WO2023217134A1

WO2023217134A1 - Biological preparation containing myocardial precursor-like cell, preparation method therefor, and application

Info

Publication number: WO2023217134A1
Application number: PCT/CN2023/092969
Authority: WO
Inventors: 周伸奥
Original assignee: 上海赛立维生物科技有限公司
Priority date: 2022-05-10
Filing date: 2023-05-09
Publication date: 2023-11-16
Also published as: CN117018033A; CN117025506A; CN117025504A; WO2023217121A1; CN117018032A; WO2023217126A1; CN117025503A; CN117025502A; WO2023217132A1; WO2023217130A1; WO2023217123A1; WO2023217129A1; WO2023217128A1; CN117025508A; CN117025505A; WO2023217125A1; CN117025507A

Abstract

The present invention provides a biological preparation containing a myocardial precursor-like cell, a preparation method therefor, and an application. The myocardial precursor-like cell positively expresses CD24 and c-Kit, and the myocardial precursor-like cell does not express MHC class II molecules. The present invention solves the problem in the prior art of immunological rejection of myocardial precursor-like cells after allotransplantation.

Description

Biological preparations containing cardiac precursor-like cells and preparation methods and applications thereof

This application requires the priority of a Chinese patent application with a filing date of May 10, 2022, an application number of 2022105036786, and an invention title of "Biological preparations, cell derivatives, preparation methods and applications". The contents of the above application are incorporated herein by reference.

Technical field

The present invention relates to the field of biotechnology, and in particular to a biological agent containing cardiac precursor-like cells and its preparation method and application.

Background technique

Heart disease is the leading cause of death in humans. During a heart attack, an adult human heart may lose up to 1 billion cardiomyocytes, and less than 1% of adult cardiomyocytes can regenerate. For heart diseases such as myocardial necrosis and heart failure, the biggest difficulty in conservative treatment is that it cannot compensate for the missing functional myocardium.

From the perspective of regenerative medicine, since mature cardiomyocytes cannot be expanded in vitro, insufficient source of seed cells is an important factor limiting the use of cell therapy to treat heart diseases. There are two specific reasons for the insufficient source of seed cells. The first is the difficulty in obtaining primary cardiomyocytes. Even if a donated heart can be obtained, it is very difficult to isolate primary cardiomyocytes. At the same time, primary cardiomyocytes cannot Expanded in vitro, they cannot be used as seed cells for cell therapy. The second aspect is that recent studies have shown that cardiac precursor-like cells and cardiomyocytes can be differentiated from pluripotent stem cells, including embryonic stem cells (Embryonic Stem cells). cells, ES) and induced pluripotent stem cells (Induced pluripotent stem cells (iPSCs), but this technical route is very complex and has the following problems: first, undifferentiated ES and undifferentiated iPSCs may have tumorigenicity during the differentiation process; second, the cells obtained through the differentiation of ES and iPSCs The purity of myocardial precursor-like cells and cardiomyocytes is difficult to guarantee, which limits the application of myocardial precursor-like cells and cardiomyocytes; thirdly, myocardial precursor-like cells derived from ES and iPSC differentiation cannot be expanded in large quantities, so each batch All applications need to be differentiated from scratch.

Currently, in the existing technology, myocardial precursor-like cells will undergo immune rejection after allogeneic transplantation, and oral anti-extrusion drugs are required to resolve the immune rejection reaction. Therefore, it is necessary to provide a biological preparation containing myocardial precursor-like cells and its preparation method and application to solve the above problems existing in the prior art.

Contents of the invention

The purpose of the present invention is to provide a biological preparation containing myocardial precursor-like cells and its preparation method and application, so as to solve the problem of immune rejection of myocardial precursor-like cells in the prior art after allogeneic transplantation.

In order to achieve the above object, the present invention provides a biological preparation containing myocardial precursor-like cells, which positively express CD24 and c-Kit, and which do not express MHC class II molecules.

The beneficial effect of the biological preparation containing myocardial precursor-like cells of the present invention is that: through the myocardial precursor-like cells positively expressing CD24 and c-Kit, and the myocardial precursor-like cells not expressing MHC class II molecules, the myocardial precursor-like cells can make the myocardium Precursor-like cells can adapt well to the human body during allogeneic transplantation, thereby solving the problem of the existing technology of myocardial precursor-like cells in allogeneic transplantation. Then there was the problem of immune rejection.

The present invention also provides a method for preparing a biological preparation containing myocardial precursor-like cells, which includes the following steps:

S0: Provide cardiac precursor-like cells;

S1: Mix the myocardial precursor-like cells with a pharmaceutically acceptable carrier to obtain the biological preparation containing the myocardial precursor-like cells, which positively express CD24 and c-Kit, The cardiac precursor-like cells do not express MHC class II molecules.

The beneficial effects of the preparation method of biological preparations containing myocardial precursor-like cells of the present invention are: the preparation method of biological preparations containing myocardial precursor-like cells is simple, and after the biological preparations containing myocardial precursor-like cells are reinfused into the human body, it can It adapts well to the human body and does not require the use of drugs to avoid immune rejection caused by allogeneic transplantation.

Preferably, the preparation method of cardiac precursor-like cells includes the following steps:

S00: Provide mature cardiomyocytes;

S01: Put the mature cardiomyocytes into a reprogramming medium for dedifferentiation culture until the confluence of the myocardial precursor-like cells is not less than 80%, and use trypsin digestion solution to treat the myocardial precursor-like cells. Digestion treatment is performed to obtain myocardial precursor-like cells, wherein the reprogramming medium includes basal medium, MEK inhibitors, inhibitory factors and protein hormones, and the reprogramming medium is used for the myocardial precursor-like cells. induction and promote the proliferation of the myocardial precursor-like cells. The beneficial effect is that the MEK inhibitor is used to induce myocardial precursor-like cells, and the inhibitory factor and the protein hormone are used to promote the proliferation of myocardial precursor-like cells.

Preferably, the preparation method of the myocardial precursor-like cells includes the following steps: S02: Put the myocardial precursor-like cells into the reprogramming medium for expansion and culture until the myocardial precursor-like cells are After the confluence is not less than 80%, the myocardial precursor-like cells are digested using the trypsin digestion solution to obtain passaged myocardial precursor-like cells. The beneficial effect is that the MEK inhibitor acts together with growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF-β signaling inhibitors and nutritional supplements to induce cardiac precursor-like cells; so The inhibitory factors and the protein hormones work together with growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF-β signaling inhibitors and nutritional supplements to promote the proliferation of cardiac precursor-like cells, which can make Cardiac precursor-like cells expand massively in vitro.

Preferably, the reprogramming medium also includes growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF-β signaling inhibitors and nutritional supplements.

Preferably, based on the volume of the basal culture medium, the content of the MEK inhibitor is 1-101M, the content of the inhibitory factor is 5-20ng/mL, and the content of the protein hormone is 1-101g/ ml.

Preferably, based on the volume of the basal culture medium, the content of the growth factor is 50-80ng/mL, the content of the ROCK kinase inhibitor is 10-501M, and the content of the Wnt signaling pathway agonist is 1-101M, the content of the TGF-β signal inhibitor is 1-101M, and the content of the nutritional supplement is 1%-10%.

Preferably, the mature cardiomyocytes are differentiated from embryonic stem cells or induced pluripotent stem cells.

Preferably, the mature cardiomyocytes are obtained from myocardial tissue through digestion.

The present invention also provides an application of a biological preparation containing myocardial precursor-like cells, using the biological preparation containing myocardial precursor-like cells to intervene in an in vivo animal model, and the animal model includes an animal model of drug-induced myocardial injury.

The beneficial effect of the application of the biological agent containing myocardial precursor-like cells of the present invention is that since the myocardial precursor-like cells do not express MHC class II molecules, there is no need to take anti-excretion medications after transplantation of the biological agent containing myocardial precursor-like cells. Drugs can reduce harm to the human body. The biological preparation containing myocardial precursor-like cells of the present invention is safer in treating heart diseases, has a wider application range, and is more acceptable to patients.

Description of the drawings

Figure 1 is a schematic photographic diagram of the morphology of primary cardiomyocytes in Example 1 of the present invention;

Figure 2 is a photographic diagram of the morphology of third-generation mature cardiomyocytes in Example 1 of the present invention;

Figure 3 is a schematic photographic diagram of the morphology of third-generation myocardial precursor-like cells cultured in Example 1 of the present invention;

Figure 4 is a schematic photographic diagram of the morphology of third-generation control myocardial precursor-like cells cultured in Example 1 of the present invention;

Figure 5 is a graph showing the growth of myocardial precursor-like cells in reprogramming culture medium and mature cardiomyocytes in cardiomyocyte culture medium in Example 1 of the present invention;

Figure 6 is a schematic diagram of the flow cytometric detection results of third-generation myocardial precursor-like cells that positively express c-Kit in Example 1 of the present invention;

Figure 7 is a schematic diagram of the flow cytometric detection results of third-generation myocardial precursor-like cells positively expressing CD24 in Example 1 of the present invention;

Figure 8 is a schematic diagram of the flow cytometric detection results of third-generation myocardial precursor-like cells negatively expressing HLA-DRPQ in Example 1 of the present invention;

Figure 9 is a schematic photograph of the cell morphology of the third-generation cardiac precursor-like cells differentiated for 7 days in Example 1 of the present invention;

Figure 10 is a schematic diagram of the flow cytometric detection results of positive expression of cTnT in third-generation cardiac precursor-like cells differentiated 7 days after differentiation in Example 1 of the present invention;

Figure 11 is a schematic diagram of the flow cytometric detection results of third-generation cardiac precursor-like cells that positively express α-Sarcometric actin after 7 days of differentiation in Example 1 of the present invention;

Figure 12 is a schematic diagram of the flow cytometric detection results of mature cardiomyocytes positively expressing cTnT in Example 2 of the present invention;

Figure 13 is a schematic diagram of the flow cytometric detection results of cardiac precursor-like cells positively expressing c-kit in Example 2 of the present invention;

Figure 14 is a schematic diagram of the flow cytometric detection results of cardiac precursor-like cells negatively expressing HLA-DRPQ in Example 2 of the present invention;

Figure 15 is a schematic diagram of the flow cytometric detection results of cardiac precursor-like cells positively expressing c-kit in Example 3 of the present invention;

Figure 16 is a schematic diagram of the flow cytometric detection results of cardiac precursor-like cells negatively expressing HLA-DRPQ in Example 3 of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention. Unless otherwise defined, technical or scientific terms used herein shall have their ordinary meaning understood by one of ordinary skill in the art to which this invention belongs. The use of "comprising" and similar words herein means that the elements or things appearing before the word include the elements or things listed after the word and their equivalents, without excluding other elements or things.

In the prior art, there are methods for preparing mature cardiomyocytes and myocardial precursor-like cells. There are three methods for preparing mature cardiomyocytes: the steps of the first preparation method include: isolating from primary heart organ tissue, and Especially neonatal tissue is preferred, and mature cardiomyocytes can be obtained; the steps of the second preparation method include: differentiation from ES or iPSCs, mature cardiomyocytes can be obtained, and the stages experienced are to differentiate from ES or iPSCs to obtain mesoderm cells, and to obtain mesoderm cells. The cells are differentiated to obtain cardiac mesoderm cells, the cardiac mesodermal cells are differentiated to obtain cardiac precursor-like cells, and the cardiac precursor-like cells are differentiated to obtain mature cardiomyocytes; the steps of the third preparation method include: from other cells, such as fibroblasts , can be transdifferentiated to obtain mature cardiomyocytes. The preparation method of myocardial precursor-like cells includes the following steps: differentiating from ES or iPSC to obtain myocardial precursor-like cells. The stages are: differentiating from ES or iPSC to obtain mesodermal cells, and differentiating from mesodermal cells to obtain cardiac mesodermal cells. , cardiomyocyte precursor-like cells are differentiated from cardiac mesoderm cells.

The deficiencies in the above existing technologies include: the first preparation method of mature cardiomyocytes has the following deficiencies: 1. Ethical issues; 2. The harvest rate is extremely low; 3. Inability to expand and meet treatment needs; 4. Immunogens Highly toxic and unsuitable for allogeneic transplantation; the second method of preparing mature cardiomyocytes has the following shortcomings: 1. ES or iPSC residues can easily cause tumors; 2. It is difficult to ensure the purity of differentiation; 3. The differentiation cycle is long and the cost is high; 4. It has high immunogenicity and is not suitable for allogeneic transplantation; the third method of preparing mature cardiomyocytes has the following shortcomings: 1. Extremely low efficiency; 2. Extremely low purity; 3. It has high immunogenicity and is not suitable for allogeneic transplantation; premyocardial cells The preparation method of somatic cells has the following shortcomings: 1. ES or iPSC residues can easily cause tumors; 2. It is difficult to ensure the purity of differentiation; 3. The differentiation cycle is long and the cost is high; 4. The cells cannot be maintained in the precursor state and will mature spontaneously. Mature cardiomyocytes are obtained; 5. In the precursor state, they cannot be expanded in large quantities; 6. They are highly immunogenic and are not suitable for allogeneic transplantation.

The present invention can use reprogramming medium to culture mature cardiomyocytes from different sources in vitro to obtain myocardial precursor-like cells. The myocardial precursor-like cells obtained by the present invention can be amplified in large quantities in vitro, wherein the different Sources of mature cardiomyocytes include mature cardiomyocytes obtained from primary cells and mature cardiomyocytes differentiated from ES or iPSCs. At the same time, the present invention can massively expand myocardial precursor-like cells differentiated from ES or iPSC in vitro, solving the problem that myocardial precursor-like cells cannot proliferate in large quantities in vitro.

Mature cardiomyocytes differentiated from ES or iPSC, or mature cardiomyocytes isolated from heart tissue through digestion, are subjected to dedifferentiation treatment in the culture medium provided by the invention to obtain myocardial precursor-like cells. Somatic-like cells have the following common characteristics: 1. Myocardial precursor-like cells can be expanded in large quantities in vitro and have industrial application prospects; 2. Myocardial precursor-like cells positively express CD24 and c-Kit; 3. Myocardial precursor-like cells possess the general characteristics of precursor cells, such as the ability to rapidly proliferate; 4. Myocardial precursor-like cells possess the functions of precursor cells and can function in the myocardium. Under the influence of differentiation medium, it can be redifferentiated into mature cardiomyocytes, with positive expression of cTnT and α-Sarcometric actin; 5. Low immunogenicity, no or almost no expression (<5%) of MHC class II antigen HLA-DP. HLA-DQ and HLA-DR are therefore suitable for allogeneic transplantation and are less likely to cause graft-versus-host disease (graft-versus-host disease in English). : GvHD); 6. Anti-inflammatory, inhibiting PBMC activation, while inhibiting M1 macrophages and promoting their transformation into M2 macrophages; 7. Anti-fibrosis, can induce stellate cells and other cells in vivo or in vitro through paracrine effects Fibrosis-derived cells undergo programmed death.

The present invention provides a biological preparation containing myocardial precursor-like cells, which positively express CD24 and c-Kit, and which do not express MHC class II molecules.

Specifically, the myocardial precursor-like cells positively express CD24 and c-Kit, and the myocardial precursor-like cells do not express MHC class II molecules, which enables the myocardial precursor-like cells to adapt well during allogeneic transplantation. human body, thereby solving the problem of immune rejection of myocardial precursor-like cells in the prior art after allogeneic transplantation.

In some embodiments of the present invention, the MHC class II molecules include HLA-DR/DP/DQ, and the HLA-DR/DP/DQ is abbreviated as HLA-DRPQ.

S0: Provide cardiac precursor-like cells;

Specifically, the preparation method of biological preparations containing myocardial precursor-like cells is simple, and after the biological preparations containing myocardial precursor-like cells are reinfused into the human body, they can adapt well to the human body and do not require the use of drugs to avoid the problems caused by allogeneic transplantation. Immune rejection.

In some embodiments of the present invention, the method for preparing cardiac precursor-like cells includes the following steps:

S00: Provide mature cardiomyocytes;

S01: Put the mature cardiomyocytes into a reprogramming medium for dedifferentiation culture until the confluence of the myocardial precursor-like cells is not less than 80%, and use trypsin digestion solution to treat the myocardial precursor-like cells. Digestion treatment is performed to obtain myocardial precursor-like cells, wherein the reprogramming medium includes basal medium, MEK inhibitors, inhibitory factors and protein hormones, and the reprogramming medium is used for the myocardial precursor-like cells. induction and promote the proliferation of the myocardial precursor-like cells. The MEK inhibitor is used to induce cardiac precursor-like cells, and the inhibitory factor and the protein hormone are used to promote the proliferation of cardiac precursor-like cells.

In some specific embodiments of the present invention, the inhibitory factor is leukemia inhibitory factor, the protein hormone is insulin, the MEK inhibitor PD0325901 is from Shanghai Lianmai Bioengineering Co., Ltd., and the leukemia inhibitory factor LIF is from Shanghai Kanglang Biology Technology Co., Ltd., the insulin comes from Ganli Pharmaceuticals.

In some embodiments of the present invention, the preparation method of the myocardial precursor-like cells includes the following steps: S02: Put the myocardial precursor-like cells into the reprogramming medium for expansion and culture until the myocardial precursors are After the fusion degree of the sample cells is not less than 80%, the myocardial precursor-like cells are digested using the trypsin digestion solution to obtain passaged myocardial precursor-like cells. The MEK inhibitor acts together with growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF-β signaling inhibitors and nutritional supplements to induce cardiac precursor-like cells; the inhibitory factor and the The above-mentioned protein hormones are used to promote the proliferation of cardiac precursor-like cells under the combined action of growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF-β signaling inhibitors and nutritional supplements, and can make cardiac precursor-like cells Extensive amplification in vitro.

In some embodiments of the present invention, the reprogramming medium further includes growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF-β signaling inhibitors and nutritional supplements.

In some specific embodiments of the present invention, the growth factors include epithelial cell growth factor EGF and basic fibroblast growth factor bFGF. The epithelial cell growth factor EGF is from the American company Peprotech, and the basic fibroblast growth factor bFGF Originated from Peprotech Company of the United States, the ROCK kinase inhibitor Y-27632 is derived from Taoshu Biotechnology, the Wnt signaling pathway agonist CHIR99021 is derived from TargetMol, the TGF-β signaling inhibitor A830 is derived from TargetMol, and the nutrition Supplements include N2 nutritional supplements derived from Invitrogen and B27 nutritional supplements derived from Invitrogen.

In some embodiments of the present invention, the content of the MEK inhibitor is 1-101M, the content of the inhibitory factor is 5-20ng/mL, and the protein content is 1-101M based on the volume of the basal culture medium. The content of white matter hormones is 1-101g/ml. In some specific embodiments, the content of the MEK inhibitor is any one of 21M, 31M, 41M, 51M, 61M, 71M, 81M and 91M based on the volume of the basal culture medium, and the inhibitory factor is The content is any one of 6ng/mL, 8ng/mL, 10ng/mL, 12ng/mL, 14ng/mL, 16ng/mL, 18ng/mL and 19ng/mL, and the content of the protein hormone is 21g/ml, Any one of 31g/ml, 41g/ml, 51g/ml, 61g/ml, 71g/ml, 81g/ml and 91g/ml.

In some embodiments of the present invention, based on the volume of the basal culture medium, the content of the growth factor is 50-80ng/mL, the content of the ROCK kinase inhibitor is 10-501M, and the Wnt signaling pathway agonist The content of the TGF-β signal inhibitor is 1-101M, and the nutritional supplement is 1%-10%. In some specific embodiments, based on the volume of the basal culture medium, the content of the growth factor is any one of 55ng/mL, 60ng/mL, 65ng/mL, 70ng/mL and 75ng/mL, and the The content of the ROCK kinase inhibitor is any one of 151M, 201M, 251M, 301M, 351M, 401M and 451M, and the content of the Wnt signaling pathway agonist is 21M, 31M, 41M, 51M, 61M, 71M, 81M and Any one of 91M, the content of the TGF-β signal inhibitor is any one of 21M, 31M, 41M, 51M, 61M, 71M, 81M and 91M, the content of the nutritional supplement is 2%, Any of 3%, 4%, 5%, 6%, 7%, 8% and 9%.

In some embodiments of the present invention, the mature cardiomyocytes are differentiated from embryonic stem cells or induced pluripotent stem cells.

In some embodiments of the present invention, the mature cardiomyocytes are processed from myocardial tissue through digestion. Understandable.

The present invention also provides an application of a biological preparation containing myocardial precursor-like cells, using the biological preparation containing myocardial precursor-like cells to intervene in an in vivo animal model, and the animal model includes an animal model of drug-induced myocardial damage.

Specifically, since myocardial precursor-like cells do not express MHC class II molecules, there is no need to take anti-excretion drugs after transplantation of biological agents containing myocardial precursor-like cells, which can reduce harm to the human body. The myocardial precursor-like cells of the present invention include Biological agents based on somatic cells are safer in the treatment of heart diseases, have a wider range of applications, and are more acceptable to patients.

The abscissas and ordinates in Figure 6, Figure 7, Figure 8, Figure 10, Figure 11, Figure 12, Figure 13, Figure 14, Figure 15 and Figure 16 have the same meaning, where the abscissa in Figure 6 represents relative fluorescence. Intensity, the ordinate represents the number of cells.

Example 1: Isolation and dedifferentiation of mouse mature cardiomyocytes to obtain myocardial precursor-like cells, and characterize mature cardiomyocytes and myocardial precursor-like cells.

Figure 1 is a schematic photographic diagram of the morphology of primary cardiomyocytes in Example 1 of the present invention; Figure 2 is a schematic photographic diagram of the morphology of third-generation mature cardiomyocytes in Example 1 of the present invention; Figure 3 is a schematic diagram of the cultured cells obtained in Example 1 of the present invention. A schematic photographic diagram of the morphology of third-generation myocardial precursor-like cells; Figure 4 is a schematic photographic diagram of the morphology of third-generation control myocardial precursor-like cells cultured in Example 1 of the present invention; Figure 5 is a schematic diagram of myocardial precursor-like cells in Example 1 of the present invention. Growth curve of somatic cells in reprogramming medium and mature cardiomyocytes in cardiomyocyte medium.

Isolation of mature mouse cardiomyocytes:

Mouse hearts 1 day after birth were used.

Reagents:

Primary cell culture medium: Based on the volume of the basic culture medium DMEM/F12, add 10% fetal bovine serum (fetal bovine serum in English) to the basic culture medium DMEM/F12 (from Yuanpei, model: L310KJ) The name is: fetal calf serum (abbreviated in English as FBS, derived from Corning) and a 1% volume content of penicillin-streptomycin double antibody solution (derived from Yuanpei, concentration 100X) to obtain the primary cell culture medium.

The composition of the digestive juice includes: based on the volume of the digestive juice, a volume content of 25% trypsin (pancreatin comes from Yuanpei, model S330JV, concentration 0.25%), a volume content of 25% collagenase II (derived from Thermo Fisher, the concentration of collagenase II is 2mg/ml), and then prepare it with a 50% volume content of D-Hanks solution (from Wuhan Pronosai Life Technology Co., Ltd.) to obtain the digestive juice, which is heated below zero Store at 20 degrees Celsius, where the pH value of D-Hanks solution is 7.2-7.8.

Cardiomyocyte culture medium: mouse cardiomyocyte culture medium, from Procell Company; mouse cardiomyocyte complete culture medium, from Wuhan Procell Life Technology Co., Ltd.

Experimental steps:

Take out the suckling rat one day after birth, disinfect the skin of the suckling rat with 75% ethanol, fix the head and limbs of the suckling rat with pins, cut the chest skin, disinfect the subcutaneous tissue of the suckling rat with 75% ethanol, and replace the forceps. and scissors, open the chest and take out the heart of the suckling rat, put the heart of the suckling rat into a plate (or penicillin bottle) containing D-Hanks solution (from Wuhan Prosai Life Technology Co., Ltd.), cut off the atrium, and cut Open the heart chamber, flush it three times with D-Hanks solution, remove the residual blood, cut the heart into 1 cubic millimeter heart fragments, and then cut the heart into pieces. Transfer the fragments to the first centrifuge tube, add 5 ml of digestive fluid, digest at 37 degrees Celsius for 5 minutes, allow natural precipitation, discard the supernatant, add 5 ml of digestive fluid, and digest at 37 degrees Celsius for 20 minutes. During the digestion process Shake the first centrifuge tube several times every 2 minutes, pipette the first centrifuge tube for 1 minute to obtain the cell suspension; suck out the undigested heart fragments in the first centrifuge tube and move them to the second centrifuge tube. Add 2 ml of 4°C pre-cooled digestion solution to the second centrifuge tube to terminate digestion. Place the second centrifuge tube into a centrifuge and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and transfer it to the second centrifuge tube. Add 2 ml of D-Hanks solution to the precipitate, then put the second centrifuge tube into the centrifuge again, centrifuge at 1500 rpm for 10 minutes, discard the supernatant, and add culture to the precipitate in the second centrifuge tube. 2 ml of solution, use a pipette to pipette into the second centrifuge tube to obtain cell suspension; add 5 ml of digestive solution to the undigested heart fragments in the second centrifuge tube and continue digestion, repeat the above operation, and combine the fragments in each centrifuge tube After removing the cell suspension, put the cell suspension into a culture bottle, and put the culture bottle into a carbon dioxide incubator for culture. The temperature of the carbon dioxide incubator is 37 degrees Celsius, and the carbon dioxide content of the carbon dioxide incubator is maintained at 5%.

Culture results of the cell suspension: After culturing the cell suspension in a carbon dioxide incubator for 24 hours, pulsating single-layer mature cardiomyocytes can be seen.

Mature cardiomyocytes degenerate into cardiac precursor-like cells in mice:

Reagents:

The composition of the reprogramming medium is as follows: basal medium DMEM/F-12 (from Wuhan Pronosai Life Technology Co., Ltd.), based on the volume of the basal medium DMEM/F-12, the content is 20 ng/ml Epithelial cell growth factor EGF, 50 ng/ml Basic Fibroblast Growth Factor bFGF, 1% N2 Nutritional Supplement (1X), 1% B27 Nutritional Supplement (1X), 10uM ROCK Kinase Inhibitor Y-27632, 3uM Wnt signaling pathway agonist CHIR99021, TGF-β signaling inhibitor A830 at 1 uM, MEK inhibitor PD0325901 at 11 M, leukemia inhibitory factor LIF at 10 ng/ml, insulin at 1 μg/ml ;

The composition of the control medium is as follows: basal medium DMEM/F-12 (from Wuhan Pronosai Life Technology Co., Ltd.), based on the volume of basal medium DMEM/F-12, containing 20 ng/ml of epithelium Cell Growth Factor EGF, Basic Fibroblast Growth Factor bFGF at 50 ng/ml, N2 Nutritional Supplement at 1% (1X), B27 Nutritional Supplement at 1% (1X) at 10uM ROCK kinase inhibitor Y-27632, 3uM Wnt signaling pathway agonist CHIR99021, 1uM TGF-β signaling inhibitor A830;

Cardiomyocyte culture medium: mouse cardiomyocyte culture medium, from Procell;

The composition of cardiomyocyte differentiation medium is as follows: basal medium α-MEM (from Shanghai Huiying Biotechnology Co., Ltd.), based on the volume of basal medium α-MEM, containing 11M dexamethasone and containing 50 nanometers g/ml vitamin C, 10mM β-glycerophosphate sodium, 3% fetal bovine serum FBS, 5ng/mL TGF-β1, 10ng/mL VEGF-A.

Experimental steps:

Primary cardiomyocytes: The cell suspension obtained by digesting myocardial tissue is seeded in a 6-well plate. Add 2 ml of DMEM/F-12+10% FBS culture medium to each well, place it in a CO ₂ incubator and culture it at 37 degrees Celsius to obtain primary cardiomyocytes, recorded as P0.

Passage myocardial precursor-like cells: When the confluence of the above-mentioned primary cardiomyocytes reaches 80%, use trypsin (sourced from Yuanbai) to digest a single layer of mature cardiomyocytes, and seed the mature cardiomyocytes at a seeding density of 20,000 cells/cm2. In a 10 cm culture dish, add 10 ml of reprogramming medium to each dish for dedifferentiation culture to obtain the first generation of myocardial precursor-like cells (marked as P1). During the expansion and culture process, the reprogramming medium is replaced every 2-3 days. When the cell confluence reaches 80%, the cells are passaged. The specific operations of passage are: digest mature cardiomyocytes with trypsin (derived from source culture). Mature cardiomyocytes were seeded into a 10 cm culture dish at a seeding density of 20,000 cells/cm², and 10 ml of reprogramming medium was added to each dish for dedifferentiation culture, and the second generation of myocardial precursor-like cells (marked as P2) were cultured. Cyclic amplification and culture process until the fifteenth generation of myocardial precursor-like cells (recorded as P15) are cultured, take pictures, count, and record the number of passages every 3 days, and draw a growth curve based on the recorded data, see Figure 5. A schematic diagram of the cell morphology of the third generation myocardial precursor-like cells is shown in Figure 3.

Passage control myocardial precursor-like cells: When the confluence of the above primary cardiomyocytes reaches 80%, digest a single layer of mature cardiomyocytes with trypsin (sourced from Yuanbai), and seed the mature cardiomyocytes at a seeding density of 20,000 cells/cm2. In a 10 cm culture dish, add 10 ml of control medium to each culture dish for dedifferentiation culture to obtain the first generation of control myocardial precursor-like cells (marked as P1). The first generation of control myocardial precursor-like cells were During the expansion and culture of the control medium, replace the control medium every 2-3 days. When the confluence of the cells reaches 80%, the cells are passaged. The specific operation of passage is: use trypsin (from source culture) Digest mature cardiomyocytes and inoculate mature cardiomyocytes into 10 cm culture dishes at a seeding density of 20,000 cells/cm². Add 10 ml of control medium to each culture dish for dedifferentiation culture. Before culturing the second generation of control myocardium, Somatic-like cells (denoted as P2) are cyclically expanded and cultured until they are cultured into third-generation control myocardial precursor-like cells (denoted as P3). A schematic diagram of the cell morphology of the third-generation control myocardial precursor-like cells is shown in Figure 4 .

Referring to Figures 3 and 4, myocardial precursor-like cells were cultured in a control medium. When the third generation of control myocardial precursor-like cells were cultured, the state of the third generation control myocardial precursor-like cells deteriorated and spread out like poached eggs. The diameter becomes larger and cannot proliferate.

Passage mature cardiomyocytes: When the confluence of the above primary cardiomyocytes reaches 80%, use trypsin (source culture) to digest a single layer of mature cardiomyocytes, and inoculate mature cardiomyocytes into a 10 cm culture medium at a seeding density of 20,000 cells/cm2. In the dish, add 10 ml of cardiomyocyte culture medium to each culture dish and culture until the confluence of mature cardiomyocytes is not less than 80% and the growth status is good. Complete the expansion culture to obtain the first generation of mature cardiomyocytes (recorded as P1). During the expansion and culture process, the cardiomyocyte culture medium is replaced every 2-3 days, and the expansion and culture process is cycled, and the second generation of mature cardiomyocytes (denoted as P2) are cultured until they are cultured into the fifth generation of mature cardiomyocytes ( Marked as P5), take pictures, count, and record the number of passages every 3 days, and draw a growth curve based on the recorded data, see Figure 5.

Referring to Figure 5, almost all of the third-generation mature cardiomyocytes obtained from mature cardiomyocytes in the cardiomyocyte culture medium died, while the third-generation myocardial precursor-like cells obtained from the reprogramming medium provided by the present invention can Stable proliferation. Compared with using cardiomyocyte culture medium to culture mature cardiomyocytes, the reprogramming medium of the present invention can stably culture myocardial precursor-like cells to the fifteenth generation, and the curve of the total cell number has been on an upward trend. It shows that the reprogramming culture medium provided by the present invention can make myocardial precursor-like cells continue to proliferate in vitro, and the total cell proliferation number increases exponentially.

Figure 6 is a schematic diagram of the flow cytometric detection results of the third generation myocardial precursor-like cells that positively express c-Kit in Example 1 of the present invention; Figure 7 is a schematic diagram of the third generation myocardial precursor-like cells that positively express CD24 in Example 1 of the present invention. Schematic diagram of flow cytometry detection results; Figure 8 is a schematic diagram of flow cytometry detection results of third-generation myocardial precursor-like cells negatively expressing HLA-DRPQ in Example 1 of the present invention; Figure 9 is a third-generation myocardial precursor in Example 1 of the present invention A schematic photograph of the cell morphology after 7 days of differentiation of the progenitor-like cells; Figure 10 is a schematic diagram of the flow cytometric detection results of positive expression of cTnT in the third-generation myocardial precursor-like cells of Embodiment 1 of the present invention after 7 days of differentiation; Figure 11 is a schematic diagram of the flow cytometry results of the third-generation cardiac precursor-like cells in Embodiment 1 of the present invention. Schematic diagram of flow cytometric detection results of third-generation cardiac progenitor-like cells that positively express α-Sarcometric actin after 7 days of differentiation.

1.0×10 ⁶ of the third generation (P3) myocardial precursor-like cells and the third generation control cardiomyocytes were taken for flow cytometry detection. The specific steps of flow cytometry detection include the following steps:

Cell surface staining:

For P3 myocardial precursor-like cells, aspirate the reprogramming medium, rinse with 5 ml of sterile PBS buffer, and then add 2 ml of trypsin digestion solution to the culture dish for digestion to obtain a cell mixture, and place the cell mixture Put the centrifuge tube into a centrifuge in a 15 ml centrifuge tube, and perform centrifugation at a speed of 200 g for 5 minutes. Discard the supernatant of the centrifuged cell mixture to obtain a cell pellet.

Add 220 μl of staining buffer to the cell pellet to resuspend the cell pellet. Transfer the resuspended cell pellet to two 1.5 ml centrifuge tubes. 100 μl/tube. Then add 5 μl of the flow cytometry antibody to be tested and mix by pipetting. Place the centrifuge tubes in the refrigerator at 2-8 degrees Celsius for 30 minutes. Add sterile PBS buffer to each centrifuge tube at a dose of 800 μl/tube, and then place the centrifuge tubes into the centrifuge. Centrifuge at 300 g for 5 minutes. After centrifugation, discard the supernatant, add 400 μl of staining buffer to each centrifuge tube to resuspend the cell pellet, then transfer it to a flow tube, and flow the resuspended cell mixture for surface markers. type detection.

The names of antibodies used in surface marker flow cytometric detection are: CD24, HLV-DRPQ.

CD24 was purchased from abcam, catalog number ab290730; HLV-DRPQ was purchased from abcam, catalog number ab7856; staining buffer was purchased from BD Biosciences, catalog number 554656.

Intracellular staining of cells:

Add 1 ml of fixed membrane solution to the cell pellet, place the centrifuge tube in a refrigerator at 2-8 degrees Celsius for 50 minutes, add 2 ml of PBS buffer to the centrifuge tube, and rotate the centrifuge tube at 300g. Centrifuge for 5 minutes. After centrifugation, add 100 μl of staining buffer to the centrifuge tube and resuspend. Add 5 microliters of the flow cytometry antibody to be tested into the above 1.5 ml centrifuge tube, mix by pipetting, and place the centrifuge tube in an incubator containing 5% carbon dioxide by volume at 37 degrees Celsius and let stand for 30 minutes. After incubation is completed, press Add sterile PBS buffer to each centrifuge tube at a dose of 800 μl/tube, then place the centrifuge tube into a centrifuge and centrifuge at a speed of 300g for 5 minutes. After centrifugation, discard the supernatant, add 400 μl of staining buffer to each centrifuge tube to resuspend the cell pellet, and then transfer it to a flow tube. The resuspended cell mixture is analyzed for intracellular markers. Flow detection.

The name of the antibody used for flow cytometric detection of intracellular markers is c-Kit. c-Kit was purchased from abcam, Cat. No. ab283653; staining buffer was purchased from BD Biosciences, Cat. No. 554656.

The results of flow cytometry are shown in Figure 6, Figure 7 and Figure 8.

Referring to Figure 6, Figure 7 and Figure 8, the third generation myocardial precursor-like cells positively expressed CD24 and c-Kit and negatively expressed HLA-DRPQ.

Take the third generation (P3) myocardial precursor-like cells and differentiate them using cardiomyocyte differentiation medium for 7 days to obtain mature cardiomyocytes. After 7 days of differentiation, the third generation myocardial precursor-like cells are The photo schematic diagram of cell morphology is shown in Figure 9. Fresh cardiomyocyte differentiation medium was replaced every 3 days, and flow cytometry was performed on the obtained mature cardiomyocytes. The test results are shown in Figures 10 and 11.

Referring to Figures 10 and 11, mature cardiomyocytes were obtained after 7 days of differentiation of the third-generation cardiomyocyte precursor-like cells. The obtained mature cardiomyocytes can positively express the markers cTnT and α-Sarcometric actin, proving that the cells are mature cardiomyocytes. cTnT was purchased from abcam, product number ab209813; α-Sarcometric actin was purchased from Biyuntian, product number AG0101.

Example 2 iPSCs are differentiated to obtain mature cardiomyocytes, which are then dedifferentiated using reprogramming medium. to obtain myocardial precursor-like cells:

Figure 12 is a schematic diagram of the flow cytometric detection results of mature cardiomyocytes that positively express cTnT in Example 2 of the present invention; Figure 13 is a schematic diagram of the flow cytometric detection results of myocardial precursor-like cells that positively express c-kit in Example 2 of the present invention; Figure 14 This is a schematic diagram of the flow cytometric detection results of cardiac precursor-like cells negatively expressing HLA-DRPQ in Example 2 of the present invention.

Reagents:

iPSC culture reagent: mTeSR1 culture medium, from Stemcell Tech;

Reagents for iPSC differentiation into mature myocardium: Myocardial differentiation reagents, from Stemcell Tech.

Experimental steps:

Put iPSCs into mTeSR1 culture medium for culture to obtain induced pluripotent stem cells. The specific operation process is carried out according to the instructions of mTeSR1 culture medium; put iPSCs into myocardial differentiation reagent for differentiation and culture to obtain mature cardiomyocytes. The specific instructions for differentiation and culture are The steps were carried out according to the instructions of the myocardial differentiation reagent. Special notes: On the 8th day of iPSC differentiation, change to maintenance medium (from Stemcell Tech) and culture for more than 15 days to obtain mature cardiomyocytes. The sign of mature cardiomyocytes is that the cardiomyocytes stop proliferating and beat independently in the culture dish. Flow cytometry was performed on mature cardiomyocytes. The specific steps of flow cytometry include:

Intracellular staining of cells:

For iPSC-differentiated mature cardiomyocytes, aspirate the reprogramming medium, rinse with 5 ml of sterile PBS buffer, and then add 2 ml of trypsin digestion solution to the culture dish for sterilization. The cell mixture was obtained by treatment. Place the cell mixture in a 15 ml centrifuge tube. Put the centrifuge tube into a centrifuge and perform centrifugation at a speed of 200g for 5 minutes. Discard the supernatant of the centrifuged cell mixture. liquid to obtain cell pellets.

Add 1 ml of fixed membrane solution to the cell pellet, place the centrifuge tube in a refrigerator at 2-8 degrees Celsius for 50 minutes, add 2 ml of PBS buffer to the centrifuge tube, and rotate the centrifuge tube at 300g. Centrifuge for 5 minutes. After centrifugation, add 100 μl of staining buffer to the centrifuge tube and resuspend. Add 5 microliters of the flow cytometry antibody to be tested into the above 1.5 ml centrifuge tube, mix by pipetting, and place the centrifuge tube in an incubator containing 5% carbon dioxide by volume at 37°C and let stand for 30 minutes. After the incubation, add sterile PBS buffer to each centrifuge tube at a dose of 800 μl/tube, then place the centrifuge tube into a centrifuge and centrifuge at a speed of 300g for 5 minutes. After centrifugation, discard the supernatant, add 400 μl of staining buffer to each centrifuge tube to resuspend the cell pellet, and then transfer it to a flow tube. The resuspended cell mixture is analyzed for intracellular markers. Flow detection.

The name of the antibody used for flow cytometric detection of intracellular markers is cTnT. cTnT was purchased from abcam, product number ab209813. The flow cytometry results are shown in Figure 12.

Referring to Figure 12, iPSCs were differentiated for 15 days to obtain mature cardiomyocytes. The flow cytometric detection of mature cardiomyocytes showed that mature cardiomyocytes positively expressed the cTnT gene.

The obtained mature cardiomyocytes are cultured in reprogramming medium to obtain myocardial precursor-like cells, and the obtained myocardial precursor-like cells are subjected to flow cytometry. The specific steps of flow cytometry include:

Cell surface staining:

For myocardial precursor-like cells, aspirate the reprogramming medium, rinse with 5 ml of sterile PBS buffer, and then add 2 ml of trypsin digestion solution to the culture dish for digestion to obtain a cell mixture. Place the cell mixture in Put the centrifuge tube into a 15 ml centrifuge tube and centrifuge it at a speed of 200 g for 5 minutes. Discard the supernatant of the centrifuged cell mixture to obtain the cell pellet.

Add 100 μl of staining buffer to the cell pellet to resuspend the cell pellet, add 5 μl of the flow cytometry antibody to be tested, and pipet to mix. Place the centrifuge tubes in the refrigerator at 2-8 degrees Celsius for 30 minutes. Add sterile PBS buffer to each centrifuge tube at a dose of 800 μl/tube, and then place the centrifuge tubes into the centrifuge. Centrifuge at 300 g for 5 minutes. After centrifugation, discard the supernatant, add 400 μl of staining buffer to each centrifuge tube to resuspend the cell pellet, then transfer it to a flow tube, and flow the resuspended cell mixture for surface markers. type detection.

The name of the antibody used for flow cytometric detection of surface markers is: HLA-DRPQ. HLA-DRPQ was purchased from abcam with the product number ab7856; the staining buffer was purchased from BD Biosciences with the product number 554656.

Intracellular staining of cells:

For myocardial precursor-like cells, aspirate the reprogramming medium, rinse with 5 ml of sterile PBS buffer, and then add 2 ml of trypsin digestion solution to the culture dish for digestion to obtain a cell mixture. Place the cell mixture in 15 ml centrifuge tube, put the centrifuge tube into a centrifuge, and centrifuge at a speed of 200g for 5 minutes, discard the centrifuge The cell pellet was obtained from the supernatant of the treated cell mixture.

Add 1 ml of fixed membrane solution to the cell pellet, place the centrifuge tube in a refrigerator at 2-8 degrees Celsius for 50 minutes, add 2 ml of PBS buffer to the centrifuge tube, and rotate the centrifuge tube at 300g. Centrifuge for 5 minutes. After centrifugation, add 100 μl of staining buffer to the centrifuge tube and resuspend. Add 5 μL of the flow cytometry antibody to be tested into the above 1.5 ml centrifuge tube, mix by pipetting, and place the centrifuge tube in an incubator containing 5% carbon dioxide by volume at 37 degrees Celsius and let stand for 30 minutes. After the incubation, add sterile PBS buffer to each centrifuge tube at a dose of 800 μl/tube, then place the centrifuge tube into a centrifuge and centrifuge at a speed of 300g for 5 minutes. After centrifugation, discard the supernatant, add 400 μl of staining buffer to each centrifuge tube to resuspend the cell pellet, and then transfer it to a flow tube. The resuspended cell mixture is analyzed for intracellular markers. Flow detection.

The name of the antibody used for flow cytometric detection of intracellular markers is c-Kit. c-Kit was purchased from abcam, product number ab283653; the staining buffer was purchased from BD Biosciences, product number 554656.

The test results are shown in Figure 13 and Figure 14. The results show that the obtained myocardial precursor-like cells positively express c-kit and negatively express HLA-DRPQ.

Example 3, Obtaining cardiac precursor-like cells derived from iPSC differentiation:

Figure 15 is a schematic diagram of the flow cytometric detection results of myocardial precursor-like cells that positively express c-kit in Example 3 of the present invention; Figure 16 is a flow cytometric detection result of myocardial precursor-like cells that negatively express HLA-DRPQ in Example 3 of the present invention. Schematic diagram.

Reagents:

iPSC culture reagent: mTeSR1 culture medium, from Stemcell Tech;

Reagents for iPSC differentiation into mature myocardium: Myocardial differentiation reagents were purchased from Stemcell Tech.

Experimental steps:

Place iPSCs in mTeSR1 culture medium for culture to obtain induced pluripotent stem cells. The specific operation process is carried out according to the instructions of mTeSR1 culture medium; put iPSCs into myocardial differentiation reagent for differentiation and culture to obtain myocardial precursor-like cells. Differentiation culture The specific steps are carried out according to the instructions of the myocardial differentiation reagent. Note: On the 8th day of differentiation, change to maintenance medium (from Stemcell Tech), which is myocardial precursor-like cells. The sign of myocardial precursor-like cells is: myocardial precursor-like cells can proliferate in a culture dish. Involuntary beating. The obtained myocardial precursor-like cells were subjected to flow cytometric detection, and the detection results are shown in Figures 15 and 16.

Referring to Figures 15 and 16, iPSC-differentiated cardiac precursor-like cells positively expressed c-kit gene and HLA-DRPQ gene.

The technical core of the present invention lies in:

1. Obtaining myocardial precursor-like cells: they can be expanded on a large scale in vitro.

Mature cardiomyocytes are isolated and cultured in reprogramming medium to obtain cardiomyocyte precursor-like cells.

iPSCs are differentiated into mature cardiomyocytes, and reprogramming medium is used to dedifferentiate and culture them to obtain myocardial precursor-like cells.

2. Application of the myocardial precursor-like cells obtained by the present invention: they do not express MHC class II molecules HLA-DR/DP/DQ that are related to immune rejection. Myocardial precursor-like cells can transdifferentiate into new mature cardiomyocytes, promote myocardial regeneration, and secrete pro-angiogenic factors to recruit capillaries and create new intracardiac blood vessels. Because the MHC class II molecule HLA-DR/DP/DQ, which is associated with immune rejection, is not expressed, patients do not need to take anti-rejection drugs after transplantation of cardiac precursor-like cells. The myocardial precursor-like cells obtained by the present invention are safer for treating heart diseases, have a wider application range, and are more acceptable to patients.

Although the embodiments of the present invention have been described in detail above, it will be obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are within the scope and spirit of the invention as described in the claims. Furthermore, the invention described herein is capable of other embodiments and of being practiced or carried out in various ways.

Claims

A biological preparation containing myocardial precursor-like cells, characterized in that the myocardial precursor-like cells positively express CD24 and c-Kit, and the myocardial precursor-like cells do not express MHC class II molecules.
A method for preparing a biological preparation containing myocardial precursor-like cells according to claim 1, characterized in that it includes the following steps:

S0: Provide cardiac precursor-like cells;

S1: Mix the myocardial precursor-like cells with a pharmaceutically acceptable carrier to obtain the biological preparation containing the myocardial precursor-like cells, which positively express CD24 and c-Kit, The cardiac precursor-like cells do not express MHC class II molecules.
The method for preparing biological agents containing myocardial precursor-like cells according to claim 2, wherein the method for preparing myocardial precursor-like cells includes the following steps:

S00: Provide mature cardiomyocytes;

S01: Put the mature cardiomyocytes into a reprogramming medium for dedifferentiation culture until the confluence of the myocardial precursor-like cells is not less than 80%, and use trypsin digestion solution to treat the myocardial precursor-like cells. Digestion treatment is performed to obtain myocardial precursor-like cells, wherein the reprogramming medium includes basal medium, MEK inhibitors, inhibitory factors and protein hormones, and the reprogramming medium is used for the myocardial precursor-like cells. induction and promote the proliferation of the myocardial precursor-like cells.
The preparation method of biological preparations containing myocardial precursor-like cells according to claim 3, characterized in that the preparation method of myocardial precursor-like cells includes the following steps:

S02: Put the myocardial precursor-like cells into the reprogramming medium for expansion and culture until the fusion degree of the myocardial precursor-like cells is not less than 80%, use the trypsin digestion solution to The myocardial precursor-like cells are digested to obtain passaged myocardial precursor-like cells.
The preparation method of biological preparations containing cardiac precursor-like cells according to claim 4, characterized in that the reprogramming medium further includes growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF-β signaling inhibitors and nutritional supplements.
The method for preparing a biological preparation containing myocardial precursor-like cells according to claim 5, wherein the content of the MEK inhibitor is 1-101 M based on the volume of the basal culture medium, and the inhibitory The content of the factor is 5-20ng/mL, and the content of the protein hormone is 1-101g/ml.
The method for preparing a biological preparation containing myocardial precursor-like cells according to claim 6, wherein the content of the growth factor is 50-80 ng/mL based on the volume of the basal culture medium, and the The content of the ROCK kinase inhibitor is 10-501M, the content of the Wnt signaling pathway agonist is 1-101M, the content of the TGF-β signaling inhibitor is 1-101M, and the content of the nutritional supplement is 1% -10%.
The method for preparing a biological preparation containing cardiac precursor-like cells according to claim 3, wherein the mature cardiomyocytes are differentiated from embryonic stem cells or induced pluripotent stem cells.
The method for preparing a biological preparation containing myocardial precursor-like cells according to claim 3, wherein the mature cardiomyocytes are obtained from myocardial tissue through digestion. get.
An application of a biological preparation containing myocardial precursor-like cells, characterized in that the biological preparation containing myocardial precursor-like cells as claimed in claim 1 is used to intervene in an in vivo animal model, the animal model includes drug-induced myocardial injury. Sexual animal models.