Preparation method and application of mesoderm-derived heart mesenchymal stem cells

Abstract

The invention discloses a preparation method and application of mesoderm-sourced heart mesenchymal stem cells, mesoderm-sourced CMSC is induced by hPSC, the differentiation path of the mesoderm-sourced CMSC is strictly limited through a specific induction culture solution, and the mesoderm-sourced CMSC is obtained after the mesoderm-sourced CMSC is subjected to the stages of mesoderm progenitor cells, cardiac progenitor cells and epicardial progenitor cells in sequence, and the mesoderm-sourced CMSC is clear in source and low in heterogeneity. The cardiac micro-tissue formed by assembling the cardiac muscle cells, the endothelial cells and the CMSC from the hPSC shows remarkable advantages in the aspects of cardiac micro-tissue polymerization capability and cardiac muscle cell maturation promotion, and related functions and repeatability of the model are improved; the requirements of a large-scale heart micro-tissue detection platform can be met; a new seed cell source is provided for cell therapy of cardiovascular diseases; a novel and high-quality cell source is provided for heart disease modeling and clinical transformation, especially for constructing heart micro-tissues, supporting heart cell transplantation, cell therapy of cardiovascular diseases and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of human pluripotent stem cell (hPSC) induced differentiation, and more specifically, to a method for preparing a cardiac mesenchymal stem cell (CMSC) derived from mesoderm and its application. Background Art

[0002] MSC is one of the most common supporting cell types at present. It is a type of adult cell with the ability of self-renewal and multi-directional differentiation. They were initially discovered in the bone marrow and have broad application prospects in tissue engineering and cell therapy. In 2006, the International Society for Cellular Therapy (ISCT) established the minimum identification criteria that MSC needs to meet: (1) When cultured under standard conditions, the cells adhere to the culture vessel and grow in a adherent-like manner; (2) The expression rates of cell surface positive markers CD73, CD90, and CD105 are > 95%, and the expression rates of negative markers CD11b, CD19, CD34, CD45, and HLA-DR are < 2%; (3) In vitro differentiation ability, which can differentiate into osteoblasts, chondrocytes, adipocytes, etc.

[0003] MSC has multiple developmental origins, including the neural crest, mesoderm, and trophoblast. They eventually colonize different adult tissues such as bone marrow, umbilical cord, adipose tissue, and heart. MSCs from different sources show their respective advantages in the fields of regenerative medicine and cell therapy due to their unique biological characteristics and functions. For example, MSCs of neural crest developmental origin have significant advantages in supporting hematopoiesis, while MSCs of mesoderm origin are more prominent in osteogenic ability (PMID: 25255216). Trophoblast-derived MSCs are considered to be a preferred stem cell source for treating pregnancy-related diseases due to their pluripotency, low immunogenicity, and ability to regulate maternal immune responses. In addition, trophoblast-derived MSCs play an important role in the development of the human placenta and are one of the key cell components for in vitro construction of trophoblast organoids (PMID: 35966876).

[0004] In the field of cardiac regeneration, cardiac MSCs exhibit excellent cardiac repair capabilities and are considered the best MSC source for treating cardiovascular diseases (PMID: 31991111). In addition, most human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) do not have a mature phenotype and are more similar to fetal cardiomyocytes in terms of cell structure, calcium handling, metabolism, contractile function, and gene expression compared to adult cardiomyocytes. This phenotypic difference limits the application of hPSC-CMs in in vitro disease modeling, drug discovery, and cell therapy.

[0005] Approximately 70% of the cells in the human heart are non-myocytes, among which cardiac mesenchymal stem cells (CMSCs) and cardiac endothelial cells constitute the majority of these cardiac support cells (PMID: 12506127). Studies have shown that cardiac support cell types play important roles in cardiomyocyte development, maturation, and cardiovascular diseases. CMSCs play a structural role and promote cardiomyocyte maturation by providing extracellular matrix molecules, mechanical signals, and paracrine factors (PMID: 19729019). A multicellular microtissue model combining hPSC-CMs with CMSCs isolated from human heart tissue and primary human endothelial cells exhibits a significant mature phenotype. Especially in compound responsiveness tests, this cardiac model shows a positive response to isoproterenol (ISO), indicating an important indicator of cardiac mature function (PMID: 27125969). However, due to the large batch differences in this multicellular microtissue prepared from CMSCs isolated from the heart, it is not conducive to biological replication in disease modeling and drug screening.

[0006] In summary, cardiac mesenchymal stem cells (CMSCs) play important roles in cardiac development, functional maturation, and regenerative repair.

[0007] In recent years, with the rapid development of hPSC technology, replacing primary human cells with hPSC-derived support cell types has many benefits in improving the relevant functions and reproducibility of multicellular microtissue models (PMID: 32459996). However, existing technologies limit the isolation of CMSCs from in situ heart tissue, which poses risks such as trauma, bleeding, and arrhythmia, and the cell quantity is limited. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have a maturation bottleneck, which limits their application in disease modeling, drug discovery, and cell therapy. Summary of the Invention

[0008] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing mesoderm-derived cardiac mesenchymal stem cells and its applications. By using a specific induction culture medium to strictly define the cell differentiation path, the present invention successfully induces mesoderm-derived cardiac mesenchymal stem cells from hPSCs with high efficiency. This method not only avoids the trauma and risks that may be caused by directly isolating CMSCs from human heart tissues, but also takes advantage of the rapid development of hPSC technology to provide an unlimited supply of CMSCs with controllable quality, effectively solving the problem of limited number of primary human cells.

[0009] Cardiac MSCs have three sources. The induction and differentiation path from mesoderm progenitor cells to epicardial progenitor cells in the present invention only takes 12 days, and the expression levels of WT and TBX18 are higher than the induction efficiency of the prior art. In addition, compared with bone marrow mesenchymal stem cells (BMSCs), CMSCs have better cardiac microtissue aggregation ability. The cardiac microtissues prepared using hPSC-derived CMSCs are closer to the characteristics of adult heart cells, can more accurately predict drug toxicity, and are better used for drug testing. The importance of CMSCs in the development, maturation, and function maintenance of the heart also enables them to provide a new source of seed cells for cell therapy of cardiovascular diseases.

[0010] In summary, the induction method of the present invention not only improves the induction efficiency and shortens the induction time, but also the entire induction and differentiation system is efficient, convenient, has high cell homogeneity, high operability, standardized procedures, stability, and homogeneity, providing a new, efficient, and safe method for the research and treatment of cardiovascular diseases.

[0011] The first object of the present invention is to provide a culture medium A.

[0012] The second object of the present invention is to provide the application of the culture medium A in inducing pluripotent stem cells into mesoderm progenitor cells.

[0013] The third object of the present invention is to provide a method for inducing pluripotent stem cells into mesoderm progenitor cells

[0014] The fourth object of the present invention is to provide a culture medium B.

[0015] The fifth object of the present invention is to provide the application of the culture medium B in inducing mesoderm progenitor cells into cardiac progenitor cells.

[0016] The sixth object of the present invention is to provide a method for inducing mesoderm progenitor cells into cardiac progenitor cells

[0017] The seventh object of the present invention is to provide a culture medium C.

[0018] The eighth object of the present invention is to provide the use of the described culture medium C in inducing cardiac progenitor cells into epicardial progenitor cells.

[0019] The ninth object of the present invention is to provide a method for inducing cardiac progenitor cells into epicardial progenitor cells.

[0020] The tenth object of the present invention is a culture medium composition.

[0021] The eleventh object of the present invention is the use of the culture medium composition in inducing pluripotent stem cells into epicardial progenitor cells.

[0022] The twelfth object of the present invention is to provide a method for inducing epicardial progenitor cells into cardiac mesenchymal stem cells.

[0023] The thirteenth object of the present invention is to provide the use of the cardiac mesenchymal stem cells prepared by the described method in the preparation of cardiac microtissues.

[0024] The fourteenth object of the present invention is to provide a culture medium composition for preparing cardiac microtissues.

[0025] The fifteenth object of the present invention is to provide the use of the described culture medium composition in the preparation of cardiac microtissues.

[0026] The sixteenth object of the present invention is to provide a method for preparing cardiac microtissues.

[0027] The seventeenth object of the present invention is to provide a cardiac microtissue.

[0028] The eighteenth object of the present invention is to provide the use of the described cardiac microtissue in serving as or preparing a cardiac model.

[0029] In order to achieve the above objects, the present invention is realized through the following technical solutions:

[0030] The present invention establishes an efficient induction system for deriving CMSC from hPSC by simulating the in vivo differentiation path, meeting the requirements of a large-scale cardiac microtissue detection platform. The cardiac microtissues assembled from three homologous purified hPSC-derived cell types (i.e., cardiomyocytes, endothelial cells, and CMSC) exhibit a higher degree of maturity closer to that in vivo. In addition, in view of the importance of CMSC in cardiac development, maturation, and function maintenance, CMSC can provide a new source of seed cells for the cell therapy of cardiovascular diseases.

[0031] The present invention strictly defines the cell differentiation pathway through a specific induction culture medium, simulates the in vivo differentiation process, and uses chemical small molecules and cytokines to finally obtain CMSC through stages such as mesoderm progenitor cells, cardiac progenitor cells, and epicardial progenitor cells, including: (1) culturing mesoderm progenitor cells in a culture medium containing a Wnt agonist; (2) further culturing cardiac progenitor cells in a culture medium containing a Wnt inhibitor, Retinoic acid (RA), BMP4, and bFGF; (3) adding a Wnt agonist, bFGF, and VEGF to the culture medium to generate epicardial progenitor cells highly expressing WT1, TBX18, TCF21, and ALDH1A; (4) further inducing CMSC in MesenCultTM-ACF Plus Culture medium to generate a population of cardiac mesenchymal stem cells.

[0032] The present invention claims protection for a culture medium A, which is a serum-free medium containing 3-6 μM CHIR99021, 50-100 μg / mL human transferrin, 100-200 μM L-ascorbic acid, 5-8 μg / ml sodium selenite, and 0.5-1% (V / V) Lip culture additive.

[0033] Preferably, it is a serum-free medium containing 5 μM CHIR99021, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive.

[0034] The application of the above-mentioned culture medium A in inducing pluripotent stem cells into mesoderm progenitor cells also belongs to the protection scope of the present invention.

[0035] The present invention also claims protection for a method of inducing pluripotent stem cells into mesoderm progenitor cells. The pluripotent stem cells are inoculated in a culture container coated with Matrigel and cultured, and then replaced with the above-mentioned culture medium A for culture.

[0036] Preferably, the pluripotent stem cells are concentrated in a culture container coated with Matrigel and cultured in a serum-free medium containing 1-10 μM Y-27632 for 0.5-1.5 days, and then replaced with the culture medium A for culture for 1.5-2.5 days.

[0037] More preferably, the pluripotent stem cells are concentrated in a culture container coated with Matrigel and cultured in mTeSR medium containing 5 μM Y-27632 for 1 day, and then replaced with the culture medium A for culture for 2 days.

[0038] Preferably, the Matrigel is Matrigel or Geltrex.

[0039] More preferably, the method for preparing the culture container coated with Matrigel is as follows: Matrigel and serum-free medium are mixed evenly at a volume ratio of 1:(90-110), covered on the culture surface of the culture container, fully reacted, and the liquid is removed before use; or Geltrex and serum-free medium are mixed evenly at a volume ratio of 1:(45-55), covered on the culture surface of the culture container, fully reacted, and the liquid is removed before use.

[0040] More preferably, before inoculating the pluripotent stem cells, they are digested with a cell digestive solution and resuspended in a serum-free cell medium containing Y-27632.

[0041] Further preferably, the cell digestive solution is Accutase.

[0042] The present invention also claims a culture medium B, which is a serum-free medium containing 1-10 μM IWP2, 0.2-1 μM Retinoic acid, 5-20 ng / ml BMP4, 2-10 ng / ml bFGF, 50-100 μg / mL human transferrin, 100-200 μM L-ascorbic acid, 5-8 μg / ml sodium selenite, and 0.5-1% (V / V) Lip culture additive.

[0043] Preferably, it is a serum-free medium containing 10 μM IWP2, 0.5 μM Retinoic acid, 15 ng / ml BMP4, 5 ng / ml bFGF, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive.

[0044] The application of the culture medium B in inducing mesoderm progenitor cells into cardiac progenitor cells also belongs to the protection scope of the present invention.

[0045] The present invention also claims a method for inducing mesoderm progenitor cells into cardiac progenitor cells, which uses the culture medium B to culture mesoderm progenitor cells.

[0046] Preferably, the mesoderm progenitor cells are cultured by the method for inducing pluripotent stem cells into mesoderm progenitor cells.

[0047] More preferably, culture for 3.5-4.5 days

[0048] Most preferably, culture for 4 days.

[0049] The present invention also claims a culture medium C, which is a serum-free medium containing 2-6 μM CHIR9902, 2-10 ng / ml bFGF, 5-20 ng / ml VEGF, 50-100 μg / mL human transferrin, 100-200 μM ascorbic acid, 5-8 μg / ml sodium selenite, and 0.5-1% (V / V) Lip culture additive.

[0050] Preferably, it is a serum-free medium containing 2 μM CHIR9902, 5 ng / ml bFGF, 10 ng / ml VEGF, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive.

[0051] The application of the described culture medium C in inducing cardiac progenitor cells into epicardial progenitor cells also belongs to the protection scope of the present invention.

[0052] A method for inducing cardiac progenitor cells into epicardial progenitor cells, using the described culture medium C to culture cardiac progenitor cells; then dissociating the cells into single cells and transferring them into culture medium C containing Y-27632 for culture; then changing to culture medium C for continuous culture.

[0053] Preferably, the cardiac progenitor cells are obtained by a method of inducing mesoderm progenitor cells into cardiac progenitor cells.

[0054] More preferably, using the described culture medium C to culture cardiac progenitor cells for 1.5-2.5 days; then dissociating the cells into single cells and transferring them into culture medium C containing 1-10 μM Y-27632 for culture for 0.5-1.5 days; then changing to the described culture medium C for continuous culture for 1.5-2.5 days.

[0055] Even more preferably, using the described culture medium C to culture cardiac progenitor cells for 2 days; then dissociating the cells into single cells and transferring them into culture medium C containing 5 μM Y-27632 for culture for 1 day; then changing to the described culture medium C for continuous culture for 2 days.

[0056] The present invention also claims a culture medium composition containing the described culture medium A, the described culture medium B, and the described culture medium C.

[0057] And the application of the described culture medium composition in inducing pluripotent stem cells into epicardial progenitor cells.

[0058] The present invention also claims a method for inducing epicardial progenitor cells into cardiac mesenchymal stem cells, which uses a serum-free medium for mesenchymal stem cells to culture epicardial progenitor cells. The epicardial progenitor cells are obtained by the method for inducing cardiac progenitor cells into epicardial progenitor cells, until the cells expressing CD29, CD90, CD73, and CD105 reach more than 98%, and the cells expressing CD34 and CD45 are less than 2%.

[0059] As a specific embodiment, the serum-free medium for mesenchymal stem cells is MesenCult TM -ACF.

[0060] Preferably, the epicardial progenitor cells are obtained by the method for inducing cardiac progenitor cells into epicardial progenitor cells.

[0061] The application of the cardiac mesenchymal stem cells prepared by the method in the preparation of cardiac microtissues also belongs to the protection scope of the present invention.

[0062] The present invention also claims a culture medium composition for preparing cardiac microtissues, which includes a first-stage culture medium and a second-stage culture medium.

[0063] The first-stage culture medium is a serum-free medium containing 1-10 μM Y-27632, 5-50 ng / mL VEGF, 5-20 ng / mL bFGF, 10-30 ng / mL IGF, 50-100 μg / mL human transferrin, 100-200 μM ascorbic acid, 5-8 μg / ml sodium selenite, and 0.5-1% (V / V) Lip culture additive.

[0064] The second-stage culture medium is a serum-free medium containing 5-50 ng / mL VEGF, 5-20 ng / mL bFGF, 10-30 ng / mL IGF, 50-100 μg / mL human transferrin, 100-200 μM ascorbic acid, 5-8 μg / ml sodium selenite, and 0.5-1% (V / V) Lip culture additive.

[0065] Preferably, it includes a first-stage culture medium and a second-stage culture medium.

[0066] The first-stage culture medium is a serum-free medium containing 5 μM Y-27632, 50 ng / mL VEGF, 15 ng / mL bFGF, 20 ng / mL IGF, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive.

[0067] The second-stage culture medium is a serum-free culture medium containing 50 ng / mL VEGF, 15 ng / mL bFGF, 20 ng / mL IGF, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive.

[0068] Also claimed is the use of the culture medium composition in the preparation of cardiac microtissues.

[0069] The present invention also claims a method for preparing a cardiac microtissue, which is prepared by using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), cardiac mesenchymal stem cells obtained by the method, and human pluripotent stem cell-derived endothelial cells.

[0070] The cardiac microtissue obtained by the preparation method is constructed based on fully human-derived cells, avoiding the species difference limitations of animal models. It is more suitable for the construction of cardiac disease models, the study of cardiac disease mechanisms, the screening and development of cardiac drugs, and the testing of personalized medical programs.

[0071] The human pluripotent stem cells are human induced pluripotent stem cells and human embryonic stem cells.

[0072] Preferably, after the cardiac mesenchymal stem cells, pluripotent stem cell-derived cardiomyocytes, and human pluripotent stem cell-derived endothelial cells obtained by the method are mixed, they are first cultured in the first-stage culture medium in the culture medium composition for preparing cardiac microtissues for 2.5 - 3.5 days, and then replaced with the second-stage culture medium in the culture medium composition for preparing cardiac microtissues, and continue to be cultured in a low-adhesion culture container for 23 - 25 days.

[0073] More preferably, after the cardiac mesenchymal stem cells, pluripotent stem cell-derived cardiomyocytes, and human pluripotent stem cell-derived endothelial cells obtained by the method are mixed, they are first cultured in the first-stage culture medium in the culture medium composition for preparing cardiac microtissues for 3 days, and then replaced with the second-stage culture medium in the culture medium composition for preparing cardiac microtissues, and continue to be cultured in a low-adhesion culture container for 24 days.

[0074] Preferably, single cells of hPSC-CMs, single cells of cardiac mesenchymal stem cells, and single cells of human pluripotent stem cell-derived endothelial cells are mixed at a cell ratio of (6 - 8):(1 - 2):(1 - 2).

[0075] More preferably, single cells of hPSC-CMs, single cells of cardiac mesenchymal stem cells, and single cells of human pluripotent stem cell-derived endothelial cells are mixed at a cell ratio of 7:1.5:1.5.

[0076] More preferably, continue the culture in a low-viscosity culture vessel and change the culture medium every 2.5 to 3.5 days.

[0077] The present invention also claims protection for a cardiac microtissue prepared by the described method.

[0078] The present invention also claims protection for the application of the cardiac microtissue in being or preparing a cardiac model.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] The present invention discloses a mesoderm-derived CMSC and its preparation method. The mesoderm-derived CMSC is induced from hPSC, and its differentiation path is strictly defined by a specific induction culture medium. After successively experiencing the stages of mesoderm progenitor cells, cardiac progenitor cells, and epicardial progenitor cells, it is obtained. Its source is clear and its heterogeneity is low. The cardiac microtissue assembled from three types of homologous purified and cryopreserved hPSC-derived cell types (namely cardiomyocytes, endothelial cells, and CMSC derived from hPSC) shows significant advantages in the aggregation ability of cardiac microtissues and promoting the maturation of cardiomyocytes compared with bone marrow mesenchymal stem cells BMSC. The CMSC obtained by the present invention improves the related functions and repeatability of the model; it can meet the needs of a large-scale cardiac microtissue detection platform; it provides a new source of seed cells for the cell therapy of cardiovascular diseases; it provides a new and high-quality cell source for cardiac disease modeling and clinical translation, especially for constructing cardiac microtissues, supporting cardiac cell transplantation, and cell therapy of cardiovascular diseases. Brief Description of the Drawings

[0081] Figure 1 It is a flow chart for the induction of mesoderm-derived cardiac mesenchymal stem cells, which is divided into 5 stages in total, namely hPSC, Mesodern Progenitor (mesoderm progenitor cells), Cardiac Progenitor (cardiac progenitor cells), EpicardialProgenitor Cell (epicardial progenitor cells), and Cardiac Mesenchymal StromalCell (cardiac mesenchymal stem cells). It takes 27 days to complete the induction.

[0082] Figure 2 The morphology of mesoderm progenitor cells with different concentrations of CHIR99021 (CHIR); A shows the concentration of CHIR99021 is 3 μM; B shows the concentration of CHIR99021 is 4 μM; C shows the concentration of CHIR99021 is 5 μM; D shows the concentration of CHIR99021 is 6 μM; the scale bar is 100 μm.

[0083] Figure 3Cell morphological changes during the induction of mesoderm-derived cardiac mesenchymal stem cells; A is the cell morphology diagram obtained after 2 days of induction of embryonic stem cells H9 in the culture medium of Example 1; B is the cell morphology diagram obtained after 4 days of induction of mesoderm progenitor cells in the culture medium of Example 2; C is the cell morphology diagram obtained after 5 days of induction of cardiac progenitor cells in the culture medium of Example 3; D is the cell morphology diagram obtained after 15 days of induction of epicardial progenitor cells in the MesenCultTM-ACF culture medium of Example 4, and the scale bar is 100 μm.

[0084] Figure 4 For fluorescence quantitative PCR to detect markers of cardiac progenitor cells and epicardial progenitor cells; A is to detect the expression of mesoderm progenitor cell markers TBXT and MIXL1 in cells induced in the mesoderm progenitor cell induction culture medium; B is the expression levels of epicardial progenitor cell markers WT1, TBX18, TCF21, and ALDH1A in cells induced in the epicardial lipid progenitor cell induction culture medium.

[0085] Figure 5 For flow cytometry to detect markers CD29, CD90, CD73, CD105, CD45, CD34, and CD19 of mesoderm-derived CMSC cardiac mesenchymal stem cells prepared.

[0086] Figure 6 For the detection of the trilineage differentiation ability (osteogenesis, adipogenesis, and chondrogenesis) of mesoderm-derived CMSC prepared from hPSC; A is the alizarin red S staining after cell osteogenic differentiation, B is the oil red O staining after adipogenic differentiation; C is the alcian blue staining after chondrogenic differentiation; the scale bar is 100 μm.

[0087] Figure 7 For the purification and identification of hPSC-CM prepared; A is the flow cytometry to detect the marker cTnT of hPSC-CM prepared and the cell morphology diagram of hPSC-CM, and the scale bar is 50 μm; B is the immunofluorescence staining to detect the expression of the myocardial marker cTnT after lactate purification, and the scale bar is 50 μm.

[0088] Figure 8 For the purification and identification of hPSC-EC prepared; A is the flow cytometry to detect the positive marker CD31, negative marker CD45 of hPSC-EC prepared and the cell morphology diagram of hPSC-EC, and the scale bar is 50 μm; B is the immunofluorescence staining to detect the expression of CD31 and CD34 in cells after flow cytometry purification, and the scale bar is 50 μm.

[0089] Figure 9Morphological diagram of the assembled cardiac microtissue; A is assembled and formed by hPSC-CM and hPSC-derived endothelial cells; B is assembled and formed by BMSC, hPSC-CM and hPSC-derived endothelial cells; Figure C is assembled and formed by CMSC, hPSC-CM and hPSC-derived endothelial cells; the scale bar is 100 μm.

[0090] Figure 10 Electron micrograph of the cardiac microtissue; A is assembled and formed by hPSC-CM and hPSC-derived endothelial cells; B is assembled and formed by BMSC, hPSC-CM and hPSC-derived endothelial cells; Figure C is assembled and formed by CMSC, hPSC-CM and hPSC-derived endothelial cells; the red arrow indicates the Z-line, and the scale bar is 0.5 μm. Detailed implementation manners

[0091] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are, unless otherwise specified, reagents and materials that can be obtained from commercial channels.

[0092] In the following embodiments, the antibodies used for flow cytometry are shown in the following table:

[0093]

[0094]

[0095] In the following embodiments, the antibodies used for immunofluorescence detection are shown in the following table:

[0096]

[0097] In the following embodiments, the specific information of the reagents used is as follows:

[0098]

[0099]

[0100] Example 1 A culture medium for inducing pluripotent stem cells into mesoderm progenitor cells

[0101] DMEM / F12 medium containing 5 μM CHIR99021, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive.

[0102] Example 2 A culture medium for inducing mesoderm into cardiac progenitor cells

[0103] DMEM / F12 medium containing 10 μM IWP2, 0.5 μM Retinoic acid, 15 ng / ml BMP4, 5 ng / ml bFGF, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive.

[0104] Example 3 A culture medium for inducing cardiac progenitor cells into epicardial progenitor cells

[0105] DMEM / F12 medium containing 2 μM CHIR9902, 5 ng / ml bFGF, 10 ng / ml VEGF, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive.

[0106] Example 4 Induction of hPSC-derived mesoderm progenitor cells

[0107] I. Experimental method

[0108] 1. Culture of human pluripotent stem cells (hPSC)

[0109] hPSC was constructed by the laboratory using the induced pluripotent stem cell technology. The hPSC was amplified using the mTeSR culture medium from STEM CELL company, and Matrigel or Geltrex was used as the basement membrane gel for pretreatment of the culture dish to maintain the undifferentiated state of the cells.

[0110] The specific operation steps are as follows

[0111] (1) Basement membrane gel plating: Thaw Matrigel or Geltrex on ice, and dilute the Matrigel stock solution (dilute the Geltrex stock solution at a ratio of 1:50) with pre-cooled DMEM-F12 at a ratio of 1:100, then add it to the culture container and incubate at room temperature for more than 2 hours for standby. When in use, aspirate the Matrigel or Geltrex in the wells, and the culture container coated with Matrigel or Geltrex is obtained.

[0112] (2) Resuscitation: Quickly thaw hPSC cells in a 37 °C water bath, transfer them to a 15 ml centrifuge tube containing 5 ml mTeSR culture medium, and centrifuge at 1100 rpm for 3 min to collect the cells.

[0113] (3) Resuspend the cells with 2 ml of mTeSR containing 5 μM ROCK inhibitor Y-27632, evenly inoculate the cells into the culture container coated with Matrigel or Geltrex, and place it in an incubator at 37 °C, 5% CO2 and 95% humidity for static culture.

[0114] (4) Replace the culture medium once a day, observe that the cells maintain an undifferentiated state, and continue culturing until the hPSC cell clones grow to a density of 80-90%, and then passage the cells.

[0115] (5) Cell passage: Wash the cells twice with PBS, add 0.5 mM EDTA, incubate at 37 °C for 3 min, discard the EDTA, gently pipette the cells with PBS until the clones divide into cell clusters containing 3-5 cells, collect the cell clusters into a 15 ml centrifuge tube, and centrifuge at 1100 rpm for 3 min to collect the cells.

[0116] (6) Discard the supernatant, resuspend the cells with 1 ml mTeSR, and evenly inoculate 100 μl of the cells per well into a culture container coated with Matrigel or Geltrex, and place it in an incubator at 37 °C, 5% CO2 and 95% humidity for static adherent culture.

[0117] (7) Repeat the above culture and amplification steps to obtain enough cells for the next differentiation induction.

[0118] 2. Induction of hPSC-derived mesoderm progenitor cells

[0119] (1) When the hPSC reaches 85% confluence, wash the cells twice with PBS, add the cell digestive solution Accutase and incubate at 37 °C for 3 min. Observe under the microscope to dissociate the cells into single cells or small cell clusters. Aspirate the cell digestive solution Accutase, and gently pipette the cells with PBS to disperse the cells evenly.

[0120] (2) Transfer the cells into a 15 ml centrifuge tube and centrifuge at 1100 rpm for 3 min to collect the cells.

[0121] (3) Resuspend the cells with mTeSR medium containing 5 μM Y-27632, and evenly inoculate the cells at a cell density of 1×10 5 / cm 2 into a culture container coated with Matrigel or Geltrex, and place it in an incubator at 37 °C, 5% CO2 and 95% humidity for static culture (as D0).

[0122] (4) On the next day (D1), replace it with the culture medium for inducing mesoderm into cardiac progenitor cells in Example 1 for culture, and 1% (V / V) penicillin-streptomycin mixture is also added to the culture medium.

[0123] Additionally, set up three groups, and replace CHIR99021 in the culture medium for inducing pluripotent stem cells into mesoderm progenitor cells in Example 1 with 3, 4, and 6 μM respectively.

[0124] (5) Incubate statically for 2 days without changing the culture medium. Culture the cells with the culture medium of Example 1 for 2 days. Detect the mesoderm progenitor cell markers TBXT and MIXL1 by real-time fluorescence quantitative PCR.

[0125] Among them, the primers for real-time fluorescence quantitative PCR are shown in Table 1:

[0126] Table 1:

[0127]

[0128] II. Experimental results

[0129] As Figure 2 shown, the largest number of mesoderm progenitor cell-like cells crawled out when cultured with the culture medium for inducing mesoderm into cardiac progenitor cells of Example 1; while when CHIR99021 was replaced with 3, 4, and 6 μM, the cell crawling out ratio was relatively low.

[0130] After culturing with the culture medium of Example 1 for 2 days (i.e., D3), the cell morphology was as shown in A of Figure 3 : the hPSC clones gradually became loose, and spindle-shaped cells spread out into the three-dimensional space at the edge. The results of detecting the mesoderm progenitor cell markers TBXT and MIXL1 by real-time fluorescence quantitative PCR were as shown in A of Figure 4 , and the obtained cells highly expressed TBXT and MIXL1.

[0131] The results showed that under the action of the culture medium of Example 1, mesoderm progenitor cells were successfully induced after 2 days of culture. This process was not only manifested morphologically as the loosening of cell clones and the spreading of spindle-shaped cells, but also proved at the molecular level. By highly expressing the mesoderm progenitor cell-specific markers TBXT and MIXL1, the mesoderm progenitor cell characteristics of the cells were further confirmed.

[0132] Induction of hPSC-derived cardiac progenitor cells in Example 5

[0133] I. Experimental method

[0134] On D3, take the mesoderm progenitor cells obtained in Example 4 and replace them with the culture medium for inducing mesoderm progenitor cells into cardiac progenitor cells of Example 2, and the culture medium also adds 1% (V / V) penicillin-streptomycin mixture.

[0135] Incubate statically for 4 days and change the culture medium every day. Culture the cells with the culture medium of Example 2 for 4 days.

[0136] II. Experimental results

[0137] Compared with D0 (A in 3), after culturing with the culture medium of Example 2 for 4 days (i.e., D7), the cell morphology changed significantly. As Figure 3As shown in B of [reference], the cells began to exhibit a cobblestone-like morphology of cardiac progenitor cells. The morphological characteristics of the induced cells were consistent with those of typical cardiac progenitor cells, indicating that under this culture condition, mesoderm progenitor cells successfully differentiated into cardiac progenitor cells.

[0138] Example 6 Induction of hPSC-derived epicardial progenitor cells

[0139] I. Experimental method

[0140] (1) Take the cardiac progenitor cells obtained in Example 5 (i.e., D7), and change the culture medium to the culture medium for inducing cardiac progenitor cells into epicardial progenitor cells in Example 3, and the culture medium is also supplemented with 1% (V / V) penicillin-streptomycin mixture.

[0141] (2) Incubate the cells statically for 2 days without changing the medium.

[0142] (3) Then (i.e., D9), wash the cells twice with PBS, add Accutase and incubate at 37 °C for 3 min. Observe under the microscope to dissociate the cells into single cells. Aspirate Accutase, and gently pipette the cells with PBS to make the cells evenly dispersed.

[0143] (4) Transfer the cells into a 15 ml centrifuge tube and centrifuge at 1100 rpm for 3 min to collect the cells.

[0144] (5) Resuspend the cell pellet with the culture medium for inducing cardiac progenitor cells into epicardial progenitor cells in Example 3 containing 5 μM Y-27632, and inoculate the cells evenly into the culture container coated with Matrigel or Geltrex at a cell density of 1×10 4 / cm 2 . Place it in an incubator at 37 °C, 5% CO2 and 95% humidity for static culture.

[0145] (6) The next day (i.e., D10), change to the culture medium for inducing cardiac progenitor cells into epicardial progenitor cells in Example 3 for culture, and the culture medium is also supplemented with 1% (V / V) penicillin-streptomycin mixture.

[0146] (7) Incubate the cells statically for 2 days without changing the medium.

[0147] For the cells cultured with the culture medium in Example 3, real-time fluorescence quantitative PCR was used to detect the markers related to endothelial progenitor cells (EPCs) (WT1, TBX18, TCF21, ALDH1A). The primers for real-time fluorescence quantitative PCR are shown in Table 2.

[0148] Table 2:

[0149]

[0150] II. Experimental Results

[0151] The cells (i.e., D12) cultured in the culture medium of Example 3 had a cell morphology as shown in Figure 3 C in [reference], and these cells presented a typical epicardial progenitor cell morphology. The cells were epithelial-like, arranged closely, and their morphology was similar to that of paving stones. Real-time fluorescence quantitative PCR showed Figure 4 B in [reference], and the expression levels of the EPCs markers WT1, TBX18, TCF21, and ALDH1A in the cells increased significantly. The expression of these markers further confirmed the epicardial progenitor cell characteristics of the cells.

[0152] Induction of hPSC-derived Cardiac Mesenchymal Stem Cells in Example 7

[0153] I. Experimental Methods

[0154] (1) Take the epicardial progenitor cells obtained in Example 6 as D12, change the culture medium to MesenCult™-ACF culture medium, and add 1% (V / V) penicillin-streptomycin mixture to the culture medium. Then, statically culture the cells in an incubator at 37 °C, 5% CO2, and 95% humidity, and change the medium every 2 - 3 days.

[0155] (2) After the cells grew to 90% confluence, add the cell digestive solution Accutase and incubate at 37 °C for 3 min. Observe under the microscope to make the cells become bright and detached, and gently pipette the cells with PBS to disperse the cells evenly.

[0156] (3) Transfer the cells into a 15 ml centrifuge tube, centrifuge at 1100 rpm for 3 min to collect the cells, discard the supernatant, and then inoculate the cells into a culture dish; at the same time, take some cells to detect the expression of cell surface markers such as CD34, CD45, CD29, CD90, CD73, and CD105.

[0157] (4) Repeat step 2 until the 27th day, and then mesoderm-derived cardiac mesenchymal stem cells (CMSC) can be obtained. Use flow cytometry to detect the proportion of cells expressing CD29, CD90, CD73, and CD105; and the proportion of cells expressing CD34, CD45, and CD19..

[0158] II. Experimental Results

[0159] The cells cultured on D27 had a cell morphology as shown in Figure 3 D in [reference], and the cells showed a typical fibroblast morphology, that is, uniform and spindle-shaped mesenchymal-like cells; use flow cytometry to detect the cells on the 27th day of differentiation (the results are shown in Figure 5) The CMSC shows low expression of hematopoietic cell line markers such as CD45, CD34, and CD19 (the expression levels are 0.46%, 0.65%, and 0.12% respectively), and high expression of fibroblast markers such as CD29, CD90, CD73, and CD105 (the expression levels are 99.99%, 99.99%, 98.26%, and 99.17% respectively), indicating that the obtained mesoderm-derived cardiac mesenchymal stem cells have low heterogeneity.

[0160] Example 8 Osteogenic, Adipogenic, and Chondrogenic Inductive Differentiation Ability of hPSC-Derived CMSC

[0161] I. Experimental Method

[0162] 1. Osteogenic inductive differentiation ability When the mesoderm-derived cardiac mesenchymal stem cells prepared in Example 7 grow to 90% density, the culture medium is replaced with osteogenic inductive differentiation culture medium (DMEM(L) supplemented with 10% (V / V) FBS, 10 mM β-glycerophosphate, 50 μg / ml L-ascorbic acid, and 100 nM dexamethasone). The culture medium is changed every 3 days, and after continuous induction culture for 21 days, alizarin red staining is performed to evaluate the osteogenic differentiation effect.

[0163] 2. Adipogenic inductive differentiation ability After the mesoderm-derived cardiac mesenchymal stem cells prepared in Example 7 grow to confluence, the culture medium is changed to adipogenic inductive differentiation medium (DMEM(H) supplemented with 10% (V / V) FBS, 100 nM indomethacin sodium pyruvate, 0.5 mM IBMX, and 1 mM dexamethasone). Similarly, the culture medium is changed every 3 days, and after continuous induction culture for 21 days, oil red O staining is performed to evaluate the adipogenic differentiation effect.

[0164] 3. Chondrogenic inductive differentiation ability First, the mesoderm-derived cardiac mesenchymal stem cells prepared in Example 7 are digested with the cell digestive solution Accutase, and the digested cells are collected and aliquoted into 15 ml centrifuge tubes at a density of 2.0 - 3.0×10 5 / 15 ml. Subsequently, centrifugation is performed at a speed of 300×g for 5 min, the supernatant is discarded, and 1 ml of chondrogenic differentiation inductive culture medium (H-DMEM supplemented with 10 ng / ml recombinant human transforming growth factor β3, 100 nM dexamethasone, 50 μg / ml L-ascorbic acid, 1 mM sodium pyruvate, 40 μg / ml proline, and ITS Supplement, premix) is added, and the cells are cultured in an incubator. The culture medium is changed every 3 days, and after continuous induction culture for 21 days, alcian blue staining is performed to evaluate the chondrogenic differentiation effect.

[0165] II. Results of the Experimental Method As Figure 6As shown, the mesoderm-derived cardiac mesenchymal stem cells (CMSCs) prepared in Example 7 were induced to differentiate into osteoblasts, adipocytes and chondrocytes, and corresponding staining assays were performed. Among them, the cells after osteogenic differentiation were stained with alizarin red S and showed ( Figure 6 A), the cells after adipogenic differentiation were stained with oil red O and showed ( Figure 6 B), and the cells after chondrogenic differentiation were stained with alcian blue and showed ( Figure 6 C).

[0166] The experimental results showed that after osteogenic differentiation of the CMSCs prepared in Example 7, the alizarin red S staining area showed dark red, indicating that the cells had been successfully induced into osteocytes and rich calcium salt deposits had been produced. After adipogenic differentiation, the oil red O staining area significantly showed orange-red to bright red, indicating that the cells had been successfully differentiated into adipocytes and lipid droplets had been formed; after chondrogenic differentiation, the alcian blue staining area showed purple-blue, indicating that the cells had been successfully induced into chondrocytes and rich cartilage matrix had been generated. The above results indicate that the mesoderm-derived cardiac mesenchymal stem cells (CMSCs) induced by the method of the present invention have the ability of trilineage differentiation.

[0167] Preparation of cardiac microtissues in Example 9

[0168] I. Experimental method

[0169] hPSCs from the same source were used to prepare hPSC-CM, hPSC-derived endothelial cells and mesoderm-derived cardiac mesenchymal stem cells respectively. Among them, the preparation method of mesoderm-derived cardiac mesenchymal stem cells was the same as that in Example 7, and the preparation methods of hPSC-CM and hPSC-derived endothelial cells were as follows:

[0170] 1. Preparation of hPSC-CM

[0171] (1) Preparation before induction: Coat a 12-well cell culture plate with Matrigel.

[0172] (2) hPSCs with a confluence of about 80% were digested with the cell digestive solution Accutase for 5 min, 5 times the volume of PBS was added, and the cells were gently pipetted until they were in a clonal state. Then they were seeded into a 12-well cell culture plate containing mTeSR plus medium with Y27632 at a cell density of 30%, which was recorded as the first day; Y27632 was removed on the second day.

[0173] (3) On the third day, the cells were cultured with a medium containing 5 μM CHIR99021, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive in DMEM / F12.

[0174] (4) From day 4 to day 8, culture with DMEM / F12 medium containing 3 μg / mL Heparin, 10 μM IWP2, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive.

[0175] (5) From day 9 to day 15, culture with DMEM / F12 medium containing 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive, and change the medium every other day.

[0176] (6) From day 15 to day 18, after purification in DMEM (Dulbecco's Modified Eagle Medium) medium containing 1 mM sodium lactate, hPSC-CMs with more than 60% of the cardiomyocytes expressing the CM-specific marker - cardiac troponin (cTnT) can be obtained and used for the preparation of cardiac microtissues.

[0177] 2. Preparation of hPSC-derived endothelial cells

[0178] Steps (1) to (3) are the same as steps (1) to (3) in "Preparation of hPSC-CM".

[0179] (4) From day 4 to day 8, culture with DMEM / F12 containing 3 μg / mL Heparin, 10 μM IWP2, 50 ng / mL VEGF, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive.

[0180] (5) From day 9 to day 21, culture with DMEM / F12 containing 20 ng / mL VEGF, 10 ng / mL FGF2, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive, and change the medium every three days.

[0181] (6) On day 22, flow-sort the CD31+CD45- cell population with CD31 (ECs marker) and CD45 (hematopoietic cell line marker) antibodies, and culture with DMEM / F12 containing 5 μM Y27632, 20 ng / mL VEGF, 10 ng / mL FGF2, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive for 1 day, then use it for the preparation of cardiac microtissues.

[0182] 3. Preparation of cardiac microtissues

[0183] (1) Take the mesoderm-derived cardiac mesenchymal stem cells (CMSCs) obtained in Example 7 and prepare cardiac microtissues at the cell composition ratio of natural hearts with the cell number ratio of hPSC-CM: CMSC: hPSC-derived endothelial cells = 7: 1.5: 1.5 as the CM+CMSC group.

[0184] According to the same method, prepare cardiac microtissues by using BMSCs to replace the mesoderm-derived cardiac mesenchymal stem cells obtained in Example 7 as the CM+BMSC group.

[0185] Prepare cardiac microtissues at the cell number ratio of hPSC-CM: hPSC-derived endothelial cells = 7: 3 as the CM group.

[0186] (2) On the first day, various cells were digested with the cell digestive solution Accutase (incubated at 37 °C for 3 min), an appropriate amount of PBS was added, and gently pipetted until the cells were completely separated into single cells. The cell suspension was collected in a 15 mL centrifuge tube, centrifuged at 1100 rpm for 3 min, and resuspended with DMEM / F12 medium containing 5 μM Y-27632, 50 ng / mL VEGF, 15 ng / mL bFGF, 20 ng / mL IGF, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive, and cell counting was performed.

[0187] (3) In each 96-well plate, the number of cells was 5000, resuspended in a V-shaped 96-well plate, centrifuged at 1100 rpm for 10 min, and 1% (V / V) penicillin-streptomycin mixture was added to the culture medium. It was statically cultured in an incubator at 37 °C, 5% CO2 and 95% humidity.

[0188] After static culture for 3 days, the medium was changed on the third day to remove Y27632 (replaced with DMEM / F12 medium containing 50 ng / mL VEGF, 15 ng / mL bFGF, 20 ng / mL IGF, 100 μg / mL human transferrin, 200 μM ascorbic acid, 7 μg / ml sodium selenite, and 1% (V / V) Lip culture additive) and transferred to a low-adhesion culture container, and cultured on an incubator shaker with the medium changed every three days.

[0189] (4) On the 27th day, the morphology and structure under electron microscopy of the obtained cardiac microtissues were evaluated.

[0190] II. Experimental Results

[0191] The identification results of hPSC-CM are as Figure 7, Flow cytometry and immunofluorescence staining results showed that before purification, 60% of the cells expressed the typical cardiac marker cTnT. After purification with lactate-free medium, the obtained CMs basically all expressed the typical cardiac markers and could be used for the preparation of cardiac microtissues.

[0192] The identification results of hPSC-derived endothelial cells (hPSC-EC) were as Figure 8 , On the 22nd day of differentiation, flow cytometry was used to detect the differentiation efficiency of ECs. Approximately 40% expressed CD31 and did not express CD45. The CD31+CD45- cell population was sorted by flow cytometry. More than 90% of the cells expressed CD31 and did not express CD45. The purified cells showed the typical polygonal morphology of ECs with serrated edges and could be used for the preparation of cardiac microtissues.

[0193] Figure 9 shown. The mesoderm-derived CMSC obtained in Example 7 was assembled with hPSC-CM and hPSC-derived endothelial cells into cardiac microtissues. Compared with the cardiac microtissues (CM+BMSC) formed by BMSC in the CM+BMSC group, the microtissues in the CM+CMSC group had regular morphology and smooth outer edges. The microtissues in the group without mesenchymal stem cells (CM group) were smaller in volume, irregular in shape, the spheres were darker and there were more dead cells attached to the outer edges ( Figure 9 A in). The cardiac microtissues in the CM+CMSC group had an increased volume, irregular shape, uneven volume size, and some tissues showed black in the middle, which might indicate poor cell state in the middle of the tissues ( Figure 9 B in). The cardiac microtissues prepared using the mesoderm-derived CMSC obtained in Example 7 showed better aggregation ability ( Figure 9 C in).

[0194] Electron microscopy analysis was performed 27 days after assembling CMSC into cardiac microtissues as shown in Figure 10 , The cardiomyocytes in the CMSC group showed more mature characteristics. The myofibers in the CM group were shorter, and the arrangement of myofibers and Z-line was relatively random, and the mitochondria presented immature round shapes ( Figure 10 A in). The myofiber length in the CM+BMSC group increased, the arrangement of myofibers and Z-line tended to be orderly, and the mitochondria were oval-shaped ( Figure 10 B in). The myofibers in the CM+CMSC group had a high degree of orderly arrangement, the Z-line was significantly increased and highly ordered, and the mitochondria presented mature long strip shapes ( Figure 10 C in), indicating that the cardiac microtissues prepared using the mesoderm-derived CMSC obtained in Example 7 were closer to the characteristics of adult heart cells, suggesting that the cardiac microtissues prepared by the present invention could more accurately predict drug toxicity and be better used for drug testing.

Claims

1. A culture medium A, characterized in that, It is a serum-free medium containing 3-6 μM CHIR99021, 50-100 μg / mL human transferrin, 100-200 μM ascorbic acid, 5-8 μg / ml sodium selenite, and 0.5-1% (V / V) Lip culture additive.

2. Use of the culture medium A described in claim 1 in the induction of pluripotent stem cells into mesoderm progenitor cells.

3. A method for inducing pluripotent stem cells into mesoderm progenitor cells, characterized in that, The pluripotent stem cells are inoculated in a culture container coated with Matrigel and cultured, and then replaced with the culture medium A described in claim 1 for culture.

4. A culture medium B, characterized in that, It is a serum-free medium containing 1-10 μM IWP2, 0.2-1 μM retinoic acid, 5-20 ng / ml BMP4, 2-10 ng / ml bFGF, 50-100 μg / mL human transferrin, 100-200 μM ascorbic acid, 5-8 μg / ml sodium selenite, and 0.5-1% (V / V) Lip culture additive.

5. Use of the culture medium B described in claim 4 in the induction of mesoderm progenitor cells into cardiac progenitor cells.

6. A method for inducing mesoderm progenitor cells into cardiac progenitor cells, characterized in that, Culture the mesoderm progenitor cells using the culture medium B described in claim 4.

7. The method according to claim 6, wherein The mesoderm progenitor cells are obtained by culturing using the method for inducing pluripotent stem cells into mesoderm progenitor cells.

8. A culture medium C, characterized in that, It is a serum-free medium containing 2-6 μM CHIR9902, 2-10 ng / ml bFGF, 5-20 ng / ml VEGF, 50-100 μg / mL human transferrin, 100-200 μM ascorbic acid, 5-8 μg / ml sodium selenite, and 0.5-1% (V / V) Lip culture additive.

9. Use of the culture medium C described in claim 8 in the induction of cardiac progenitor cells into epicardial progenitor cells.

10. A method for inducing cardiac progenitor cells into epicardial progenitor cells, characterized in that, Culture the cardiac progenitor cells using the culture medium C described in claim 8; then dissociate the cells into single cells and transfer them to the culture medium C containing Y-27632 for culture; then replace it with the culture medium C for continuous culture.

11. The method according to claim 10, characterized in that, The cardiac progenitor cells are obtained by using the method for inducing the mesoderm progenitor cells described in claim 6 into cardiac progenitor cells.

12. A culture medium composition, characterized in that, Containing the culture medium A described in claim 1, the culture medium B described in claim 4, and the culture medium C described in claim 8.

13. Use of the culture medium composition described in claim 12 in the induction of pluripotent stem cells into epicardial progenitor cells.

14. A method for inducing epicardial progenitor cells into cardiac mesenchymal stem cells, characterized in that, Culture the epicardial progenitor cells using a mesenchymal stem cell serum-free medium. The epicardial progenitor cells are obtained by the method for inducing the cardiac progenitor cells described in claim 10 until the cells expressing CD29, CD90, CD73, and CD105 reach more than 98%, and the cells expressing CD34 and CD45 are less than 2%.

15. Use of the cardiac mesenchymal stem cells prepared by the method described in claim 14 in the preparation of cardiac microtissues.

16. A culture medium composition for preparing cardiac microtissues, characterized in that, Comprising a first-stage culture medium and a second-stage culture medium, The first-stage culture medium is a serum-free medium containing 1-10 μM Y-27632, 5-50 ng / mL VEGF, 5-20 ng / mL bFGF, 10-30 ng / mL IGF, 50-100 μg / mL human transferrin, 100-200 μM ascorbic acid, 5-8 μg / ml sodium selenite, and 0.5-1% (V / V) Lip culture additive; The second-stage culture medium is a serum-free medium containing 5-50 ng / mL VEGF, 5-20 ng / mL bFGF, 10-30 ng / mL IGF, 50-100 μg / mL human transferrin, 100-200 μM ascorbic acid, 5-8 μg / ml sodium selenite, and 0.5-1% (V / V) Lip culture additive.

17. Use of the culture medium composition according to claim 16 in the preparation of cardiac microtissues.

18. A method for preparing a cardiac microtissue, characterized in that, Prepared from cardiac mesenchymal stem cells, cardiomyocytes derived from human pluripotent stem cells, and endothelial cells derived from human pluripotent stem cells prepared by the method according to claim 14.

19. The method according to claim 18, characterized in that, After mixing cardiac mesenchymal stem cells, cardiomyocytes derived from human pluripotent stem cells, and endothelial cells derived from human pluripotent stem cells prepared by the method according to claim 14, first culture with the first-stage culture medium in the culture medium composition for preparing cardiac microtissues according to claim 16 for 2.5-3.5 days, and then replace it with the second-stage culture medium in the culture medium composition for preparing cardiac microtissues according to claim 16, and continue to culture in a low-adhesion culture container for 23-25 days.

20. A cardiac microtissue, characterized in that, Prepared according to the method described in claim 18 or 19.

21. Use of the cardiac microtissue according to claim 20 as or in the preparation of a cardiac model.

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