationship between small molecule inhibitors and reprogramming from mature / young cardiomyocytes to cardiac stem / progenitor cells has not been reported so far.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a method for efficiently reprogramming mature and juvenile cardiomyocytes into cardiac stem / progenitor cells without genetic modification. In another aspect, the present invention aims 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 aims to provide a method for suppressing and / or defibrosing fibrosis of fibroblasts. In yet another aspect, the present invention aims to provide a method for suppressing the onset and / or aggravation of cardiovascular diseases and activating the development and / or function of the cardiovascular system.

Means for Solving the Problems

[0009] In order to achieve the above object, the inventors repeatedly studied low molecular weight compounds that can contribute to the reprogramming of cardiomyocytes. As a result, when cardiomyocytes were cultured in the presence of a Rho kinase inhibitor, it was found that the expression of undifferentiated markers was maintained and the increase in the expression of mature cardiomyocyte markers, senescence markers, and / or endothelial cell markers could be suppressed. Thus, successful reprogramming of cardiomyocytes was achieved using the low molecular weight compound, a Rho kinase inhibitor. In addition, since cardiomyocytes cultured in the presence of a Rho kinase inhibitor can proliferate over a long period while maintaining their state as stem cells, it was found that the Rho kinase inhibitor brings about the maintenance of the undifferentiated state. Further, it was found that in cardiomyocytes treated with the ROCK inhibitor of the present invention, signal transduction pathways related to cardiovascular diseases are suppressed and signal transduction pathways related to cardiovascular development and / or function are activated. Furthermore, it was found that co-culture of cardiomyocytes and fibroblasts cultured in the presence of these low molecular weight compounds or culture of fibroblasts in the presence of exosomes derived from the cardiomyocytes can suppress fibrosis. Also, it was found that the exosomes can defibrillate fibrotic cells.

Advantages of the Invention

[0010] According to the present invention, cardiomyogenic stem / progenitor cells having self-renewal ability can be safely and rapidly induced from various cardiomyocytes without genetic modification. Further, according to the present invention, it is possible to stably culture cardiomyocytes containing cardiomyogenic stem / progenitor cells for a long period of time. Further, by treating with the ROCK inhibitor of the present invention, it can be expected that cardiomyocytes will suppress the onset and / or aggravation of cardiovascular diseases and / or activate the development and / or function of the cardiovascular system. Further, cardiomyocytes treated with the ROCK inhibitor of the present invention or a secretome or exosome derived from the cardiomyocytes can safely and rapidly suppress the fibrosis of fibroblasts and / or defibroticate them. The method of the present invention enables the supply of cardiomyocytes in autologous / heterologous transplantation, and can be used for the treatment and prevention of cardiomyopathy, myocarditis, and other diseases related to myocardial fibrosis. Furthermore, it can be used for the preparation of these model cells, the evaluation of therapeutic drugs, and the evaluation of cardiotoxicity. Furthermore, since the method of the present invention can safely and rapidly induce and / or maintain cardiomyogenic stem / progenitor cells from cardiomyocytes without genetic modification, it can be applied to cardiac function regenerative medicine.

Brief Description of Drawings

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Mode for Carrying Out the Invention

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

[0013] The "cardiomyocytes" used in the myocardial reprogramming method of the present invention may be either mature cardiomyocytes or young cardiomyocytes. For example, the cardiomyocytes may be cells that express 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, for example, human, rat, mouse, guinea pig, rabbit, sheep, horse, pig, cow, monkey, etc., preferably human, rat, mouse, and most preferably human.

[0015] Cardiac cells may be cardiomyocytes isolated from the heart excised from a mammal (primary cardiomyocytes), immortalized cardiomyocytes (for example, cardiomyocytes into which the SV40 large T antigen has been introduced), pluripotent stem cells such as embryonic stem cells (ES cells) or iPS cells, or mesenchymal stem cells, cardiomyocytes obtained by known differentiation induction methods (for example, 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), etc., but are preferably primary cardiomyocytes. Alternatively, the cardiomyocytes may be cardiomyocytes present in the heart in the body of a mammal.

[0016] When using primary cardiomyocytes, for example, in the case of rodents, it is preferable to use the heart excised from an adult at 10 - 20 weeks of age, but the heart of a juvenile individual at 8 weeks of age or younger may also be used. In the case of humans, it is preferable to use adult heart tissue pieces excised by surgery, but the heart excised from a dead fetus may also be used. Alternatively, it is also possible to use cryopreserved cells (cryopreserved cardiomyocytes) of cardiomyocytes isolated and purified from these excised hearts. As a method for obtaining cardiomyocytes from the heart or a tissue piece thereof of a mammal, a method of digesting the myocardial tissue with collagenase and removing non-parenchymal cells and cell fragments by filtration, centrifugation, etc. can be used.

[0017] As used herein, "cardiac muscle stem / precursor cells" (hereinafter also referred to as "CMSC") refers to cells having unipotency committed to differentiation into cardiac muscle and self-renewal ability. Preferably, the CMSC herein has a high expression level of stem cell markers. For example, the expression level of the stem cell markers of the CMSC herein may be higher compared to mature cardiac muscle cells. As used herein, "stem cell markers" refers to one or more markers selected from Pdx1, Nkx6.1, Gata4, Vcam1, Hes1, Sox9, Foxa2, CK19, and CD133, and preferably, Gata4 and / or Vcam1. Also preferably, the CMSC herein has a low expression level of mature markers. For example, the expression level of the mature markers of the CMSC herein may be lower compared to fully differentiated cardiac muscle cells. As used herein, "mature markers" refers to the mature markers of cardiac muscle cells, and examples include Myl2, Myl7, Myh7, Herg, K cnq1, Tcap, Vcam1, Sirpa, etc. can be mentioned. Also, the CMSC herein has less cell death upon treatment with a ROCK inhibitor. Preferably, the CMSC herein has a low expression level of senescence markers. For example, the expression level of the senescence markers of the CMSC herein may be lower compared to mature cardiac muscle cells or cardiac muscle cells untreated with a ROCK inhibitor. As used herein, "senescence markers" refers to the senescence markers of cardiac muscle cells, and examples include CDKN1A, CDKN2A, p53, senescence-associated acidic β-galactosidase (SA-β-Gal), etc. The levels of the stem cell markers, mature markers, senescence markers, and endothelial cell markers may be at the mRNA level or the protein level.

[0018] In this specification, a ROCK inhibitor is a substance known to have an action of inhibiting the function of Rho - associated kinase. For example, 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), 4 - methyl - 5 - [[(2S) - 2 - methyl - 1,4 - diazepan - 1 - yl]sulfonyl]isoquinoline (H - 1152), etc. may be mentioned, and preferably Y - 27632. These ROCK inhibitors may be used singly or in combination of two or more compounds. The ROCK inhibitor may be in the free form, in the form of salts such as hydrochloride, sulfate, or in the form of a solvate or hydrate.

[0019] Also, in the reprogramming method of the present invention, a small - molecule signal transduction pathway inhibitor other than the ROCK inhibitor may be used in combination with the ROCK inhibitor. Such inhibitors include, for example, but are not limited to, GSK3 inhibitors, MEK inhibitors, etc.

[0020] When the reprogramming method of the present invention is carried out in vitro, it can be carried out by culturing cardiomyocytes in the presence of the inhibitor. Specifically, these inhibitors are added at an effective concentration in the medium for culturing. Here, the medium may be any medium that can be used for culturing cardiomyocytes. Examples of commercially available media include, for example, CMC medium, etc. Also, as described in the examples, Myocyte basal medium supplemented with Supplement Pack Myocyte Cell GM (5% FBS, 5μg / mL It is also possible to use a medium supplemented with Insulin, 2 ng / mL FGF-b, 0.5 ng / mL EGF) and 1% Antibiotic x Antimitotic. The concentration of the ROCK inhibitor added to the medium can be appropriately selected from, for example, the range of 0.01 - 500 μM, 0.1 - 100 μM, 1 - 50 μM, and more preferably is 10 μM.

[0021] Culture vessels used for culturing include, for example, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, etc. As the culture vessel, a culture vessel for suspension culture of cells can be used. Alternatively, in the case of an adherent culture, a culture vessel whose inner surface is coated with a cell-supporting substrate for the purpose of improving the adhesiveness to cells can be used. Examples of such cell-supporting substrates include collagen, atelocollagen, gelatin, Matrigel, poly-L-lysine, laminin, fibronectin, and the like.

[0022] Cardiomyocytes are 10 2 -10 6 cells / cm 2 , preferably 10 3 -10 5 cells / cm 2 and can be seeded on the culture vessel at a cell density of. The culture of cardiomyocytes can be carried out at 30 - 40 °C (preferably about 37 °C) in an atmosphere of a CO2 incubator (preferably a CO2 concentration of about 5%). The culture period can be, for example, 1 - 4 weeks, preferably 1 - 3 weeks. Also, the medium can be replaced with fresh medium (which may contain the inhibitor) every 1 - 3 days. The induction of CMSC can be confirmed by the expression of the stem cell marker in the cultured cardiomyocytes. Also, the obtained CMSC can be isolated by a method using the stem cell marker (for example, FACS, etc.) as needed.

[0023] When subculturing, when the cultured cells reach 80% confluence, the cells are trypsinized to dissociate them, and on a new culture vessel, 10 3 -10 5 cells / cm 2 are seeded at a density of. Preferably, it is exchanged with a medium containing a ROCK inhibitor. Stable CMSCs can be obtained after about 4 to 6 passages. After more than 10 passages, it may be cloned by a conventional method. As a method for confirming that CMSCs are maintained after subculture, as an example, at a low density (for example, 10 2 -10 3 cells / cm 2 ), cells are seeded in the above culture vessel, and the cell morphology or number may be observed or measured over time, or the expression of CMSC markers may be confirmed.

[0024] By performing the treatment of cardiomyocytes with a ROCK inhibitor in vivo, direct reprogramming can be performed on cardiomyocytes present in the heart in vivo. In this case, it is performed by treating cardiomyocytes in the heart of a mammal with a ROCK inhibitor. The ROCK inhibitor may be administered locally to the heart or the target site in the heart, or may be administered systemically.

[0025] The present invention also relates to CMSCs obtained by culturing cardiomyocytes in the presence of a ROCK inhibitor. The CMSCs can also be used as a fibrotic inhibitor of fibroblasts described below, and can also be prepared as transplanted cardiomyocytes through proliferation and redifferentiation. For example, CMSCs can be redifferentiated into cardiomyocytes by a known differentiation induction method (for example, 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, for the evaluation of the cardiotoxicity of a test substance, the preparation of transplanted myocardium, the source of the secreted secretome, and the inhibitor of cardiomyocyte fibrosis.

[0026] A method for evaluating the cardiotoxicity of a test substance includes culturing CMSC or redifferentiated cardiomyocytes in the presence of the test substance. Treatment of CMSC or cardiomyocytes with the test substance is usually carried out by adding the test substance to the medium or culture solution for culturing CMSC or cardiomyocytes, but is not limited to this method. For example, when the test substance is a protein or the like, the DNA vector expressing the protein may be introduced into the cells for treatment. The method for evaluating the cardiotoxicity of a test substance may further include measuring or observing the impairment of CMSC treated with the test substance, and determining that the test substance has cardiotoxicity when the impairment of CMSC is confirmed.

[0027] For example, a method for evaluating the cardiotoxicity of a test substance may include obtaining CMSC by treating cardiomyocytes with a ROCK inhibitor, treating the obtained CMSC with the test substance, measuring or observing the impairment of the CMSC treated with the test substance, and determining that the test substance has cardiotoxicity when the impairment of the CMSC is confirmed. Alternatively, a method for evaluating the cardiotoxicity of a test substance may include obtaining CMSC by treating cardiomyocytes with a ROCK inhibitor, redifferentiating the obtained CMSC into cardiomyocytes, treating the redifferentiated cardiomyocytes with the test substance, measuring or observing the impairment of the cardiomyocytes treated with the test substance, and determining that the test substance has cardiotoxicity when the impairment of the cardiomyocytes is confirmed. The degree of impairment may be, for example, based on the survival rate, morphology of CMSC or cardiomyocytes, or apoptosis and necrosis markers. Specifically, for example, when the survival rate of CMSC or cardiomyocytes decreases by adding the test substance to the culture solution of CMSC or cardiomyocytes, the test substance is determined to have cardiotoxicity, and when there is no significant change in the survival rate, the test substance is determined not to have cardiotoxicity.

[0028] In addition, the CMSC of the present invention can be used for the preparation of transplanted myocardium. The method for preparing transplanted myocardium may include culturing and growing CMSC, redifferentiating the grown CMSC into cardiomyocytes, and preparing transplanted myocardium from the redifferentiated cardiomyocytes. Thus, in one aspect, the present invention relates to transplanted myocardium containing the CMSC of the present invention or cardiomyocytes derived from the CMSC of the present invention. Further, the transplanted myocardium can be used as a therapeutic or prophylactic agent for myocardial disorders. Myocardial disorder refers to a state in which some abnormality occurs in the myocardium and the function of the heart is abnormal, including acute cardiovascular diseases and chronic cardiovascular diseases. Examples of chronic cardiovascular diseases include cardiomyopathy (such as dilated cardiomyopathy), myocarditis, myocardial infarction, cardiac hypertrophy, hypertension, and the like. CMSC can be suspended in an appropriate isotonic buffer (for example, PBS) and used. The CMSC suspension varies depending on the type of heart disease, the severity of myocardial disorder, etc. For example, in the case of an adult, 10 8 -10 11 cells can be transplanted, for example, by directly injecting them into the myocardium or directly injecting them into the myocardium from the atrium using a catheter. In addition, the transplanted myocardium may be a myocardial sheet obtained by co-culturing cardiomyocytes differentiated from the CMSC of the present invention with vascular endothelial cells and vascular wall cells. The myocardial sheet is transplanted by attaching it to the treatment site of the mammalian heart.

[0029] 2. Method for culturing cardiomyocytes, method for maintaining and culturing CMSC In another aspect, the present invention relates to a method for culturing cardiomyocytes, which includes culturing cardiomyocytes in the presence of a ROCK inhibitor. In yet another aspect, the present invention relates to a method for maintaining and culturing CMSC, which includes culturing CMSC in the presence of a ROCK inhibitor.

[0030] The culturing of cardiomyocytes and CMSCs can be carried out by subculturing according to the above culturing method. In particular, the culturing method of the present invention enables long-term culturing while maintaining the stem cell properties / precursor cell properties of CMSCs. In this specification, the maintenance of CMSCs or the maintenance of stem cell properties / precursor cell properties may mean that the level of mature markers is low and / or the level of stem cell markers is high. For example, after a long-term culturing period, the level of mature markers expressed by the cardiomyocytes or CMSCs may be lower than the level of mature markers expressed by cardiomyocytes cultured in the absence of a ROCK inhibitor. Also, after a long-term culturing period, the level of stem cell markers expressed by the cardiomyocytes or CMSCs may be higher than the level of stem cell markers expressed by cardiomyocytes cultured in the absence of a ROCK inhibitor. In this specification, "long-term" means that the culturing period during which cardiomyocytes or induced CMSCs proliferate is long compared to cardiomyocytes not treated with a ROCK inhibitor, and may mean 20 days or more, 1 month or more, 40 days or more, or 2 months or more.

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

[0032] In another aspect, the present invention relates to the use of a ROCK inhibitor for the manufacture of an agent for inducing CMSCs from cardiomyocytes. The present invention further relates to a method for inducing CMSCs from cardiomyocytes, which includes 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 a method for inducing CMSCs from cardiomyocytes.

[0033] 4. Secretome and exosome In one aspect, the present invention is an exosome or secretome derived from cardiomyocytes cultured in the presence of a ROCK inhibitor. As used herein, "secretome" is a general term for useful components secreted into the cell culture supernatant, and includes, for example, protein components such as various cytokines and chemokines, extracellular matrix such as ECM, and fine particles such as extracellular vesicles. Also, as used herein, "exosome" is a vesicle derived from an endosome membrane formed during the endocytosis process, having a diameter of about 20 to 200 nm (preferably 50 to 150 nm) released from various cells, and is mainly composed of lipids, proteins, and nucleic acids (microRNA, messenger RNA, DNA).

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

[0035] The secretome of the present invention can be obtained as a culture solution (for example, culture supernatant) obtained by culturing cardiomyocytes or CMSCs in the presence of a ROCK inhibitor. The culture of cardiomyocytes and CMSCs can be performed according to the above description.

[0036] The exosome of the present invention can be recovered from the secretome. The method for recovering exosomes from the culture solution or secretome can be performed using any known method or commercially available kit. For example, ultracentrifugation (for example, Thery C., Curr. Protoc. Cell Biol. (2006) Chapter 3: Unit 3.22.), polymer precipitation method, immunoprecipitation method, FACS method, ultrafiltration method, gel filtration method, HPLC method, and a method of adsorbing to a carrier such as beads using an antibody or lectin can be mentioned. Also, exosomes may be recovered using a commercially available exosome isolation kit.

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

[0038] The presence of exosomes can be measured by a nanoparticle tracking system (for example, a device such as NanoSight). Further, on the surface of exosome particles, for example, molecules such as CD9, CD63, and CD81, which are tetraspanins, are present, 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 by immunological measurement methods (for example, Western blot, etc.).

[0039] 5. Fibrosis suppression method, fibrosis suppressor, defibrillation method, and defibrillation agent The secretome or exosomes released by cardiomyocytes or CMSCs treated with a ROCK inhibitor can suppress the fibrosis of cardiomyocytes and defibrillate fibrotic cardiomyocytes. Therefore, in another aspect, the present invention relates to a fibrosis suppression method and a defibrillation method 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, the ROCK inhibitor may be directly administered to cardiomyocytes in vivo to act on the ROCK inhibitor to release the secretome or exosomes, or treated with the ROCK inhibitor It is also possible to use the following cardiomyocytes, or to isolate and / or purify and use the selectome or exosomes released by cardiomyocytes treated with a ROCK inhibitor. In addition, fibrosis of cardiomyocytes, or defibrosis of fibrotic cardiomyocytes, may be brought about, for example, by activation of fibrotic cells or suppression of genes included in a signal transduction pathway related to fibrosis by microRNA contained in the selectome or exosomes released by cardiomyocytes treated with a ROCK inhibitor. Examples of such signal transduction pathways include TGFB1, E2F1, EGF, HRAS, AGT, etc., and preferably TGFB1.

[0040] When directly treating cardiomyocytes in vivo by administering a ROCK inhibitor, the ROCK inhibitor can act on cardiomyocytes already located in the vicinity of fibroblasts. Thus, the present invention includes a method for suppressing fibrosis and a method for defibrosis of fibroblasts, including treating cardiomyocytes present at a position capable of exerting a paracrine action on fibroblasts with a ROCK inhibitor.

[0041] When using cells, it may be carried out by using cardiomyocytes previously treated with a ROCK inhibitor, localizing the cardiomyocytes in the vicinity of fibroblasts, or co-culturing both cells. Alternatively, cardiomyocytes 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 suppressing fibrosis of fibroblasts, including treating cardiomyocytes with a ROCK inhibitor and localizing the cardiomyocytes with fibroblasts at a position where the cardiomyocytes can exert a paracrine action.

[0042] The term "paracrine action" means that a substance secreted from cardiomyocytes or CMSCs treated with a ROCK inhibitor acts on cells and tissues around the cardiomyocytes or CMSCs. Therefore, the "position capable of exerting paracrine action" is a position 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 present in the vicinity or adjacent to the fibroblasts. When cardiomyocytes pre-treated with a ROCK inhibitor are allowed to act on fibroblasts in vivo, the cardiomyocytes can be locally transplanted to the heart or a target site within the heart. That is, by transplanting the cardiomyocytes or CMSCs treated with the ROCK inhibitor of the present invention into the heart, a secretome can be released locally in the heart, and fibrosis by surrounding fibroblasts can be suppressed by paracrine action.

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

[0044] The culture supernatant of cardiomyocytes or CMSCs treated with a ROCK inhibitor and the exosomes separated and purified from the culture supernatant suppress fibrosis of cardiomyocytes. Therefore, the fibrosis suppression method of the present invention can also be carried out using the secretome or exosomes released from cardiomyocytes or CMSCs treated with a ROCK inhibitor. Therefore, the present invention relates to a method for suppressing fibrosis of fibroblasts, which includes treating fibroblasts with a secretome 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 fibrosis of fibroblasts, which includes culturing cardiomyocytes in the presence of a ROCK inhibitor, recovering the secretome or exosomes from the cultured cardiomyocytes, and treating the fibroblasts with the recovered secretome or exosomes.

[0045] In this specification, the method for suppressing fibrosis of fibroblasts may also be a method for preventing or treating diseases accompanied by myocardial fibrosis, and the agent for suppressing fibrosis of fibroblasts may also be a preventive or therapeutic agent for diseases accompanied by myocardial fibrosis. Diseases accompanied by myocardial fibrosis include diseases caused by myocardial fibrosis and diseases aggravated by myocardial fibrosis. For example, myocardial injury, myocardial fibrosis, myocardial infarction, heart failure, cardiac hypertrophy, hypertension, etc. can be mentioned.

[0046] 6. Method for suppressing onset and / or aggravation of cardiovascular diseases, and inhibitor MicroRNAs encapsulated in exosomes derived from cardiomyocytes treated with a ROCK inhibitor target genes associated with cardiovascular diseases, specifically genes associated with cardiovascular diseases such as cardiac necrosis and cell death, cardiac dilation, heart failure, and cardiac arrest. Therefore, by treating cardiomyocytes with a ROCK inhibitor, the onset and / or exacerbation of cardiovascular diseases can be suppressed. In addition, by treating cardiomyocytes with a ROCK inhibitor, signal transduction pathways related to the development and / or function of the cardiovascular system can be activated. Thereby, for example, cell motility, angiogenesis, vasculogenesis, and the development of vascular structures of endothelial cells can be promoted. Therefore, in another aspect, the present invention relates to an inhibitor for suppressing the onset and / or exacerbation of cardiovascular diseases, a therapeutic agent for cardiovascular diseases, an agent for promoting cell motility of endothelial cells, an agent for promoting angiogenesis, an agent for promoting vasculogenesis, and an agent for promoting the development of vascular structures, which comprises treating cardiomyocytes with a ROCK inhibitor, cardiomyocytes cultured in the presence of a ROCK inhibitor, a secretome or exosomes released from cardiomyocytes treated with a ROCK inhibitor. In this method, the ROCK inhibitor may be directly administered to cardiomyocytes in vivo to act on the ROCK inhibitor to release a secretome or exosomes, cardiomyocytes treated with a ROCK inhibitor may be used, or a secretome or exosomes released from cardiomyocytes treated with a ROCK inhibitor may be isolated and / or purified and used. Further, this method may include suppressing the signal transduction pathway related to the cardiovascular disease and / or activating the signal transduction pathway related to the development and / or function of the cardiovascular system by microRNAs encapsulated in exosomes derived from cardiomyocytes treated with a ROCK inhibitor. Examples of cardiovascular diseases include the above-mentioned acute and chronic cardiovascular diseases (such as cardiomyopathy (e.g., dilated cardiomyopathy), 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 disease, arterial occlusion, congestive heart failure, and heart failure.

[0047] In another aspect, the present invention relates to a method for suppressing the onset and / or exacerbation of cardiovascular diseases or a method for treating the same, which comprises administering to a patient in need thereof an effective amount of a ROCK inhibitor, cardiomyocytes treated with a ROCK inhibitor, or a secretome or exosome released from cardiomyocytes treated with a ROCK inhibitor. The present invention also relates to the use of a ROCK inhibitor, cardiomyocytes treated with a ROCK inhibitor, or a secretome or exosome released from cardiomyocytes treated with a ROCK inhibitor for producing an agent for suppressing the onset and / or exacerbation of cardiovascular diseases. Alternatively, the present invention relates to a ROCK inhibitor, cardiomyocytes treated with a ROCK inhibitor, or a secretome or exosome released from cardiomyocytes treated with a ROCK inhibitor for suppressing the onset and / or exacerbation of cardiovascular diseases or for treating the same.

[0048] 7. Administration method and formulation The selectome and exosome can be obtained from cardiomyocytes treated with a ROCK inhibitor by the above-described method. When the treatment with the selectome or exosome is carried out in vitro, it can be carried out by culturing cardiomyocytes or fibroblasts in the presence of the selectome or exosome. When carried out in vivo, the selectome or exosome may be administered locally to the heart or a target site within the heart, or may be administered systemically.

[0049] The above-described drug may contain, as an active ingredient, a ROCK inhibitor, cardiomyocytes treated with a ROCK inhibitor, or a secretome or exosome released from cardiomyocytes treated with a ROCK inhibitor alone, or may contain other components as necessary. For example, When the agent is for administration to animals, other components may be pharmaceutically acceptable additives such as sterilized water, physiological saline, buffers, excipients, binders, disintegrants, emulsifiers, surfactants, stabilizers, lubricants, diluents, fluidity promoters, flavoring agents, coloring agents, and fragrances. For example, when the agent of the present invention is used for administration to animals, those agents can be administered in an oral dosage form or a parenteral dosage form such as an injection or a drip infusion. Also, those agents may be orally administered as tablets, powders, granules, syrups, etc., or may be parenterally administered as injections or drip infusions. The dosage amount may be an amount effective to achieve the purpose and can be determined according to symptoms, age, gender, body weight, dosage form, etc.

[0050] The method of the present invention can be carried out in vivo, ex vivo, or in vitro, except when such an interpretation is inconsistent.

[0051] The present invention will be described in more detail below using examples, which do not limit the scope of the present invention. The documents cited throughout this specification are incorporated herein by reference in their entirety.

Examples

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

[0053] The culture medium for PC-100-021 was prepared as follows: Vascular A Smooth Muscle Cell growth Kit (containing 5% FBS, 5% L-Glutamine, 50 μg / mL Ascorbic acid, 5 ng / mL EGF, 5 μg / mL Insulin, 5 ng / mL FGF-b) (ATCC, Cat#PCS-100-042) and 1% Antibiotic x Antimitotic were added.

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

[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 culture medium (CMM) suitable for cardiomyocyte culture and culture medium for PC-100-021, respectively, and were cultured in a planar manner in an incubator (37 °C, 5% CO2 / 95% air).

[0056] (2) Subculture The above-mentioned primary cultured human cardiomyocytes were treated with TrypLE Express (ThermoFisher) and recovered, and were seeded at 9×10 cells / cm2 into a culture vessel (2 μg / cm2, 150 mm dish) coated with collagen I, and were cultured in Vascular Smooth Muscle Cell growth medium or Myocyte basal medium. The cultured cells were prepared into a frozen stock using CELLBANKER (registered trademark) 1 (Takara Bio). 2 ,150mm dish) 3 cells / cm 2

[0057] (Example 2) Effects of TGFβ receptor inhibitor and ROCK inhibitor in cardiomyocyte culture (1) Culture in the presence of the test small molecule compound The TGFβ receptor inhibitor, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A-83-01:A) (final concentration 1 μM), which is a low molecular weight compound, the ROCK inhibitor, (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide (Y-27632:Y) (final concentration 10 μM), or 3 mL of Vascular Smooth Muscle Cell growth medium or Myocyte basal medium containing or not containing both compounds of A-83-01 and Y-27632 were plated on a 35 mm plate (IWAKI) containing the medium, and subcultured human cardiomyocytes were seeded at 0.5×10 2 cells / cm 2 . On the third day, 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 in the same manner every three days.

[0058] (2) Low-speed imaging at low cell density After medium replacement, low-speed imaging was performed using a BZ9000 all-in-one fluorescence microscope (Keyence). Also, individual cells were tracked throughout the imaging period (110 days), and the final cell number derived from each cell was counted.

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

[0060] (3) Quantitative RT-PCR Total RNA was isolated from each cardiomyocyte after 1 month and 2 months of culture in the presence of the test low molecular weight compound using the miRNeasy Mini Kit (QIAGEN). The reverse transcription reaction was carried out using the High-Capacity cDNA Reverse Transcription Kit Lifetechnologies according to the manufacturer's guidelines. PCR was performed using Taqman Probe (ThermoFisher) with the obtained cDNA as a template. The expression level of the target gene was normalized with β-actin, an endogenous control.

[0061] Figure 2 shows the expression levels of the mRNAs of cardiomyocyte progenitor markers (GATA4, VCAM-1) and MYL2, a cardiomyocyte maturation marker, after culturing HCM cells and PC-100-021 cells in the presence of A-83-01 (A) or Y-27632 (Y). In both HCM cells and PC-100-021 cells, in the untreated group (N.T.), differentiation progressed with continuous culture, resulting in a decrease in the expression of progenitor markers (GATA4, VCAM-1) and an increase in the expression of the maturation marker (MYL2). The decrease in these progenitor markers (GATA4, VCAM-1) and the increase in the maturation marker (MYL2) were suppressed by treating HCM cells and PC-100-021 cells with Y-27632. On the other hand, treatment with A-83-01 was shown to promote the decrease in progenitor markers (GATA4, VCAM-1) and the increase in the maturation marker (MYL2).

[0062] Figure 3A shows the expression levels of the mRNAs of cardiomyocyte progenitor markers (GATA4, VCAM-1), MYL2, a cardiomyocyte maturation marker, and the mRNAs of senescence markers (CDKN1A, CDKN2A) after culturing HCM cells and PC-100-021 cells in the presence of A-83-01 (A) or Y-27632 (Y). HCM cells and PC-1 In the case of both HCM cells and PC-100-021 cells in the untreated group (N.T.), differentiation progressed by continuing the culture, the expression of progenitor cell markers (GATA4, VCAM-1) decreased, and the expression of the mature marker (MYL2) increased. The decrease in these progenitor cell markers (GATA4, VCAM-1) and the increase in the mature marker (MYL2) were suppressed by treating HCM cells and PC-100-021 cells with Y-27632. On the other hand, treatment with A-83-01 was shown to promote the increase in the mature marker (MYL2). Furthermore, the senescence markers (CDKN1A, CDKN2A) were hardly expressed in HCM cells and PC-100-021 cells treated with A-83-01 (A) or Y-27632 (Y). Therefore, it was shown that there was little cell death due to treatment with A-83-01 (A) or Y-27632 (Y). Also, from Figure 3B, in HCM cells and PC-100-021 cells treated with A-83-01 (A) or Y-27632 (Y), the endothelial cell marker (CD31) was not expressed, and the muscle cell marker was expressed, and it was confirmed that these cells contained only muscle cells and no endothelial cells were mixed in the primary culture.

[0063] (Example 3) Characteristics of the signal transduction pathway of cardiomyocytes treated with a ROCK inhibitor When cardiomyocytes were treated with Y-27632 (Y), the signal transduction pathway that changed compared to untreated cardiomyocytes was estimated. For this estimation, Ingenuity® Pathway Analysis (IPA) (QIAGEN) was used according to the manufacturer's instructions.

[0064] In cardiomyocytes treated with the ROCK inhibitor Y-27632 (Y), compared with untreated cardiomyocytes, the pathways related to cardiovascular diseases were inhibited. Specifically, it was presumed that the signal transduction pathways related to ventricular dysfunction, left ventricular dysfunction, left heart damage, heart failure, familial cardiovascular disease, cerebrovascular dysfunction, left ventricular abnormality, ventricular abnormality, peripheral vascular disease, atherosclerosis, arteriosclerosis, vascular occlusion, vascular occlusion disease, arterial occlusion, congestive heart failure, and heart failure were suppressed (Figure 4A). Therefore, it was suggested that the signal transduction pathways related to cardiovascular diseases decreased in cardiomyocytes treated with the ROCK inhibitor. Also, when cardiomyocytes were treated with Y-27632 (Y), the signal transduction pathways related to cardiac contraction, blood pressure, and myocardial contraction were suppressed, and it was presumed that the signal transduction pathways related to endothelial cell motility, vasculogenesis, angiogenesis, and the development of vascular structures were activated (Figure 4B). Therefore, it was suggested that the signal transduction pathways related to the development and function of the cardiovascular system were activated in cardiomyocytes treated with the ROCK inhibitor. Furthermore, from such signal system changes, it was suggested that cardiomyocytes treated with the ROCK inhibitor exhibited regenerative characteristics and were less likely to undergo malignant transformation.

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

[0066] Figure 5 shows the number of particles (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 tracking system (Nanosight LM-10). Furthermore, it shows the presence of CD9 molecule, CD63 molecule, and CD81 molecules contained in the exosomes identified by Western blotting. This indicates that exosomes were actually recovered.

[0067] (Example 5) Marker expression in TGFβ-stimulated fibroblasts co-cultured with cardiomyocytes treated with a TGFβ receptor inhibitor and a ROCK inhibitor Human cardiac fibroblasts were activated with TGFβ for 24 hours. The activated fibroblasts were co-cultured with cardiomyocytes treated with A-83-01 or Y-27632 for 48 hours or more. Total RNA and proteins were extracted for fibrosis activation marker analysis.

[0068] Figure 6 is a graph showing the mRNA levels (A, C) of ACTA2, a fiber-related gene, and the protein levels (B, D) of αSMA, a fibrosis marker, in TGFβ-stimulated fibroblasts co-cultured with cardiomyocytes treated with A-83-01 and Y-27632. The expression levels of ACTA2 and αSMA can be used as indicators of cell fibrosis. The expression levels of ACTA2 and αSMA increased with the fibrosis of human cardiac fibroblasts induced by TGFβ treatment, but decreased with co-culture with cardiomyocytes, indicating that the activation of fibroblasts was suppressed. Therefore, it was shown that the secretome purified from cardiomyocytes treated with a TGFβ receptor inhibitor and a ROCK inhibitor has the effect of suppressing the activation of fibroblasts.

[0069] Figure 7 is a graph showing the mRNA level (A, C) of ACTA2, a fiber-related gene, and the protein level (B, D) of αSMA, a fibrosis marker, in TGFβ-stimulated fibroblasts co-cultured with cardiomyocytes treated with A-83-01 and Y-27632. The graph shows the mean ± standard deviation. The expression levels of ACTA2 and αSMA can be used as indicators of cell fibrosis. The expression levels of ACTA2 and αSMA increased with the fibrosis of human cardiac fibroblasts by TGFβ treatment, but decreased with co-culture with cardiomyocytes, indicating that the activation of fibroblasts was suppressed. Therefore, the effect of suppressing the activation of fibroblasts by the secretome purified from cardiomyocytes treated with TGFβ receptor inhibitor and ROCK inhibitor was shown.

[0070] (Example 6) Marker Expression in TGFβ-Stimulated Fibroblasts Cultured in the Presence of Exosomes Derived from Cardiomyocytes Treated with TGFβ Receptor Inhibitor and ROCK Inhibitor Human cardiac fibroblasts were activated with TGFβ for 24 hours. Next, exosomes (EV) derived from cardiomyocytes treated with Y-27632 were added, and the fibroblasts were cultured for an additional 48 hours. Total RNA and protein were extracted for fibrosis activation marker analysis.

[0071] Figure 8 is a graph showing the mRNA level (A, C) of ACTA2 and the protein level (B, D) of αSMA expressed by fibroblasts that incorporated exosomes purified from cardiomyocytes treated with A-83-01 and Y-27632 into fibroblasts activated by TGFβ treatment. The expression levels of ACTA2 and αSMA increased with the fibrosis of human cardiac fibroblasts by TGFβ treatment, but decreased significantly with the addition of exosomes, indicating that the activation of fibroblasts was suppressed. Therefore, the effect of suppressing the activation of fibroblasts by exosomes purified from cardiomyocytes treated with A-83-01 and Y-27632 was shown.

[0072] Figure 9 is a graph showing the mRNA levels (A, C) of ACTA2 and the protein levels (B, D) of αSMA expressed by fibroblasts that incorporated exosomes purified from cardiomyocytes treated with A-83-01 and Y-27632 into fibroblasts activated by TGFβ treatment. The graph shows the mean ± standard deviation. The expression levels of ACTA2 and αSMA increase with the fibrosis of human cardiac fibroblasts by TGFβ treatment, but the levels were significantly decreased by the addition of exosomes, indicating that the activation of fibroblasts was suppressed. Therefore, the effect of suppressing the activation of fibroblasts by exosomes purified from cardiomyocytes treated with A-83-01 and Y-27632 was shown to be

[0073] (Example 7) Immunostaining Similar to Example 5 or Example 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 (EV) derived from the HCM cells. Next, the cells were washed twice with PBS and then fixed by adding 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) at room temperature for 1 hour. The secondary antibody conjugated to AlexaFluor 594 was further incubated for 1 hour. The nucleus was stained with DAPI (Vectashield).

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

[0075] Figure 11 shows the 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. It was found that the expression of αSMA, fibronectin, and collagen I was significantly suppressed by exosomes derived from HCM treated with a ROCK inhibitor, and the inhibitory effect on the fibrosis of cardiac fibroblasts was confirmed.

[0076] Figure 12 shows typical 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. The graph shows the mean ± standard deviation. It was found that TGFβ treatment induced the expression of αSMA and increased the number of αSMA-positive cells, but further addition of exosomes derived from HCM treated with Y-27632 suppressed the expression by TGFβ and decreased the number of αSMA-positive cells. Similarly, it was found that the expression of fibronectin and collagen I was also significantly suppressed by exosomes derived from HCM treated with Y-27632, and the inhibitory effect on the fibrosis of cardiac fibroblasts was confirmed.

[0077] (Example 8) Changes in signal transduction pathways in fibroblasts cultured in the presence of exosomes derived from cardiomyocytes treated with a ROCK inhibitor In activated fibroblasts and fibroblasts treated with exosomes derived from cardiomyocytes treated with Y-27632 (Y), signal transduction pathways that changed compared to untreated cardiomyocytes were estimated. For this estimation, IPA was used according to the manufacturer's instructions.

[0078] In activated fibroblasts, the related signal transduction pathways were presumed to be promoted in the order of hepatic fibrosis / hepatic stellate cell activation, CREB signaling in neurons, axon guidance signaling, cardiac hypertrophy signaling (increase), breast cancer control by Stathmin1, atherosclerotic signaling, hepatic fibrosis signaling pathway, STAT3 pathway, roles of macrophages, fibroblasts, endothelium, and osteoarthritis pathway (Figure 13A). On the other hand, in fibroblasts treated with exosomes derived from cardiomyocytes treated with Y-27632 (Y), the related signal transduction pathways were presumed to be promoted in the order of CREB signaling in neurons, sperm motility, breast cancer control by Stathmin1, cardiac hypertrophy signaling, STAT3 pathway, estrogen receptor signaling, roles of macrophages, fibroblasts, endothelium, IL-15 production, hepatic fibrosis / hepatic stellate cell activation, and PTEN signaling (Figure 13B).

[0079] (Example 9) Changes in activation signal transduction factors in fibroblasts cultured in the presence of exosomes derived from cardiomyocytes treated with a ROCK inhibitor For the signal transduction pathways presumed in Example 8, in order to presume more specific signal transduction factors, in activated fibroblasts, the signal transduction pathways that were suppressed or promoted compared to untreated cardiomyocytes, and the signal transduction pathways that were suppressed or promoted compared to untreated cardiomyocytes when activated fibroblasts were treated with exosomes derived from HCM cells treated with a ROCK inhibitor were presumed. For this presumption, IPA was used according to the manufacturer's instructions.

[0080] In activated fibroblasts, the related signal transduction pathways were presumed to be suppressed in the order of NFκB (complex), HGF, Vegf, IL17A, IL1B, CSF2, CHUK, EGF, Tlr, TLR4, and were presumed to be promoted in the order of TGFB1, Tgf beta, TGFB3, NUPR1, NORAD, TAZ, MRTFA, TGFBR1, TGFB2, DRD2 (Figure 14A, B).

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

[0082] (Example 10) Functional analysis of microRNA For exosomes derived from HCM cells treated with a ROCK inhibitor, an exosome group in which the encapsulated microRNA was most highly expressed was selected (see Fig. 15A), and the target genes of the microRNA were identified. Specifically, the culture medium of HCM cells treated with a ROCK inhibitor was replaced with a serum-free culture medium, and after culturing for 72 hours, 500 ml of the culture supernatant was collected. The culture supernatant was centrifuged at 10,000×g for 30 minutes to remove cell debris and the like, and then ultracentrifuged at 100,000×g at 4°C for 70 minutes using a Beckman Coulter ultracentrifuge (Optima-XE-90). The pellet was dissolved in PBS(-), and after further ultracentrifugation at 100,000×g at 4°C for 70 minutes, it was dissolved in an appropriate amount of PBS(-). From this exosome fraction, microRNA was recovered using the miRNeasy kit (QIAGEN), and 8 ng of it was subjected to RNA analysis using a next-generation sequencer (DNAchip Research Institute).

[0083] As a result, it was shown that 18.5% of the 513 genes targeted by the selected microRNA were related to cardiovascular diseases such as cardiac necrosis and cell death, cardiac dilation, and heart failure (see Fig. 15B). In addition, it was shown that the target genes of the selected microRNA were deeply involved in the TGFB1 signaling pathway (see Fig. 15C).

Claims

1. A method for producing cardiomyocyte stem / progenitor cells, comprising treating cardiomyocytes with a ROCK inhibitor.

2. A method for culturing cardiomyocytes, comprising culturing cardiomyocytes in the presence of a ROCK inhibitor.

3. The culture period is 1 month or longer, and / or after the lapse of the culture period, the level of one or more markers selected from cardiomyocyte maturation markers, senescence markers, and endothelial cell markers expressed by the cardiomyocytes is lower than the level of the corresponding markers of cardiomyocytes cultured in the absence of a ROCK inhibitor. The culture method according to claim 2, characterized in that.

4. The culture method according to claim 3, wherein the maturation marker is MYL2, and / or the senescence marker is CDKN1A and / or CDKN2A, and / or the endothelial cell marker is CD31.

5. The culture method according to claim 3 or claim 4, characterized in that after the lapse of the culture period, the level of the cardiomyocyte progenitor cell marker expressed by the cardiomyocytes is higher than the level of the corresponding marker of cardiomyocytes cultured in the absence of a ROCK inhibitor.

6. A method for maintaining and culturing cardiomyocyte stem / progenitor cells, comprising culturing cardiomyocyte stem / progenitor cells in the presence of a ROCK inhibitor.

7. A method for preparing a secretome or exosome, comprising recovering a secretome or exosome from cardiomyocytes cultured by the method according to claims 2 to 5.

8. A method for inhibiting fibrosis of fibroblasts, comprising localizing cardiomyocytes treated with a ROCK inhibitor and fibroblasts at a position where the cardiomyocytes can exert a paracrine effect.

9. A method for inhibiting fibrosis of fibroblasts, comprising treating cardiomyocytes present at a position where a paracrine action can be exerted on fibroblasts with a ROCK inhibitor.

10. A method for inhibiting fibrosis of fibroblasts, comprising treating fibroblasts with a secretome or exosome extracted from cardiomyocytes cultured in the presence of a ROCK inhibitor.

11. Culturing cardiomyocytes in the presence of a ROCK inhibitor, Recovering a secretome or exosome from the cultured cardiomyocytes, The method for inhibiting fibrosis of fibroblasts according to claim 8, comprising treating fibroblasts with the recovered secretome or exosome.

12. The method according to any one of claims 1 to 11, wherein the ROCK inhibitor is (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide.

13. The method according to any one of claims 1 to 12, wherein the cardiomyocytes are human cardiomyocytes.

14. An inducer of cardiac stem / progenitor cells or a maintenance culture agent for cardiac stem / progenitor cells, containing a ROCK inhibitor as an active ingredient.

15. Cardiac stem / progenitor cells obtained by culturing cardiomyocytes in the presence of a ROCK inhibitor.

16. Exosomes or selectomes derived from cardiomyocytes cultured in the presence of a ROCK inhibitor.

17. The exosomes or selectomes according to claim 16, containing microRNA targeting a gene related to cardiovascular disease.

18. The exosomes or selectomes according to claim 17, wherein the gene related to cardiovascular disease is a gene involved in the TGFβ1 signaling pathway.

19. A fibrosis inhibitor or defibrosis agent, containing a ROCK inhibitor, cardiomyocytes cultured in the presence of a ROCK inhibitor, or the exosomes or selectomes according to any one of claims 16 to 18 as an active ingredient.

20. The inducer of cardiac stem / progenitor cells or the maintenance culture agent for cardiac stem / progenitor cells according to claim 14, the cardiac stem / progenitor cells according to claim 15, the exosomes or selectomes according to any one of claims 16 to 18, or the fibrosis inhibitor or defibrosis agent according to claim 19, wherein the ROCK inhibitor is (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide.

21. An inhibitor or therapeutic agent for the onset and / or exacerbation of cardiovascular disease, containing a ROCK inhibitor acting on cardiomyocytes as an active ingredient.

22. The inhibitor or therapeutic agent according to claim 21, wherein the cardiovascular disease is at least one disease selected from the group consisting of ventricular dysfunction, left ventricular dysfunction, left heart disorder, heart failure, familial cardiovascular disease, cerebrovascular dysfunction, left ventricular abnormality, ventricular abnormality, peripheral vascular disease, atherosclerosis, arteriosclerosis, vascular occlusion, vascular occlusion disease, arterial occlusion, congestive heart failure, and heart failure.

23. An agent for promoting cell motility, angiogenesis, vasculogenesis, and vascular structure formation of endothelial cells, which contains a ROCK inhibitor acting on cardiomyocytes as an active ingredient.

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