Method for culturing cardiac progenitor cells with dual differentiation potential into myocardium and epicardium and applications thereof

Abstract

The present application relates to a culture method and application of cardiac progenitor cells with myocardial and epicardial dual differentiation potential. Specifically, the present application provides cardiac progenitor cells with myocardial and epicardial dual differentiation potential, and provides a culture medium and culture method for inducing human pluripotent stem cells to differentiate into the dual potential cardiac progenitor cells, and further provides a method for differentiating the dual potential cardiac progenitor cells into myocardial cells and / or epicardial cells. The present application provides a new cell model for the development process and principle research of early cardiac lineage, and has a wide application prospect in the field of cell therapy and regenerative medicine related to heart diseases.

Description

Technical Field

[0001] This invention relates to the field of cell culture, and more specifically to a method and application for culturing cardiac progenitor cells with dual differentiation potential of myocardium and epicardium. Background Technology

[0002] Cardiovascular disease is the leading cause of death worldwide, causing approximately 19 million deaths annually, accounting for about 32% of all deaths globally—nearly twice as many as cancer. The adult mammalian and human heart loses its regenerative capacity after birth; therefore, heart-related diseases such as end-stage heart failure, dilated cardiomyopathy, restrictive cardiomyopathy, hypertrophic cardiomyopathy, and complex congenital heart disease typically require heart transplantation. However, in practice, due to the extremely limited number of donors and the requirements for immunological matching, the transplant success rate is quite low.

[0003] Human pluripotent stem cells (hPSCs) possess the self-renewal and differentiation capabilities to differentiate into cardiomyocytes (CMs) or cardiac-related progenitor cells. These cells can then be transplanted to replace damaged myocardium, offering significant potential for treating common heart diseases caused by cardiomyocyte loss or damage, such as myocardial infarction, myocarditis, cardiomyopathy, and arrhythmias. Stem cells have a wide range of sources, originating from patients themselves or donors, making immunological matching easier to achieve.

[0004] Besides cardiomyocytes, another important functional cell type in the heart is the epicardial cell (Epi). During embryonic development, epicardial cells secrete factors that promote the proliferation and differentiation of cardiomyocytes. They also act as progenitor cells, producing other functional cardiac cells, including cardiac fibroblasts, cardiac smooth muscle cells, and pericytes. In adults, when the heart is damaged, the epicardium plays a vital role in maintaining myocardial survival in mammals where the heart cannot regenerate. However, in some vertebrates with regenerative hearts, such as zebrafish, epicardial cells are essential for cardiomyocyte regeneration. Therefore, epicardial cells have been extensively studied as a promising candidate for cardiac transplantation.

[0005] Constructing cardiac progenitor cells derived from pluripotent stem cells in vitro, capable of simultaneously generating cardiomyocytes and epicardial cells, would be an ideal source of cells for heart transplantation. However, current induction differentiation protocols produce cardiac progenitor cells that primarily differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in subsequent differentiation. Cardiac progenitor cells originate from mesodermal precursor cells that differentiate via primitive streaks during early gastrulation. Due to limitations in research samples, our understanding of early human heart development is very limited; it remains unclear when and from which progenitor cells cardiomyocytes and epicardial cells separate.

[0006] Therefore, there is an urgent need in this field to develop differentiation protocols for the efficient in vitro generation of cardiac progenitor cells with bidirectional differentiation potential in the myocardium and epicardium. Summary of the Invention

[0007] The purpose of this invention is to provide a culture medium and a culture method for producing cardiac progenitor cells with bidirectional differentiation potential of myocardium and epicardium in vitro, as well as a culture medium and a method for further producing myocardial cells and epicardial cells.

[0008] In a first aspect of the invention, an in vitro cell population is provided, characterized in that at least 80% of the cells in the cell population are bipotent cardiac progenitor cells, which have the potential to differentiate into cardiomyocytes and epicardial cells.

[0009] In another preferred embodiment, at least 85%, at least 90%, or at least 95% of the cells in the cell population are said to be bipotent cardiac progenitor cells.

[0010] In another preferred embodiment, the dual-potential cardiac progenitor cells are GATA6-positive, MEIS1-positive, and / or PDGFRA-positive cells.

[0011] In another preferred embodiment, the proportion of GATA6 positive cells in the cell population is ≥90%, preferably ≥95%, and more preferably ≥98%.

[0012] In another preferred embodiment, the proportion of MEIS1 positive cells in the cell population is ≥90%, preferably ≥95%, and more preferably ≥98%.

[0013] In another preferred embodiment, the proportion of PDGFRA-positive cells in the cell population is ≥85%, preferably ≥90%, and more preferably ≥92%.

[0014] In another preferred embodiment, the dual-potential cardiac progenitor cells are HDAC2-positive, COL1A2-positive, SPON1-positive, and / or PSAT1-positive cells.

[0015] In another preferred embodiment, the dual-potential cardiac progenitor cells are ISL1-negative, NKX2-5-negative, HAND2-negative, and / or MESP1-negative cells.

[0016] In another preferred embodiment, the bipotent cardiac progenitor cells express low, essentially no, or no ISL1, NKX2-5, HAND2, and / or MESP1 genes.

[0017] In another preferred embodiment, the cell population also contains other cells besides the bipotent cardiac progenitor cells; preferably, the other cells include: endoderm-derived cells and pluripotent stem cells.

[0018] In another preferred embodiment, the proportion of the other cells in the cell population is ≤20%, preferably ≤15%, and more preferably ≤10%.

[0019] In another preferred embodiment, the cell population is generated from in vitro induced culture of human pluripotent stem cells (hPSCs).

[0020] In another preferred embodiment, the human pluripotent stem cells include: human embryonic stem cells and human induced pluripotent stem cells.

[0021] In another preferred embodiment, the induction culture is carried out in the presence of CHIR99021 and BI-1347.

[0022] In another preferred embodiment, the cell population is obtained by the following method: (1) Human pluripotent stem cells are cultured in a first culture medium, which includes: basal culture medium, CHIR99021 and BI-1347, and the culture time is 2 ± 0.5 days, preferably 2 days; (2) The cells obtained in step (1) are cultured in a second culture medium, which includes a basal culture medium and a culture time of 1 ± 0.5 days, preferably 1 day.

[0023] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% B27.

[0024] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% insulin-removed B27.

[0025] In another preferred embodiment, the first culture medium comprises: basal culture medium, 3-10 μM CHIR99021 and 0.5-3.0 μM BI-1347.

[0026] In another preferred embodiment, the first culture medium comprises: basal culture medium, 4-6 μM CHIR99021 and 0.5-2.0 μM BI-1347.

[0027] In another preferred embodiment, the first culture medium comprises: basal culture medium, 5 μM CHIR99021 and 0.5 μM BI-1347.

[0028] In another preferred embodiment, the second culture medium is the basal culture medium.

[0029] In another preferred embodiment, the second culture medium comprises: a basal culture medium, a Wnt signaling pathway inhibitor, and optionally retinoic acid; preferably, the Wnt signaling pathway inhibitor is IWP2.

[0030] In another preferred embodiment, the second culture medium comprises: a basal culture medium, 3-10 μM IWP2, and optionally 0.2-1.0 μM retinoic acid.

[0031] In another preferred embodiment, the second culture medium comprises: basal medium, 5 μM IWP2, and optionally 0.5 μM retinoic acid.

[0032] In a second aspect of the invention, a culture medium combination for inducing pluripotent stem cell differentiation to generate bipotent cardiac progenitor cells is provided, the culture medium combination comprising: an initial induction medium and a second-stage induction medium; The initial induction medium includes: basal medium and initial induction factors; the initial induction factors include: CHIR99021 and BI-1347; The second-stage induction medium includes: basal medium and optional second-stage induction factor.

[0033] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% B27.

[0034] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% insulin-removed B27.

[0035] In another preferred embodiment, the initial inducing factors include 3-10 μM CHIR99021 and 0.5-3.0 μM BI-1347.

[0036] In another preferred embodiment, the initial inducing factors include: 4-6 μM CHIR99021 and 0.5-2.0 μM BI-1347.

[0037] In another preferred embodiment, the initial inducing factors are 5 μM CHIR99021 and 0.5 μM BI-1347.

[0038] In another preferred embodiment, the second-stage inducing factor includes: a Wnt signaling pathway inhibitor and optionally retinoic acid; preferably, the Wnt signaling pathway inhibitor is IWP2.

[0039] In another preferred embodiment, the second-stage inducing factor includes 3-10 μM IWP2 and optionally 0.2-1.0 μM retinoic acid.

[0040] In another preferred embodiment, the second-stage inducing factor includes: 5 μM IWP2, and optionally 0.5 μM retinoic acid.

[0041] In a third aspect of the invention, a reagent combination for inducing cardiac progenitor cells to differentiate into cardiomyocytes is provided, the reagent combination comprising: (i) basal culture medium, and (ii) a Wnt signaling pathway inhibitor.

[0042] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% B27.

[0043] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% insulin-removed B27.

[0044] In another preferred embodiment, the Wnt signaling pathway inhibitor is IWP2.

[0045] In a fourth aspect of the invention, a reagent combination for inducing cardiac progenitor cell differentiation into epicardial cells is provided, the reagent combination comprising: (i) basal culture medium, (ii) a Wnt signaling pathway inhibitor; and (iii) retinoic acid.

[0046] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% B27.

[0047] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% insulin-removed B27.

[0048] In another preferred embodiment, the Wnt signaling pathway inhibitor is IWP2.

[0049] In a fifth aspect of the invention, a method for generating bipotent cardiac progenitor cells in vitro is provided, characterized in that the bipotent cardiac progenitor cells have the potential to differentiate into cardiomyocytes and epicardial cells, the method comprising the steps of: S1. Provide human pluripotent stem cells; S2. Providing an initial induction medium comprising: a basal medium and initial induction factors comprising: CHIR99021 and BI-1347; culturing the human pluripotent stem cells in the initial induction medium; S3. Provide a second-stage induction medium, which includes a basal medium and an optional second-stage induction factor; culture the cells in S2 in the second-stage induction medium to obtain the cell population.

[0050] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% B27.

[0051] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% insulin-removed B27.

[0052] In another preferred embodiment, in step S2, the initial induction time is 2-3 days, preferably 2 days.

[0053] In another preferred embodiment, the initial inducing factors include 3-10 μM CHIR99021 and 0.5-3.0 μM BI-1347.

[0054] In another preferred embodiment, the initial inducing factors include: 4-6 μM CHIR99021 and 0.5-2.0 μM BI-1347.

[0055] In another preferred embodiment, the initial inducing factors are 5 μM CHIR99021 and 0.5 μM BI-1347.

[0056] In another preferred embodiment, in step S3, the induction time of the second stage is 12-36 hours, preferably 24 ± 2 hours.

[0057] In another preferred embodiment, the second-stage inducing factor includes: a Wnt signaling pathway inhibitor, and optionally retinoic acid.

[0058] In another preferred embodiment, the Wnt signaling pathway inhibitor is IWP2.

[0059] In another preferred embodiment, the second-stage inducing factor includes 3-10 μM IWP2 and optionally 0.2-1.0 μM retinoic acid.

[0060] In another preferred embodiment, the second-stage inducing factor includes: 5 μM IWP2, and optionally 0.5 μM retinoic acid.

[0061] In another preferred embodiment, the method further includes the steps of: S4. The cell population obtained in step S3 is further cultured to obtain cardiomyocytes and / or epicardial cells.

[0062] In another preferred embodiment, in step S4, the culture is carried out in the basal culture medium for a period of 3 days or more, preferably 3 to 7 days.

[0063] In a sixth aspect of the invention, a method for generating cardiomyocytes through in vitro culture is provided, the method comprising the steps of: Y1. Human pluripotent stem cells are cultured in an initial induction medium, the initial induction medium comprising: basal medium and initial induction factor; Y2. The cells obtained in step Y1 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors for 48±12h, preferably 48±2h. Y3. Culture the cells obtained in step Y2 in basal culture medium for at least 2 days, preferably 2 to 6 days, more preferably 2 days; harvest the cell population; or, Y1. Human pluripotent stem cells are cultured in an initial induction medium, the initial induction medium comprising: basal medium and initial induction factor; Y2. Culture the cells obtained in step Y1 in basal culture medium for 24±6h, preferably 24±2h; Y3. The cells obtained in step Y2 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors for 48±12h, preferably 48±2h. Y4. Culture the cells obtained in step Y3 in a basal culture medium for more than 1 day, preferably 1 to 5 days, more preferably 1 day; harvest the cell population.

[0064] In another preferred embodiment, the Wnt signaling pathway inhibitor is IWP2.

[0065] In another preferred embodiment, the concentration of IWP2 is 3-10 μM, preferably 4-6 μM, and more preferably 5 μM.

[0066] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% B27.

[0067] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% insulin-removed B27.

[0068] In another preferred embodiment, the initial inducing factors include CHIR99021 and BI-1347.

[0069] In another preferred embodiment, the initial inducing factor comprises 3-10 μM CHIR99021 and 0.5-3.0 μM BI-1347; preferably, the initial inducing factor comprises 4-6 μM CHIR99021 and 0.5-2.0 μM BI-1347; more preferably, the initial inducing factor is 5 μM CHIR99021 and 0.5 μM BI-1347.

[0070] In another preferred embodiment, the proportion of cardiomyocytes in the harvested cell population is ≥80%, preferably ≥86%.

[0071] In a seventh aspect of the invention, a method for generating epicardial cells in vitro is provided, characterized in that the method comprises the steps of: Z1. Human pluripotent stem cells are cultured in an initial induction medium comprising: a basal medium and an initial induction factor; Z2. The cells obtained in step Z1 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors and retinoic acid for 48±12 h, preferably 48±2 h. Z3. Culture the cells obtained in step Z2 in basal medium for at least 4 days, preferably 4 to 6 days, more preferably 4 days; harvest the cell population. or, Z1. Human pluripotent stem cells are cultured in an initial induction medium comprising: a basal medium and an initial induction factor; Z2. The cells obtained in step Z1 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors for 24±6 h, preferably 24±2 h. Z3. The cells obtained in step Z3 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors and retinoic acid for 24±6 h, preferably 24±2 h. Z4. The cells obtained in step Z3 are cultured in basal medium in the presence of retinoic acid for 24±6 h, preferably 24±2 h. Z5. Culture the cells obtained in step Z4 in a basal culture medium for at least 3 days, preferably 3 to 5 days, more preferably 3 days; harvest the cell population.

[0072] In another preferred embodiment, the Wnt signaling pathway inhibitor is IWP2.

[0073] In another preferred embodiment, the concentration of IWP2 is 3-10 μM, preferably 4-6 μM, and more preferably 5 μM.

[0074] In another preferred embodiment, the concentration of retinoic acid is 0.2-1.0 μM, preferably 0.4-0.6 μM, and more preferably 0.5 μM.

[0075] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% B27.

[0076] In another preferred embodiment, the basal culture medium is a combination of advanced RPMI 1640 medium and 2% insulin-removed B27.

[0077] In another preferred embodiment, the initial inducing factors include CHIR99021 and BI-1347.

[0078] In another preferred embodiment, the initial inducing factor comprises 3-10 μM CHIR99021 and 0.5-3.0 μM BI-1347; preferably, the initial inducing factor comprises 4-6 μM CHIR99021 and 0.5-2.0 μM BI-1347; more preferably, the initial inducing factor is 5 μM CHIR99021 and 0.5 μM BI-1347.

[0079] In another preferred embodiment, the proportion of epicardial cells in the harvested cell population is ≥55%, preferably ≥60%.

[0080] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0081] Figure 1 These are heatmaps and feature maps showing the expression of retinoic acid signaling-related genes in different cell lineages (based on a human gastrulation single-cell sequencing dataset). a) Heatmap showing the expression levels of genes related to the retinoic acid signaling pathway during the human gastrulation stage (based on the Richard CV Tyser et al. dataset). b) Top right image: UMAP visualization of heart development-related cell types in human gastrulation stage embryos. Bottom image: Featured distribution map showing the expression of specified genes in human gastrulation stage embryos.

[0082] Figure 2 This is a schematic diagram of a functional experiment to verify related signals in the differentiation of bipotent cardiac progenitor cells.

[0083] Figure 3 The images show staining diagrams (a) and statistical diagrams (b) of bipotent cardiac progenitor cells produced by pluripotent stem cells under different differentiation conditions.

[0084] Figure 4This is an analysis diagram of single-cell sequencing of bipotent cardiac progenitor cells generated under different differentiation conditions. a is a UMAP dimensionality reduction plot showing the cell clusters and cell type definitions obtained from single-cell transcription (scRNA-seq) under three induced differentiation conditions; b is a violin plot of gene expression levels of cardiac progenitor-related clusters; c is the positivity rate of representative cardiac progenitor genes under different conditions, corresponding to b; d is a proportion of cell types generated under the three induced differentiation conditions; e is a detailed proportion of cell types generated under the three induced differentiation conditions, corresponding to d; f is a violin plot of expression levels of other characteristic genes related to cardiac progenitor clusters.

[0085] Figure 5 This is a schematic diagram of a functional experiment to verify related signals in the differentiation of myocardial / epicardial cells into bipotent cardiac progenitor cells.

[0086] Figure 6 These are staining images of myocardial (CM) and epicardial (Epi) cells produced by bispotent cardiac progenitor cells under different differentiation conditions.

[0087] Figure 7 These are statistical results of cardiomyocytes (TNNT2+) and epicardial cells (WT1+) produced by dypotent cardiac progenitor cells under different differentiation conditions. Method (2) represents the differentiation conditions for producing cardiomyocytes, and method (3) represents the differentiation conditions for producing epicardial cells.

[0088] Figure 8 Yes Figure 5 The analysis diagram of single-cell sequencing of cells generated by method (2) on day 10. a is the Uniform Manifold Approximation and Projection Map (UMAP) of all cells, showing the generated clusters and cell types, including atrial cardiomyocytes (aCM), ventricular cardiomyocytes (vCM), and atrioventricular cardiomyocytes (avc-CM); b is a dot plot of cell-specific marker genes used to define each cell cluster; c is a scale diagram of each cell type.

[0089] Figure 9 Yes Figure 5 The analysis diagram of single-cell sequencing of cells generated by method (3) on day 10. a is the uniform manifold approximation and projection (UMAP) of all cells, showing the generated clusters and cell types, including epicardial cells (Epi), epicardial progenitor cells (EPDCs), and epicardial fibroblasts (Epi-Fibro); b is a dot plot of cell-specific marker genes used to define each cell cluster; c is a scale diagram of each cell type.

[0090] Figure 10 This is a schematic diagram of a series of functional experiments designed to verify related signals under different differentiation conditions for the differentiation of pluripotent stem cells into bipotent cardiac progenitor cells.

[0091] Figure 11 Yes Figure 10 Staining images of myocardial and epicardial cells produced under different differentiation conditions.

[0092] Figure 12 Yes Figure 10 Statistical results of myocardial (TNNT2+) and epicardial cells (WT1+) produced under different differentiation conditions.

[0093] Figure 13 This is a schematic diagram of a series of functional experiments designed to verify related signals under different differentiation conditions for the differentiation of bipotent cardiac progenitor cells into myocardial / epicardial cells.

[0094] Figure 14 Yes Figure 13 Staining images of myocardial and epicardial cells produced by bispotent cardiac progenitors under different differentiation conditions.

[0095] Figure 15 Yes Figure 13 Statistical results of cardiac progenitor cells (TNNT2+) and epicardial cells (WT1+) produced under different differentiation conditions.

[0096] Figure 16 This is a schematic diagram of a functional experiment to verify related signals for the differentiation of cardiac progenitor cells into myocardial / epicardial cells. BMS493 is an inhibitor of retinoic acid.

[0097] Figure 17 Yes Figure 16 Staining images of myocardial and epicardial cells produced by cardiac progenitor cells under different differentiation conditions.

[0098] Figure 18 Yes Figure 16 Statistical results of cardiac progenitor cells (TNNT2+) and epicardial cells (WT1+) produced under different differentiation conditions.

[0099] Figure 19 This is a schematic diagram of a functional experiment to verify related signals in cardiomyocyte differentiation targeting cardiac progenitor cells.

[0100] Figure 20 Yes Figure 19 Staining images of myocardial and epicardial cells produced by cardiac progenitor cells under different differentiation conditions.

[0101] Figure 21 Yes Figure 19 Statistical results of cardiac progenitor cells (TNNT2+) and epicardial cells (WT1+) produced under different differentiation conditions.

[0102] Figure 22This is a schematic diagram of a functional experiment to verify related signals in the differentiation of epicardial cells from bipotent cardiac progenitor cells.

[0103] Figure 23 Yes Figure 22 Staining images of myocardial and epicardial cells produced by bispotent cardiac progenitors under different differentiation conditions.

[0104] Figure 24 Yes Figure 22 Statistical results of myocardial and epicardial cells produced by bispotent cardiac progenitor cells under different differentiation conditions. Detailed Implementation

[0105] Through extensive and in-depth research, the inventors have provided a cardiac progenitor cell with bipotent differentiation potential into both cardiomyocytes and epicardium. They have also provided a method for inducing human pluripotent stem cells to differentiate into the aforementioned bipotent cardiac progenitor cells, and a method for further differentiating these bipotent cardiac progenitor cells into cardiomyocytes and epicardial cells, as well as the induction culture medium used during the induction process. This invention can rapidly (e.g., within 3 days) and efficiently induce human pluripotent stem cells to differentiate into bipotent cardiac progenitor cells. Under cardiomyocyte differentiation conditions, the bipotent cardiac progenitor cells can generate over 80% cardiomyocytes within 3 days; under epicardial cell differentiation conditions, they can generate nearly 60% epicardial cells within 5 days, with epicardium and epicardial-derived cells accounting for over 80%. This invention was completed based on these findings.

[0106] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0107] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0108] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0109] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may occur but are not required to occur.

[0110] Bipotent cardiac progenitor cells Cardiac progenitor cells are a class of pluripotent stem cells or precursor cells capable of differentiating into cardiac lineage cells, including but not limited to cardiomyocytes, epicardial cells, endocardial cells, and smooth muscle cells. Cardiac progenitor cells can originate from embryonic stem cells, induced pluripotent stem cells, fetal heart tissue, or adult heart tissue. Unlike pluripotent stem cells, they do not possess the long-term capacity for self-renewal and expansion, but they can differentiate into functional cardiac cells in vitro or in vivo.

[0111] Cardiac cardiomyocytes (CM) are highly differentiated terminal cells in the heart tissue responsible for contractile function. They are usually the main effector cells that play a role in repairing cardiac contractile function after transplantation.

[0112] Epicardial cells (Epi) are cell types derived from mesothelial cells that cover the outer surface of the heart. Epicardial cells not only constitute the epicardial epithelial layer, but can also differentiate into cardiac fibroblasts, smooth muscle cells, and pericytes after heart development or injury. They can be used for combined transplantation with cardiomyocytes or cardiac progenitor cells to promote angiogenesis, inhibit fibrosis, and improve the integration and function of transplanted cells.

[0113] As used herein, the term "bipotent cardiac progenitor cell" refers to a cardiac progenitor cell possessing the dual differentiation potential of cardiomyocytes and epicardial cells. In conventional in vitro induced pluripotent stem cell-derived cardiac progenitor cell protocols, the resulting cardiac progenitor cells typically exhibit a tendency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells. There are no reports in this art of cardiac progenitor cells possessing the dual differentiation potential of cardiomyocytes and epicardial cells.

[0114] In this invention, through exploration of culture medium components and culture methods, cardiac progenitor cells with bidirectional differentiation potential for cardiomyocytes and epicardial cells were obtained, namely, the bipotent cardiac progenitor cells of this invention. The bipotent cardiac progenitor cells of this invention are GATA6-positive, MEIS1-positive, and / or PDGFRA-positive, and specifically express… HDAC2 , COL1A2 , SPON1 and PSAT1 They rarely or never express representative genes of cardiac progenitor cells that have been reported to have the potential to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells. ISL1 , NKX2-5 , HAND2 and MESP1 .

[0115] The bipotent cardiac progenitor cells of the present invention can generate cardiomyocytes and / or epicardial cells through spontaneous differentiation and induced differentiation.

[0116] In some embodiments, the dual-potential cardiac progenitor cell population of the present invention can spontaneously differentiate to simultaneously generate cardiomyocytes and epicardial cells. For example, the differentiated cardiomyocytes account for approximately 5-85%, and / or the differentiated epicardial cells account for approximately 5-65%. "Spontaneous differentiation" refers to differentiation in a basal culture medium without the addition of Wnt inhibitors and inducing factors such as retinoic acid.

[0117] In some embodiments, the dual-potential cardiac progenitor cell population of the present invention can generate cardiomyocytes through induced differentiation, for example, the proportion of differentiated cardiomyocytes is ≥20%, such as ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, and epicardial cells are generated without differentiation or with almost no differentiation.

[0118] The method for inducing the differentiation of the bipotent cardiac progenitor cell population of the present invention into cardiomyocytes includes: culturing the bipotent cardiac progenitor cells in the presence of cardiomyocyte inducing factors. The cardiomyocyte inducing factors include: a Wnt signaling pathway inhibitor; in a preferred embodiment, the Wnt signaling pathway inhibitor is IWP2 (CAS No.: 686770-61-6).

[0119] In some embodiments, the bipotent cardiac progenitor cell population of the present invention can generate epicardial cells through induced differentiation, for example, the proportion of differentiated epicardial cells is ≥20%, such as ≥30%, ≥40%, ≥50%, ≥60%, and cardiomyocytes are generated without differentiation or with almost no differentiation.

[0120] The method for inducing the differentiation of the bipotent cardiac progenitor cell population of the present invention into epicardial cells includes culturing the bipotent cardiac progenitor cells in the presence of epicardial cell inducing factors. The epicardial cell inducing factors include a Wnt signaling pathway inhibitor and retinoic acid (RA); in a preferred embodiment, the Wnt signaling pathway inhibitor is IWP2 (CAS No.: 686770-61-6).

[0121] Culture medium and culture method for generating bipotent cardiac progenitor cells In this invention, a culture medium and a culture method are provided for inducing pluripotent stem cell differentiation to generate the bipotent cardiac progenitor cells described herein.

[0122] The bipotent cardiac progenitor cells of this invention are generated from in vitro induced pluripotent stem cells (hPSCs). In this invention, the source of the human pluripotent stem cells is not limited, and may include, for example, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In some embodiments, the human pluripotent stem cells are provided in the form of cell spheroids or embryoid bodies. In this invention, the method for forming cell spheroids of human pluripotent stem cells is known in the art, for example, by culturing in an embryonic stem cell culture medium containing 10 μM Y27632 for 24 hours, preferably the embryonic stem cell culture medium being TeSR™-E8™ medium.

[0123] This invention provides a culture medium kit for inducing pluripotent stem cell differentiation to generate bipotent cardiac progenitor cells, referred to as cardiac progenitor cell differentiation medium, which includes an initial induction medium and a second-stage induction medium.

[0124] The initial induction medium and the second-stage induction medium of the present invention are both derived from the basal medium. In some embodiments, the basal medium of the present invention is a combination of advanced RPMI 1640 medium and 2% B27. In some embodiments, the basal medium of the present invention is a combination of advanced RPMI 1640 medium and 2% insulin-free B27. Preferably, it is a combination of advanced RPMI 1640 medium and 2% B27, also known as aRPMI-B27 medium. The basal medium in the initial induction medium and the second-stage induction medium of the present invention can be the same or different, preferably the same.

[0125] The initial induction medium of the present invention is used for the first stage of induction culture, and the culture time of the first stage is about 2 to 3 days, preferably about 2 days.

[0126] The initial induction medium consists of: basal medium and initial induction factors, including CHIR99021 and BI-1347. CHIR99021 is a GSK-3 inhibitor and Wnt signaling pathway activator, with CAS number 252917-06-9. BI-1347 is a CDK8 inhibitor, with CAS number 2163056-91-3.

[0127] In the initial induction medium of the present invention, the concentrations of the initial inducing factors are as follows: 3-10 μM CHIR99021 and 0.5-3.0 μM BI-1347. In some embodiments, the concentrations of the initial inducing factors are as follows: 4-6 μM CHIR99021 and 0.5-2.0 μM BI-1347. In some embodiments, the initial inducing factors are 5 μM CHIR99021 and 0.5 μM BI-1347.

[0128] In a preferred embodiment, the initial induction medium of the present invention comprises the following components: aRPMI-B27 medium and added components 5 μM CHIR99021 and 0.5-2 µM BI-1347, wherein the preferred concentration of BI-1347 is 0.5 µM.

[0129] The second-stage induction medium of the present invention is used for the second-stage induction culture, and the culture time of the second stage is about 12-36 hours, preferably about 24 hours.

[0130] The components of the second-stage induction medium are selected from one of the following three groups: (1) basal medium; (2) basal medium and added Wnt signaling pathway inhibitor; (3) basal medium and added Wnt signaling pathway inhibitor and retinoic acid. The basal medium is preferably aRPMI-B27 medium. When adding a Wnt signaling pathway inhibitor, the Wnt signaling pathway inhibitor is preferably IWP2, with a concentration of 3-10 μM, preferably 4-6 μM, and more preferably 5 μM. When adding retinoic acid, the concentration of retinoic acid is 0.2-1.0 μM, preferably 0.4-0.6 μM, and more preferably 0.5 μM.

[0131] In a preferred embodiment, the second-stage induction culture medium of the present invention is any one of the following three: (1) aRPMI-B27 culture medium; (2) aRPMI-B27 culture medium with added ingredient 5 μM IWP2; (3) aRPMI-B27 culture medium with added ingredients 5 μM IWP2 and 0.5 μM retinoic acid.

[0132] In the specific implementation process, human pluripotent stem cell spheres are provided, and a two-stage induction culture is adopted: in the first stage, the initial induction medium in the cardiac progenitor cell differentiation medium is used for 48 hours of induction culture; in the second stage, any one of the three media in the second stage induction medium of the cardiac progenitor cell differentiation medium is selected for further induction culture for 24 hours.

[0133] Preferably, in the second stage, the aRPMI-B27 medium, which is the second-stage induction medium in cardiac progenitor cell differentiation culture medium, is used to continue induction culture for 24 hours.

[0134] In the above implementation scheme, after two-stage induction, more than 90% of bipotent cardiac progenitor cells can be generated on the third day after the start of induction, with a small number of endoderm-derived cells and undifferentiated pluripotent stem cells remaining.

[0135] In some embodiments, the resulting pluripotent stem cell spheres are induced for 48 hours with the initial induction medium, and then cultured further in aRPMI-B27 medium, the second-stage induction medium for cardiac progenitor cell differentiation. On the eighth day after induction begins, cardiomyocytes and epicardial cells can be generated simultaneously, with the proportion of cardiomyocytes and epicardial cells being randomized. In this invention, "day n after induction begins" is counted from the start of the first stage.

[0136] Culture medium and culture method for producing cardiomyocytes Based on obtaining the bipotent cardiac progenitor cells of the present invention, the present invention further provides culture reagents and culture methods for generating cardiomyocytes.

[0137] The reagent combination used to induce cardiac progenitor cell differentiation into cardiomyocytes includes: (i) basal culture medium and (ii) a Wnt signaling pathway inhibitor, such as IWP2. In some embodiments, culturing the bipotent cardiac progenitor cells of the present invention in basal culture medium in the presence of a Wnt signaling pathway inhibitor yields cardiomyocytes comprising ≥20%, for example ≥50%, ≥60%, ≥70%, ≥80%, or ≥90%.

[0138] In a specific implementation, the components of the cardiomyocyte induction culture medium, referred to simply as the cardiomyocyte differentiation culture medium, are selected from the following group: (1) Combination 1: aRPMI-B27 medium and added component 5 μM IWP2; (2) Combination 2: First use aRPMI-B27 medium, then use aRPMI-B27 medium and add 5 μM WP2.

[0139] The method for inducing cardiac progenitor cells to differentiate into cardiomyocytes is selected from one of the following two schemes: Option 1: Y1. Human pluripotent stem cells are cultured in an initial induction medium, the initial induction medium comprising: basal medium and initial induction factor; Y2. The cells obtained in step Y1 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors for 48±12h, preferably 48±2h. Y3. Culture the cells obtained in step Y2 in a basal culture medium for more than 2 days, preferably 2 to 6 days, more preferably 2 days; harvest the cell population.

[0140] In the specific implementation plan, the pluripotent stem cell spheres formed are induced with the initial induction medium for 48 hours, and then induced and cultured for another 48 hours with aRPMI-B27 medium and 5 μM IWP2. After that, they are cultured with aRPMI-B27 medium. On the 6th day after the start of induction, more than 80% of cardiomyocytes can be produced, and few or no epicardial cells are produced.

[0141] Option 2: Y1. Human pluripotent stem cells are cultured in an initial induction medium, the initial induction medium comprising: basal medium and initial induction factor; Y2. Culture the cells obtained in step Y1 in basal culture medium for 24±6h, preferably 24±2h; Y3. The cells obtained in step Y2 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors for 48±12h, preferably 48±2h. Y4. Culture the cells obtained in step Y3 in a basal culture medium for more than 1 day, preferably 1 to 5 days, more preferably 1 day; harvest the cell population.

[0142] In the specific implementation plan, the formed pluripotent stem cell spheres are induced with the initial induction medium for 48 hours, and then cultured with combination two of the myocardial differentiation medium, namely aRPMI-B27 medium, for 24 hours; then induced and cultured with aRPMI-B27 medium and the added component 5 μM IWP2 for another 48 hours; then cultured with aRPMI-B27 medium for another 6 days after the start of induction. More than 80% of cardiomyocytes can be generated, and few or no epicardial cells are generated.

[0143] In the above implementation scheme, more than 80% of the cells are cardiomyocytes, a small number of cardiac fibroblasts are produced, few or no epicardial cells are produced, and a small number of endoderm-derived cells and endothelial cells are also produced.

[0144] Culture medium and culture method for producing epicardial cells Based on obtaining the bipotent cardiac progenitor cells of the present invention, the present invention further provides culture reagents and culture methods for generating epicardial cells.

[0145] The reagent combination used to induce cardiac progenitor cell differentiation into epicardial cells includes: (i) basal culture medium, (ii) a Wnt signaling pathway inhibitor, such as IWP2; and (iii) retinoic acid. In some embodiments, culturing the bipotent cardiac progenitor cells of the present invention in basal culture medium in the presence of a Wnt signaling pathway inhibitor and retinoic acid yields epicardial cells in a proportion of ≥20%, for example ≥30%, ≥40%, ≥50%, or ≥60%.

[0146] In a specific implementation, the components of the epicardial cell induction culture medium, referred to simply as the epicardial differentiation culture medium, are selected from the following group: (1) Combination 1: aRPMI-B27 medium and added components 5 μM IWP2 and 0.5 μM retinoic acid; (2) Combination 2: First use aRPMI-B27 medium with 5 μM IWP2, then use aRPMI-B27 medium with 5 μM IWP2 and 0.5 μM retinoic acid, then use aRPMI-B27 medium with 0.5 μM retinoic acid.

[0147] The method used to induce cardiac progenitor cells to differentiate into epicardial cells is selected from one of the following two schemes: Option 1: Z1. Human pluripotent stem cells are cultured in an initial induction medium comprising: a basal medium and an initial induction factor; Z2. The cells obtained in step Z1 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors and retinoic acid for 48±12 h, preferably 48±2 h. Z3. Culture the cells obtained in step Z2 in basal medium for at least 4 days, preferably 4 to 6 days, more preferably 4 days; harvest the cell population. In the specific implementation plan, the formed pluripotent stem cell spheres are induced with the initial induction medium for 48 hours, and then further induced with aRPMI-B27 medium and the added components 5 μM IWP2 and 0.5 μM retinoic acid for another 48 hours. After that, they are further cultured with aRPMI-B27 medium. On the 8th day after the start of induction, more than 60% of epicardial cells can be produced, and few or no cardiomyocytes are produced.

[0148] Option 2: Z1. Human pluripotent stem cells are cultured in an initial induction medium comprising: a basal medium and an initial induction factor; Z2. The cells obtained in step Z1 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors for 24±6 h, preferably 24±2 h. Z3. The cells obtained in step Z3 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors and retinoic acid for 24±6 h, preferably 24±2 h. Z4. The cells obtained in step Z3 are cultured in basal medium in the presence of retinoic acid for 24±6 h, preferably 24±2 h. Z5. Culture the cells obtained in step Z4 in a basal culture medium for at least 3 days, preferably 3 to 5 days, more preferably 3 days; harvest the cell population.

[0149] In the specific implementation plan, the formed pluripotent stem cell spheres are induced with an initial induction medium for 48 hours, and then further induced with aRPMI-B27 medium and 5 μM IWP2 for 24 hours; then further induced with aRPMI-B27 medium and 5 μM IWP2 and 0.5 μM retinoic acid for 24 hours; then further induced with aRPMI-B27 medium and 0.5 μM retinoic acid for 24 hours; and finally further induced with aRPMI-B27 medium. On the 8th day after the start of induction, more than 60% of epicardial cells can be produced, with little or no cardiomyocytes produced.

[0150] In the above implementation scheme, more than 80% of the cells are epicardial-related cells, including about 60% epicardial cells and about 20% epicardial-derived cells, with few or no cardiomyocytes, accompanied by a small number of endoderm-derived cells and endothelial cells.

[0151] The beneficial effects of this invention include: 1. This invention is the first to culture and identify cardiac progenitor cells with bidirectional differentiation potential of cardiomyocytes and epicardial cells in vitro.

[0152] 2. This invention develops a culture medium kit, which provides a method for efficiently inducing pluripotent stem cells to differentiate into bipotent cardiac progenitor cells in vitro and for inducing bipotent cardiac progenitor cells to further differentiate into myocardial or epicardial cells.

[0153] 3. This invention can rapidly (e.g., within 3 days) and efficiently induce pluripotent stem cells to differentiate into bipotent cardiac progenitor cells. Under cardiomyocyte differentiation conditions, bipotent cardiac progenitor cells can generate more than 80% of cardiomyocytes. Under epicardial cell differentiation conditions, bipotent cardiac progenitor cells can generate more than 80% of epicardial-related cells, including epicardial and epicardial-derived cells.

[0154] 4. This invention provides a new cell model for studying the developmental process and principles of early cardiac lineages.

[0155] 5. Given the important functions of bipotent cardiac progenitor cells, cardiomyocytes, and epicardial cells in the heart, this invention has broad application prospects in the fields of cell therapy and regenerative medicine for heart-related diseases.

[0156] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions. In this invention, “%” represents volume percentage.

[0157] Example 1: Single-cell transcriptome analysis of early human embryonic heart development Based on the dataset of Richard CV Tyser et al., we analyzed single-cell transcriptome data of human embryos during the gastrula stage, the initial stage of human heart development.

[0158] The results are as follows Figure 1 As shown. Figure 1 A shows a heatmap of gene expression levels related to the retinoic acid signaling pathway during the human embryonic gastrulation period. Figure 1 b shows the uniform manifold approximation and projection (UMAP) of heart development-related cell types in the gastrula and the characteristic point map of retinoic acid-related gene expression.

[0159] The results showed differential expression and reception of retinoic acid (RA) signaling across different germ layers. Notably, high RA responsiveness was observed in both neonatal and late mesoderm populations, which originated from the cardiogenic mesoderm.

[0160] Example 2: Inducing human pluripotent stem cell differentiation to generate bipotent cardiac progenitor cells In this embodiment, a culture medium and a culture method for inducing human pluripotent stem cells to differentiate into bipotent cardiac progenitor cells are provided.

[0161] (I) Culture medium The aRPMI-B27 culture medium used in this embodiment is: advanced RPMI 1640 and 2% B27.

[0162] Advanced RPMI 1640 was purchased from Gibco, part number 12633012; B27 was purchased from Gibco, part number 17504-044; TeSR TM -E8 TMThe basal medium was purchased from Stem Cell, catalog number 5990. All reagents and instruments used, unless otherwise specified, were readily available through legitimate market channels. In the following examples, "%" refers to volume percentages in the culture media.

[0163] 1. Initial induction medium: aRPMI-B27 medium with added components 5 μM CHIR99021 and 0.5 μM BI-1347; 2. The second-stage induction medium shall be any one of the following three: Method (1): aRPMI-B27 medium; Method (2): aRPMI-B27 medium and 5 μM IWP2 added; Method (3): aRPMI-B27 medium with added components 5 μM IWP2 and 0.5 μM retinoic acid.

[0164] (II) Cultivation Steps A schematic diagram of the differentiation culture process for hESCs to differentiate into cardiac progenitor cells is shown below. Figure 2 Where (1)-(3) represent three different methods of inducing differentiation, and the specific steps are as follows: 1. Preparation of pluripotent stem cell spheres Human pluripotent stem cells were digested into single cells using 33% TrypLE, centrifuged to remove the supernatant, and then treated with TeSR containing 10 μM Y27632. TM -E8 TM Cells were resuspended in culture medium and counted. Cells were seeded into 96-well low-adsorption U-shaped plates at a rate of 4000 cells / 100 μL / well, centrifuged at 300g for 3 minutes to allow the cells to aggregate to the bottom of the wells, and then cultured in a CO2 incubator for 24 hours to form cell spheroids. 2. First stage of induced differentiation On day 0 (d0), the TeSR in the orifice was completely sucked out. TM -E8 TM Culture medium: Add initial induction medium and incubate in a CO2 incubator for 48 hours.

[0165] 3. Second stage of induced differentiation On day 2 (d2), the initial induction medium in the wells was aspirated, and one of the second-stage induction media was added. The cells were then cultured in a CO2 incubator for 24 hours. The resulting cells were cardiac progenitor cells.

[0166] Method (1) The second-stage induction medium used was aRPMI-B27 medium; Method (2) uses the second-stage induction medium: aRPMI-B27 medium with 5 μM IWP2 added; Method (3) uses the second-stage induction medium: aRPMI-B27 medium with 5 μM IWP2 and 0.5 μM retinoic acid added.

[0167] (III) Training Results The cardiac progenitor cells cultured using the above three methods were characterized and identified, and the results are as follows: On day 3 after the start of differentiation induction, cardiac progenitor cells generated under different differentiation conditions were stained, and the results are as follows: Figure 3 On day 3 after the start of differentiation induction, single-cell sequencing was performed on cardiac progenitor cells generated under different differentiation conditions, and the results are as follows: Figure 4 As shown.

[0168] The results showed that on the third day after the induction began, more than 90% of bipotent cardiac progenitor cells were generated under all three differentiation conditions, accompanied by a small number of endoderm-derived cells and undifferentiated cells. Among them, method (3) produced the highest proportion of bipotent cardiac progenitor cells, reaching 96%.

[0169] Example 3: Induction of bipotent cardiac progenitor cells into cardiomyocytes and / or epicardial cells In this embodiment, a culture medium and method are provided for inducing hPSCs to differentiate into bipotent cardiac progenitor cells and further differentiate into cardiomyocytes and / or epicardial cells, including three schemes: spontaneous differentiation, cardiomyocyte lineage differentiation, and epicardial lineage differentiation. A flowchart is shown below. Figure 5 The details are as follows: (I) Training Program Method (1): Spontaneous Differentiation Scheme ① Preparation of pluripotent stem cell spheres: Human pluripotent stem cells were digested into single cells using 33% TrypLE, centrifuged to remove the supernatant, and then treated with TeSR containing 10 μM Y27632. TM -E8 TM Cells were resuspended in culture medium and counted. Cells were seeded into 96-well low-adsorption U-shaped plates at a rate of 4000 cells / 100 μL / well, centrifuged at 300g for 3 minutes to allow the cells to aggregate to the bottom of the wells, and then cultured in a CO2 incubator for 24 hours to form cell spheroids. ② First stage of induced differentiation: TeSR in the aspiration well TM -E8 TM The culture medium was the same as that used in Example 2, and the culture was carried out in a CO2 incubator for 48 hours. ③ Second stage of induction differentiation: The initial induction medium in the wells was aspirated, and aRPMI-B27 medium from the second stage induction medium was added. The aRPMI-B27 medium was changed every 48 hours. On the 8th day after the start of induction, myocardial and epicardial cells were produced simultaneously, and the proportion of myocardial and epicardial cells to the total number of cells was random.

[0170] Method (2): Myocardial lineage differentiation protocol ① Same as step ① in method (1); ② Same as step ② in method (2); ③ Second-stage induction differentiation: The initial induction medium in the wells was exhausted, and aRPMI-B27 medium and 5 μM IWP2 additive from the second-stage induction medium were added. The cells were cultured in a CO2 incubator for 48 hours. Then, the medium was replaced with aRPMI-B27 medium without additives, and thereafter the aRPMI-B27 medium was replaced every 48 hours. On day 6 after induction began, more than 80% of the cells were cardiomyocytes, with little or no epicardial cells produced.

[0171] Method (3): Epicardial lineage differentiation protocol ① Same as step ① in method (1); ② Same as step ② in method (2); ③ Second-stage induction differentiation: The initial induction medium in the wells was exhausted, and then aRPMI-B27 medium, along with the additives 5 μM IWP2 and 0.5 μM retinoic acid, was added. The cells were cultured in a CO2 incubator for 48 hours. Afterward, the medium was replaced with aRPMI-B27 medium without additives, and thereafter the aRPMI-B27 medium was changed every 48 hours. On day 8 after induction began, more than 60% of the cells were epicardial cells, with little or no cardiomyocytes produced.

[0172] (II) Training Results The myocardial and epicardial cells generated by the above three differentiation methods were characterized and identified, and the results are as follows: On day 8 after induction began, myocardial and epicardial cells derived from cardiac progenitors under different differentiation conditions were stained, and the results are as follows: Figure 6 As shown, Figure 6 The statistical results are shown in Figure 7 On day 10 after the start of induced differentiation, single-cell sequencing was performed on cells generated by the myocardial lineage differentiation protocol of method (2), and the results are as follows. Figure 8 As shown. On day 10 after the start of induced differentiation, single-cell sequencing was performed on cells generated by the epicardial lineage differentiation protocol of method (3), and the results are as follows. Figure 9 As shown.

[0173] The results showed that, under the spontaneous differentiation scheme of method (1), both cardiomyocytes and epicardial cells were generated simultaneously, with the proportions of the two cell types being random; under the cardiomyocyte lineage differentiation scheme of method (2), more than 80% of cardiomyocytes were generated, including atrial cardiomyocytes, ventricular cardiomyocytes and atrioventricular canal cardiomyocytes, with very few epicardial cells, accompanied by a small number of cardiac fibroblasts, endoderm-derived cells and endothelial cells; under the epicardial lineage differentiation scheme of method (3), more than 80% of epicardial-related cells were generated, including proliferating epicardium, epicardium, epicardial-derived progenitor cells and epicardial-derived fibroblasts, with very few cardiomyocytes, accompanied by a small number of endoderm-derived cells and endothelial cells.

[0174] Example 4: The role of BI-1347 in the differentiation of pluripotent stem cells into bipotent cardiac progenitor cells In this embodiment, the role of BI-1347 in the differentiation of pluripotent stem cells into bipotent cardiac progenitor cells was detected by a BI-1347 concentration gradient experiment.

[0175] (I) Training Program Under otherwise identical conditions, different initial induction media were used for culturing. A schematic diagram of the experimental procedure is shown below. Figure 10 .

[0176] Methods (1) to (5) used the following initial induction media respectively: Method (1): aRPMI-B27 medium and 5 μM CHIR99021 added; Method (2): aRPMI-B27 medium with added components 5 μM CHIR99021 and 0.2 μM BI-1347; Method (3): aRPMI-B27 medium and added components 5 μM CHIR99021 and 0.5 μM BI-1347; Method (4): aRPMI-B27 medium with added components 5 μM CHIR99021 and 1 μM BI-1347; Method (5): aRPMI-B27 medium with added components 5 μM CHIR99021 and 2 μM BI-1347; The specific cultivation steps are as follows: ① Preparation of pluripotent stem cell spheres: Human pluripotent stem cells were digested into single cells using 33% TrypLE. After centrifugation and removal of supernatant, the cells were resuspended in TeSR™-E8™ medium containing 10 μM Y27632 and counted. Cells were seeded into 96-well low-adsorption U-shaped plates at a rate of 4000 cells / 100 μL / well, centrifuged at 300g for 3 minutes to allow the cells to aggregate at the bottom of the wells, and then cultured in a CO2 incubator for 24 hours to form cell spheres. ② First stage of induction differentiation: the TeSR™-E8™ medium in the well was exhausted, and different initial induction media were added according to methods (1) to (5) respectively. The culture was induced in a CO2 incubator for 48 hours. ③ Second stage of induction differentiation: Remove the initial induction medium from the wells, add aRPMI-B27 medium and continue culturing. Replace the aRPMI-B27 medium every 48 hours thereafter.

[0177] (II) Training Results The myocardial and epicardial cells generated by the above five methods were characterized and identified, and the results are as follows: On day 8 after induction began, myocardial and epicardial cells derived from cardiac progenitors under different differentiation conditions were stained, and the results are as follows: Figure 11 As shown, Figure 11 The statistical results are as follows Figure 12 As shown.

[0178] The results showed that without the addition of BI-1347 in the first-stage induction process, bipotent cardiac progenitor cells could not be generated, as subsequent differentiation only produced epicardial cells and not cardiomyocytes. With the addition of 0.2 μM BI-1347, some samples generated both cardiomyocytes and epicardial cells in the later stages of differentiation, but the number of cardiomyocytes was very small. With the addition of 0.5-2 μM BI-1347, both cardiomyocytes and epicardial cells were generated simultaneously in the later stages. This indicates that a certain concentration of BI-1347 is crucial for promoting the generation of bipotent cardiac progenitor cells, with 0.5 μM BI-1347 being the preferred concentration.

[0179] Example 5: The role of retinoic acid in the differentiation of dual-potency cardiac progenitor cells into myocardium or epicardium In this embodiment, the role of retinoic acid in the differentiation of bipotent cardiac progenitor cells into myocardium or epicardium was detected by a retinoic acid concentration gradient experiment.

[0180] (I) Training Program Under otherwise identical conditions, different second-stage induction media were used for culturing. A schematic diagram of the experimental procedure is shown below. Figure 13 .

[0181] Initial induction medium: aRPMI-B27 medium with added components 5 μM CHIR99021 and 0.5 μM BI-1347.

[0182] The second-stage induction medium used in methods (1) to (9) is as follows: (1) aRPMI-B27 medium and added component 5 μM IWP2; (2) aRPMI-B27 medium and added components 5 μM IWP2 and 0.02 μM retinoic acid; (3) RPMI-B27 medium and added components 5 μM IWP2 and 0.05 μM retinoic acid; (4) RPMI-B27 medium and added components 5 μM IWP2 and 0.1 μM retinoic acid; (5) RPMI-B27 medium and added components 5 μM IWP2 and 0.2 μM retinoic acid; (6) RPMI-B27 medium and added components 5 μM IWP2 and 0.5 μM retinoic acid; (7) aRPMI-B27 medium and added components 5 μM IWP2 and 1 μM retinoic acid; (8) RPMI-B27 medium and added components 5 μM IWP2 and 2 μM retinoic acid; (9) RPMI-B27 medium and added components 5 μM IWP2 and 5 μM retinoic acid; The specific cultivation steps are as follows: ① Preparation of pluripotent stem cell spheres: Human pluripotent stem cells were digested into single cells using 33% TrypLE. After centrifugation and removal of supernatant, the cells were resuspended in TeSR™-E8™ medium containing 10 μM Y27632 and counted. Cells were seeded into 96-well low-adsorption U-shaped plates at a rate of 4000 cells / 100 μL / well, centrifuged at 300g for 3 minutes to allow the cells to aggregate at the bottom of the wells, and then cultured in a CO2 incubator for 24 hours to form cell spheres. ② First stage of differentiation induction: Exhaust the TeSR™-E8™ medium in the wells, add the initial induction medium, and incubate in a CO2 incubator for 48 hours; ③ Second stage induction differentiation: The initial induction medium in the well was aspirated, and the second stage induction medium was added to the wells in methods (1) to (9) respectively. The wells were incubated in a CO2 incubator for 48 hours. Then the medium was replaced with aRPMI-B27 medium without additives. The aRPMI-B27 medium was replaced every 48 hours thereafter.

[0183] (II) Training Results The myocardial and epicardial cells generated by the nine methods were characterized and identified, and the results are as follows: On day 8 after induction began, myocardial and epicardial cells derived from cardiac progenitors under different differentiation conditions were stained, and the results are as follows: Figure 14 As shown, Figure 14 The statistical results are shown in Figure 15 .

[0184] The results showed that the number of cardiomyocytes decreased in a dose-dependent manner with increasing retinoic acid concentration; while the number of epicardial cells increased in a dose-dependent manner when the retinoic acid concentration was ≤0.5 μM, but decreased with increasing retinoic acid concentration when the concentration was >0.5 μM. This indicates that a certain concentration of retinoic acid promotes the differentiation of bipotent cardiac progenitor cells into epicardial cells but is detrimental to their differentiation into cardiomyocytes. Among these, 0.5 μM retinoic acid showed the best promoting effect on epicardial cell differentiation of bipotent cardiac progenitor cells.

[0185] Example 6: The relationship between retinoic acid and epicardial lineage differentiation of cardiac progenitor cells In this embodiment, by adding the inverse agonist BMS493 of the pan-retinoic acid receptor to inhibit retinoic acid signaling, the relationship between retinoic acid and the differentiation of cardiac progenitor cells into the epicardial lineage was investigated.

[0186] (I) Experimental Scheme Under otherwise identical conditions, different second-stage induction media were used for culturing. A schematic diagram of the experimental procedure is shown below. Figure 16 .

[0187] Initial induction medium: aRPMI-B27 medium with added components 5 μM CHIR99021 and 0.5 μM BI-1347.

[0188] The second-stage induction media used in methods (1) to (3) are as follows: (1) RPMI-B27 medium containing 5 μM IWP2 and 0.5 μM retinoic acid; (2) aRPMI-B27 medium and added components 5 μM IWP2, 0.5 μM retinoic acid and 1 μM BMS493; (3) aRPMI-B27 culture medium and added components 5 μM IWP2, 0.5 μM retinoic acid and 2 μM BMS493.

[0189] The specific steps are as follows: ① Preparation of pluripotent stem cell spheres: Human pluripotent stem cells were digested into single cells using 33% TrypLE. After centrifugation and removal of supernatant, the cells were resuspended in TeSR™-E8™ medium containing 10 μM Y27632 and counted. Cells were seeded into 96-well low-adsorption U-shaped plates at a rate of 4000 cells / 100 μL / well, centrifuged at 300g for 3 minutes to allow the cells to aggregate at the bottom of the wells, and then cultured in a CO2 incubator for 24 hours to form cell spheres. ② First stage of differentiation induction: Exhaust the TeSR™-E8™ medium in the wells, add the initial induction medium, and incubate in a CO2 incubator for 48 hours; ③ Second stage induction differentiation: The initial induction medium in the well was exhausted. Different second stage induction media were added according to methods (1) to (3) and cultured in a CO2 incubator for 48 hours. Then it was replaced with aRPMI-B27 medium without additives. The aRPMI-B27 medium was replaced every 48 hours thereafter.

[0190] (II) Training Results The myocardial and epicardial cells generated by the above three methods were characterized and identified, and the results are as follows: On day 8 after induction began, myocardial and epicardial cells derived from cardiac progenitors under different differentiation conditions were stained, and the results are as follows: Figure 17 As shown, Figure 17 The statistical results are shown in Figure 18 .

[0191] The results showed that, compared with the control group without retinoic acid inhibitors, the addition of 1 μM BMS493 significantly reduced the number of epicardial cells, while 2 μM BMS493 completely inhibited epicardial cell production. This indicates that retinoic acid is a key factor in promoting the differentiation of bipotent cardiac progenitor cells into epicardial cells, and inhibiting retinoic acid signaling inhibits epicardial cell production.

[0192] Example 7: The critical time period for WNT pathway inhibition signaling to determine the subsequent myocardial fate of cardiac progenitors In this embodiment, by adding the WNT pathway inhibitor IWP2 at different time points, the key time period in which the WNT pathway inhibition signal determines the subsequent myocardial fate of cardiac progenitor cells was determined.

[0193] (I) Training Program Under otherwise identical conditions, IWP2 was added at different times during the second induction phase. See the experimental flowchart below. Figure 19 .

[0194] Method (1): Comparison method ① Preparation of pluripotent stem cell spheres: Human pluripotent stem cells were digested into single cells using 33% TrypLE. After centrifugation and removal of supernatant, the cells were resuspended in TeSR™-E8™ medium containing 10 μM Y27632 and counted. Cells were seeded into 96-well low-adsorption U-shaped plates at a rate of 4000 cells / 100 μL / well, centrifuged at 300g for 3 minutes to allow the cells to aggregate at the bottom of the wells, and then cultured in a CO2 incubator for 24 hours to form cell spheres. ② First stage of differentiation induction: Exhaust the TeSR™-E8™ medium in the wells, add the initial induction medium, and incubate in a CO2 incubator for 48 hours; ③ Second stage of induction differentiation: The initial induction medium in the wells was exhausted, and aRPMI-B27 medium and 5 μM IWP2 were added to the second stage induction medium. The medium was then cultured in a CO2 incubator for 48 hours. After that, the medium was replaced with aRPMI-B27 medium without additives. The aRPMI-B27 medium was then replaced every 48 hours.

[0195] Methods (2)~(3): Delay the IWP2 addition time ① Same as step ① in method (1); ② Same as step ② in method (1); ③ Second stage induction differentiation: The initial induction medium in the well was aspirated, and aRPMI-B27 medium from the second stage induction medium was added. Method (2) was cultured in a CO2 incubator for 24 hours, and method (3) was cultured in a CO2 incubator for 48 hours. Then it was replaced with aRPMI-B27 medium from the second stage induction medium and 5 μM IWP2 was added. It was cultured in a CO2 incubator for 48 hours. Then it was replaced with aRPMI-B27 medium without additives. The aRPMI-B27 medium was replaced every 48 hours thereafter.

[0196] (II) Training Results The myocardial and epicardial cells generated by the above three methods were characterized and identified, and the results are as follows: On day 8 after induction began, myocardial and epicardial cells derived from cardiac progenitors under different differentiation conditions were stained, and the results are as follows: Figure 20 As shown, Figure 20 The statistical results are shown in Figure 21 .

[0197] The results showed that inhibiting the WNT pathway on day 2 or 3 after induction resulted in the production of over 80% of cardiomyocytes, while inhibiting the WNT pathway on day 4 resulted in the production of both cardiomyocytes and epicardial cells, with randomized proportions of each cell type. This indicates that the cardiomyocyte-promoting effect of WNT pathway inhibition mainly occurs on day 3 after induction, i.e., the stage of bipotent cardiac progenitor cell production.

[0198] Example 8: The critical time period in which retinoic acid determines the subsequent epicardial fate of cardiac progenitor cells In this embodiment, by adding retinoic acid at different time points, the key time period in which retinoic acid determines the subsequent epicardial fate of cardiac progenitor cells was determined.

[0199] (I) Training Program Under otherwise identical conditions, retinoic acid was added at different times during the second induction phase. See the schematic diagram for the experimental procedure. Figure 22 .

[0200] Method (1): Comparison method ① Preparation of pluripotent stem cell spheres: Human pluripotent stem cells were digested into single cells using 33% TrypLE. After centrifugation and removal of supernatant, the cells were resuspended in TeSR™-E8™ medium containing 10 μM Y27632 and counted. Cells were seeded into 96-well low-adsorption U-shaped plates at a rate of 4000 cells / 100 μL / well, centrifuged at 300g for 3 minutes to allow the cells to aggregate at the bottom of the wells, and then cultured in a CO2 incubator for 24 hours to form cell spheres. ② First stage of differentiation induction: Exhaust the TeSR™-E8™ medium in the wells, add the initial induction medium, and incubate in a CO2 incubator for 48 hours; ③ Second stage of induction differentiation: The initial induction medium in the wells was exhausted, and aRPMI-B27 medium and 5 μM IWP2 were added to the second stage induction medium. The medium was then cultured in a CO2 incubator for 48 hours. After that, the medium was replaced with aRPMI-B27 medium without additives. The aRPMI-B27 medium was then replaced every 48 hours.

[0201] Methods (2) to (6): Based on method (1), according to Figure 22 The proposed method involves adding retinoic acid at different times and treating for 48 hours.

[0202] (II) Training Results The myocardial and epicardial cells generated by the above six methods were characterized and identified, and the results are as follows: On day 8 after induction began, myocardial and epicardial cells derived from cardiac progenitors under different differentiation conditions were stained, and the results are as follows: Figure 23 As shown, Figure 23 The statistical results are shown in Figure 24 .

[0203] The results showed that adding retinoic acid on the second or third day after induction resulted in the production of over 60% epicardial cells. However, adding retinoic acid on the fourth day or later resulted primarily in the production of cardiomyocytes, accounting for approximately 80%, with no epicardial cells produced. This indicates that the role of retinoic acid in determining the epicardial fate of bipotent cardiac progenitor cells mainly occurs on the third day after induction, i.e., during the bipotent cardiac progenitor cell production stage.

[0204] In summary, this invention provides a method and culture medium for inducing human pluripotent stem cells to differentiate into bipotent cardiac progenitor cells, and identifies retinoic acid as a key signal determining the differentiation of bipotent cardiac progenitor cells into epicardial cells. Furthermore, this invention identifies a key time window for differentiation signals to regulate the fate of bipotent cardiac progenitor cells in the later stages of myocardial or epicardial differentiation.

[0205] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An isolated cell population, characterized in that, At least 80% of the cells in the cell population are bipotent cardiac progenitor cells, which have the potential to differentiate into cardiomyocytes and epicardial cells.

2. The cell population as described in claim 1, characterized in that, The cell population was obtained by the following method: (1) Human pluripotent stem cells are cultured in a first culture medium, which includes: basal culture medium, CHIR99021 and BI-1347, and the culture time is 2 ± 0.5 days, preferably 2 days; (2) The cells obtained in step (1) are cultured in a second culture medium, which includes a basal culture medium and a culture time of 1 ± 0.5 days, preferably 1 day.

3. The cell population as described in claim 2, characterized in that, The basal culture medium is a combination of advanced RPMI 1640 medium and 2% B27.

4. The cell population as described in claim 2, characterized in that, The second culture medium includes: basal culture medium, Wnt signaling pathway inhibitor, and optional retinoic acid; preferably, the Wnt signaling pathway inhibitor is IWP2.

5. A culture medium composition for inducing pluripotent stem cell differentiation to generate bipotent cardiac progenitor cells, characterized in that, The culture medium combination includes: an initial induction medium and a second-stage induction medium; The initial induction medium includes: basal medium and initial induction factors; the initial induction factors include: CHIR99021 and BI-1347; The second-stage induction medium includes: basal medium and optional second-stage induction factor.

6. A reagent combination for inducing the differentiation of bipotent cardiac progenitor cells into cardiomyocytes, characterized in that, The reagent combination includes: (i) basal culture medium and (ii) Wnt signaling pathway inhibitor.

7. A reagent combination for inducing bipotent cardiac progenitor cells to differentiate into epicardial cells, characterized in that, The reagent combination includes: (i) basal culture medium, (ii) Wnt signaling pathway inhibitors, and (iii) retinoic acid.

8. A method for generating bipotent cardiac progenitor cells in vitro, characterized in that, The dual-potential cardiac progenitor cells have the potential to differentiate into cardiomyocytes and epicardial cells, and the method includes the following steps: S1. Provide human pluripotent stem cells; S2. Providing an initial induction medium comprising: a basal medium and initial induction factors comprising: CHIR99021 and BI-1347; culturing the human pluripotent stem cells in the initial induction medium; S3. Provide a second-stage induction medium, which includes a basal medium and an optional second-stage induction factor; culture the cells in S2 in the second-stage induction medium to obtain the cell population.

9. A method for generating cardiomyocytes through in vitro culture, characterized in that, The method includes the following steps: Y1. Human pluripotent stem cells are cultured in an initial induction medium, the initial induction medium comprising: basal medium and initial induction factor; Y2. The cells obtained in step Y1 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors for 48±12h, preferably 48±2h. Y3. Culture the cells obtained in step Y2 in basal culture medium for at least 2 days, preferably 2 to 6 days, more preferably 2 days; harvest the cell population; or, Y1. Human pluripotent stem cells are cultured in an initial induction medium, the initial induction medium comprising: basal medium and initial induction factor; Y2. Culture the cells obtained in step Y1 in basal culture medium for 24±6h, preferably 24±2h; Y3. The cells obtained in step Y2 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors for 48±12h, preferably 48±2h. Y4. Culture the cells obtained in step Y3 in a basal culture medium for more than 1 day, preferably 1 to 5 days, more preferably 1 day; harvest the cell population.

10. A method for generating epicardial cells in vitro, characterized in that, The method includes the following steps: Z1. Human pluripotent stem cells are cultured in an initial induction medium comprising: a basal medium and an initial induction factor; Z2. The cells obtained in step Z1 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors and retinoic acid for 48±12 h, preferably 48±2 h. Z3. Culture the cells obtained in step Z2 in basal medium for at least 4 days, preferably 4 to 6 days, more preferably 4 days; harvest the cell population. or, Z1. Human pluripotent stem cells are cultured in an initial induction medium comprising: a basal medium and an initial induction factor; Z2. The cells obtained in step Z1 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors for 24±6 h, preferably 24±2 h. Z3. The cells obtained in step Z3 are cultured in basal medium in the presence of Wnt signaling pathway inhibitors and retinoic acid for 24±6 h, preferably 24±2 h. Z4. The cells obtained in step Z3 are cultured in basal medium in the presence of retinoic acid for 24±6 h, preferably 24±2 h. Z5. Culture the cells obtained in step Z4 in a basal culture medium for at least 3 days, preferably 3 to 5 days, more preferably 3 days; harvest the cell population.

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