A method for improving the yield and purity of the directed induction of cardiomyocytes from pluripotent stem cells

Abstract

The present application provides a method for improving the yield and purity of cardiomyocytes derived from pluripotent stem cells, which comprises one or more of the following steps: EB formation culture; cardiac progenitor cell induction and differentiation culture; cardiomyocyte induction and differentiation culture; cardiomyocyte maturation culture; and cardiomyocyte purification culture. In another aspect, the present application also provides a culture medium, a medium combination, a cell culture system, and a kit for improving the yield and purity of cardiomyocytes derived from pluripotent stem cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for improving the yield and purity of cardiomyocytes induced from pluripotent stem cells. BACKGROUND

[0002] Cardiovascular diseases remain the leading cause of death worldwide. For humans and mammals, cardiomyocytes have the ability to proliferate before birth, but this ability decreases rapidly after birth. Adult cardiomyocytes have little ability to proliferate. When a heart tissue necrotic disease such as myocardial infarction occurs, the adult cardiomyocytes have lost the ability to proliferate, and the necrotic tissue cannot be repaired by cardiomyocyte regeneration, so the heart function decline caused by such diseases is irreversible. Although the use of drugs can increase myocardial contractility and improve the pumping capacity of the heart, the increased burden on the heart may worsen the condition.

[0003] Transplanting normal cardiomyocytes to replace the dead cells is one of the ways to fundamentally treat such heart diseases. Cell transplantation therapy aimed at repairing injured heart tissue stem cells can greatly improve the current status of heart treatment. Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the ability to proliferate indefinitely in vitro and to generate cells that form the heart, opening up a new era of cardiovascular research, drug discovery, toxicology testing, and myocardial therapy. Obtaining a large number of highly purified hPSC-derived cardiomyocytes is essential for basic heart research and clinical translation applications. The differentiation of hPSCs into cardiovascular cells, especially into cardiomyocytes (CMS), smooth muscle cells (SMCs), and endothelial cells (ECs), has made significant progress.

[0004] The various methods reported for differentiating cardiomyocytes from stem cells mainly have the following defects: the efficiency of inducing cardiomyocyte differentiation is not high, and the obtained cardiomyocytes are a mixed cell population of pacemaker cells, atrial cardiomyocytes, and ventricular cardiomyocytes, and accurate directional differentiation cannot be achieved.

[0005] There are differences between different hPSCs in the yield and purity of tissue cells produced by induced pluripotent stem cells. The tumorigenic potential of residual undifferentiated cells makes the safety of hPSC-derived cardiovascular cells more serious. In order to solve these problems, we have developed a high-yield, high-efficiency, and high-purity cardiomyocyte induction method by adjusting the cell morphology before induction, key developmental signaling pathways, and respiratory patterns after induction.

[0006] Experiments prove that the method provided by the application can induce pluripotent stem cells to differentiate into myocardial cells, and the culture medium used in the method can not only make pluripotent stem cells quickly and efficiently differentiate into myocardial cells, but also can produce myocardial cells on a large scale, and the quality is stable and the safety is high, which provides a large number of cell sources for tissue engineering, drug research and development and cell therapy. The application has great application value. SUMMARY

[0007] The application provides a method for improving the yield and purity of pluripotent stem cells directed to induce myocardial cells, a culture medium, a culture medium combination, a cell culture system and a kit.

[0008] Culture medium

[0009] EB formation medium

[0010] In one aspect, the application provides an EB formation medium.

[0011] Preferably, the EB formation medium is an IPS base medium added with an organic compound PVA and / or an organic compound MC.

[0012] Preferably, the IPS base medium is selected from one or more of the following: TeSR-E8, mTESR1, E8, Essential 8 TMMedium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, a-Minimal Essential Medium (a-MEM), G- Minimal Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), AmnioMax, Amino Max II complete Medium (Gibco, Newyork, USA), Chang's Medium, MesemCult-XF Medium (STEMCELL Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM / F12, Ham's F-12 K Medium, HepatoZYME-SFM, William's E Medium, Waymouth's Medium, or Hepatocyte Culture Medium;

[0013] Preferably, the IPS basal medium comprises one or more of TeSR-E8, mTESR1 or E8.

[0014] Preferably, the IPS basal medium comprises TeSR-E8.

[0015] Most preferably, the IPS basal medium is TeSR-E8.

[0016] Preferably, the concentration of the organic compound PVA is 1-8 mg / ml; preferably, the concentration of the organic compound PVA is 2-5 mg / ml, specifically 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml; most preferably, the concentration of the organic compound PVA is 4 mg / ml.

[0017] Preferably, the concentration of the organic compound MC is 0.1%-1%; preferably, the concentration is 0.2%-0.8%; preferably, the concentration is 0.3%-0.5%.

[0018] In one embodiment, the IPS basal medium is a commercialized medium; preferably, the TeSR-E8, mTESR1 or E8 mentioned above is a commercialized medium of Stemcell company.

[0019] Cardiac progenitor cell induction and differentiation medium

[0020] In another aspect, the present application provides a cardiac progenitor cell induction and differentiation medium.

[0021] Preferably, the cardiac progenitor cell induction and differentiation medium is formulated by adding cytokines and / or GSK-3 inhibitors into a cardiac progenitor cell induction and differentiation basal medium.

[0022] Preferably, the cytokines include TGF-β, activin, bone morphogenetic protein (BMP), growth differentiation factor (GDF); preferably, the cytokines include bone morphogenetic protein; preferably, the cytokines include bone morphogenetic protein 4 (BMP4); preferably, the cytokines are bone morphogenetic protein 4 (BMP4).

[0023] Preferably, the concentration of BMP4 is 10-40 ng / ml; preferably, the concentration of BMP4 is 20-30 ng / ml, specifically, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml, 25 ng / ml, 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, 30 ng / ml; preferably, the concentration of BMP4 is 25 ng / ml.

[0024] Preferably, the GSK-3 inhibitor is GSK-3 inhibitor CHIR99021; preferably, the concentration of CHIR99021 can be 1 μM-8 μM; preferably, 3 μM-5 μM; specifically, 3 μM, 4 μM, 5 μM.

[0025] In one embodiment, the main component in the cardiac progenitor cell induction and differentiation basal medium is a first cell culture medium and / or glutamine.

[0026] Preferably, the first cell culture medium is selected from one or more of the following: TeSR-E8, mTESR1, E8, Essential 8 TMMedium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, a-Minimal Essential Medium (a-MEM), G- Minimal Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), AmnioMax, Amino Max II complete Medium (Gibco, Newyork, USA), Chang's Medium, MesemCult-XF Medium (STEMCELL Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM / F12, Ham's F-12 K Medium, HepatoZYME-SFM, William's E Medium, Waymouth's Medium, or Hepatocyte Culture Medium.

[0027] Preferably, the first cell culture medium is DMEM / F-12 medium.

[0028] Preferably, the glutamine is a substitute thereof; preferably, the substitute is GlutaMAX TM Supplement.

[0029] In one embodiment, the cardiac progenitor cell induction differentiation basal medium further comprises one or more of thio-glycerol, L-ascorbic acid, catalase, reduced glutathione, human insulin, superoxide dismutase, transferrin, T3, L-carnitine, ethanolamine, galactose, putrescine, sodium selenite, corticosterone, linoleic acid, linolenic acid, progesterone, DL-a tocopherol, DL-a-tocopherol acetate, oleic acid, pipemidic acid, and biotin.

[0030] Preferably, the cardiomyocyte induction and differentiation medium further comprises thio-glycerol, L-ascorbic acid, catalase, reduced glutathione, human insulin, superoxide dismutase, transferrin, T3, L-carnitine, ethanolamine, galactose, putrescine, sodium selenite, corticosterone, linoleic acid, linolenic acid, progesterone, DL-a tocopherol, DL-a-tocopherol acetate, oleic acid, pipemidic acid and biotin in the cardiomyocyte induction and differentiation basal medium.

[0031] Cardiomyocyte induction and differentiation medium

[0032] In another aspect, the present application provides a cardiomyocyte induction and differentiation medium; preferably, the cardiomyocyte induction and differentiation medium is a cardiomyocyte induction and differentiation basal medium supplemented with one or more of a cytokine, a Wnt pathway inhibitor and an additive.

[0033] Preferably, the cytokine comprises TGF-β, activin, bone morphogenetic protein (BMP), growth differentiation factor (GDF); preferably, the cytokine comprises bone morphogenetic protein; preferably, the cytokine comprises bone morphogenetic protein 4 (BMP4); preferably, the cytokine is bone morphogenetic protein 4 (BMP4).

[0034] Preferably, the Wnt pathway inhibitor comprises one or more of IWR-1, IWP-2, Pyrivinium pamoate, Salinomycin, Salinomycin sodium salt, FH535, Wogonin, PNU-74654, Echinacoside, Prinaberel, IWP-4, SKI II, Triptonide, Gigantol, Hematein, Specnuezhenide, Prodigiosin, Ginkgetin, XAV939, KY02111 and C59.

[0035] Preferably, the Wnt pathway inhibitor comprises C59 and / or IWR-1.

[0036] Preferably, the Wnt pathway inhibitor is C59 and / or IWR-1

[0037] More preferably, the Wnt pathway inhibitor is C59.

[0038] Preferably, the concentration of BMP4 is 0-20 ng / ml; preferably, the concentration of BMP4 is 5-15 ng / ml, in particular 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml; preferably, the concentration of BMP4 is 10 ng / ml.

[0039] In one embodiment, the concentration of C59 is 0.1-10 μM; preferably, the concentration of C59 is 0.5-8 μM; preferably, the concentration of C59 is 1-5 μM; in particular including 1 μM, 2 μM, 3 μM, 4 μM, 5 μM; preferably, the concentration of C59 is 1.5-2.5 μM; in particular including 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM; preferably, the concentration of C59 is 2 μM.

[0040] In one embodiment, the concentration of IWR-1 is 0.1-10 μM; preferably, the concentration of IWR-1 is 0.5-8 μM; preferably, the concentration of IWR-1 is 3-8 μM; in particular including 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM; preferably, the concentration of IWR-1 is 4.5-5.5 μM; in particular including 4.6 μM, 4.7 μM, 4.8 μM, 4.9 μM, 5M, 5.1 μM, 5.2 μM, 5.3 μM, 5.4 μM, 5.5 μM; preferably, the concentration of IWR-1 is 5 μM.

[0041] Preferably, the additive is one or more of retinol, catalase, reduced glutathione, superoxide dismutase, transferrin, T3, L-carnitine, ethanolamine, galactose, putrescine, sodium selenite, corticosterone, linoleic acid, linolenic acid, progesterone, DL-a tocopherol, DL-a-tocopherol acetate, oleic acid, pipemidic acid and biotin.

[0042] In one embodiment, the main component of the cardiomyocyte induction and differentiation basal medium is a second cell culture medium and / or glutamine.

[0043] Preferably, the cardiomyocyte induction and differentiation basal medium further comprises penicilin, streptomycin, dactinomycin, ampicillin, carbenicillin, streptomycin sulfate, polymyxin B sulfate, polymyxin B sulfate, neomycin sulfate, kanamycin sulfate, gentamicin sulfate.

[0044] Preferably, the cardiomyocyte induction and differentiation basal medium comprises two antibiotics.

[0045] Preferably, the cardiomyocyte induction and differentiation basal medium comprises Penicilin and streptomycin.

[0046] Preferably, the second cell culture medium is selected from one or more of: TeSR-E8, mTESR1, E8, Essential 8 TM Medium, Dulbecco’s Modified Eagle’s Medium, Minimal Essential Medium, Basal Medium Eagle, F-10, F-12, α-Minimal Essential Medium, Glasgow’s Minimal Essential Medium, Iscove’s Modified Dulbecco’s Medium, AmnioMax, Amino Max II complete Medium, Gibco, Newyork, USA, Chang’s Medium, MesemCult-XF Medium, STEMCELL Technologies, Vancouver, Canada, RPMI 1640, Ham’s F12, DMEM / F12, Ham's F-12 K Medium, HepatoZYME-SFM, William’s E Medium, Waymouth’s Medium, or Hepatocyte Culture Medium.

[0047] Preferably, the cell culture medium is RPMI-1640.

[0048] Preferably, the glutamine is a substitute thereof; preferably, the substitute is GlutaMAX TM Supplement.

[0049] Cardiomyocyte maturation medium

[0050] In another aspect, the present application provides a cardiomyocyte maturation medium.

[0051] Preferably, the cardiomyocyte maturation medium is identical to the cardiomyocyte induction-differentiation basal medium composition.

[0052] In one embodiment, the cardiomyocyte maturation medium further comprises one or more of the following added to the cardiomyocyte induction-differentiation basal medium: retinol, catalase, reduced glutathione, superoxide dismutase, transferrin, T3, L-carnitine, ethanolamine, galactose, putrescine, sodium selenite, corticosterone, linoleic acid, linolenic acid, progesterone, DL-alpha tocopherol, DL-alpha-tocopheryl acetate, oleic acid, pipemidic acid, biotin, and human insulin.

[0053] Cardiomyocyte purification medium

[0054] In another aspect, the present application provides a cardiomyocyte purification medium.

[0055] Preferably, the cardiomyocyte purification medium is L-lactic acid and / or gentamicin sulfate added to a third cell culture medium.

[0056] Preferably, the third cell culture medium is selected from one or more of the following: TeSR-E8, mTESR1, E8, Essential 8 TMMedium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, a-Minimal Essential Medium (a-MEM), G- Minimal Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), AmnioMax, Amino Max II complete Medium (Gibco, Newyork, USA), Chang's Medium, MesemCult-XF Medium (STEMCELL Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM / F12, Ham's F-12 K Medium, HepatoZYME-SFM, William's E Medium, Waymouth's Medium, or Hepatocyte Culture Medium; preferably, the third cell culture medium is DMEM medium.

[0057] Preferably, the DMEM medium is a medium containing less than 1000 mg / L glucose or a medium containing no glucose.

[0058] Preferably, the volume percentage of L-lactic acid is 0.5%-5%, specifically 0.5%, 1%, 2%, 3%, 4%, 5%; preferably, the volume percentage of L-lactic acid is 1%.

[0059] Preferably, the concentration of gentamicin sulfate in the medium is 15-35 μg / ml, preferably, the concentration of gentamicin sulfate in the medium is 20-30 μg / ml, specifically, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, 24 μg / ml, 25 μg / ml, 26 μg / ml, 27 μg / ml, 28 μg / ml, 29 μg / ml, 30 μg / ml; preferably, the concentration of gentamicin sulfate in the medium is 25 μg / ml.

[0060] Method

[0061] Method of inducing embryoid body formation

[0062] In another aspect, the present application provides a method of inducing embryoid body formation, the method comprising the step of inducing pluripotent stem cells to form embryoid bodies.

[0063] Preferably, the method comprises the step of culturing pluripotent stem cells using an EB formation medium;

[0064] Preferably, the culturing is for a period of 1-5 days.

[0065] Preferably, the culturing is for a period of 2-4 days.

[0066] More preferably, the culturing is for a period of 3 days.

[0067] Preferably, the pluripotent stem cells comprise one or more of ESCs, iPSCs, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, adipose stem cells, cord blood stem cells.

[0068] Preferably, the pluripotent stem cells comprise ESCs and / or iPSCs.

[0069] Preferably, the pluripotent stem cells are ESCs and / or iPSCs.

[0070] More preferably, the pluripotent stem cells are IPSC-NFLs.

[0071] In one embodiment, the method of inducing embryoid body formation further comprises the step of detecting the degree of cell aggregation before and / or after inducing embryoid body formation.

[0072] In one embodiment, the inducing embryoid body formation begins passaging after the pluripotent stem cells are expanded to 50-95% confluency.

[0073] Preferably, the inducing embryoid body formation begins passaging after the pluripotent stem cells are expanded to 65-90% confluency.

[0074] Preferably, the inducing embryoid body formation begins passaging after the pluripotent stem cells are expanded to 75-85% confluency.

[0075] Method of inducing cardiac progenitor cells

[0076] In another aspect, the present application provides a method of inducing cardiac progenitor cells, the method comprising the step of inducing embryoid bodies or pluripotent stem cells to form cardiac progenitor cells.

[0077] Preferably, the method comprises the step of culturing embryoid bodies or pluripotent stem cells using a cardiac progenitor cell inducing differentiation medium.

[0078] Preferably, the culturing is for a period of 1-5 days.

[0079] Preferably, the culturing is for 2-4 days.

[0080] Preferably, the culturing is for 3 days.

[0081] Preferably, the culturing is replaced every 24-60 hours; preferably, the culturing is replaced every 36-48 hours; preferably, the culturing is replaced every 40-60 hours; preferably, the culturing is replaced every 48 hours.

[0082] Preferably, the embryoid bodies are prepared by the method of inducing embryoid body formation described above.

[0083] Preferably, the pluripotent stem cells comprise one or more of ESCs, iPSCs, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, adipose stem cells, cord blood stem cells.

[0084] Preferably, the pluripotent stem cells comprise ESCs and / or iPSCs.

[0085] Preferably, the pluripotent stem cells are ESCs and / or iPSCs.

[0086] More preferably, the pluripotent stem cells are IPSC-NFLs.

[0087] Method of inducing cardiomyocytes

[0088] In another aspect, the present application provides a method of inducing cardiomyocytes, the method comprising the step of inducing cardiac progenitor cells to form cardiomyocytes; preferably, the cardiomyocytes are immature.

[0089] Preferably, the cardiac progenitor cells are prepared by the method of inducing cardiac progenitor cells described above.

[0090] Preferably, the method comprises the step of culturing the cardiac progenitor cells using cardiomyocyte induction differentiation medium.

[0091] Preferably, the method is culturing the cardiac progenitor cells using cardiomyocyte induction differentiation medium.

[0092] Preferably, the culturing is for 1-5 days.

[0093] Preferably, the culturing is for 2-3 days.

[0094] Preferably, the culturing is for 2-3 days.

[0095] Preferably, the culturing is replaced every 24-60 hours.

[0096] Preferably, the culture is changed every 36-48 hours.

[0097] Preferably, the culture is changed every 48 hours.

[0098] Method of promoting maturation of immature cardiomyocytes

[0099] In another aspect, the present application provides a method for promoting maturation of immature cardiomyocytes, wherein the immature cardiomyocytes are obtained by the aforementioned method for inducing cardiomyocytes.

[0100] Preferably, the method comprises the step of culturing the immature cardiomyocytes using a cardiomyocyte maturation medium.

[0101] Preferably, the culturing lasts for 1-15 days.

[0102] Preferably, the culturing lasts for 2-12 days.

[0103] Preferably, the culturing lasts for 7-10 days.

[0104] Preferably, the culturing lasts for 8-9 days.

[0105] Preferably, the culture is changed every 12-60 hours.

[0106] Preferably, the culture is changed every 36-60 hours during the first 1-6 days of culturing.

[0107] Preferably, the culture is changed every 48 hours during the first 1-6 days of culturing.

[0108] Preferably, the culture is changed every 6-36 hours during the 7-10 days of culturing.

[0109] Preferably, the culture is changed every 24 hours during the 7-10 days of culturing.

[0110] Preferably, the cardiomyocyte maturation refers to the stepwise formation of cell network structure by the cells, followed by the initiation of beating, and finally the formation of myocardial fiber network.

[0111] Preferably, the cardiomyocyte maturation is further confirmed by immunological or molecular detection methods for the formation of cardiomyocytes; preferably, the molecular detection is for one or more of TNNT, NKX2-5, TBX5, β-MHC, PDGFRα, SLC8A, MYL3, MLC2v, KCNH2 and KCNJ2; preferably, the immunological detection is for cTNT and / or α-actinin.

[0112] Method of purifying cardiomyocytes

[0113] In another aspect, the present application provides a method for purifying cardiomyocytes, wherein the cardiomyocytes are mature cardiomyocytes; preferably, the mature cardiomyocytes are obtained from the aforementioned method for promoting maturation of immature cardiomyocytes.

[0114] Preferably, the method comprises a step of culturing the mature cardiomyocytes using a cardiomyocyte purification medium.

[0115] Preferably, the culturing lasts for 1-15 days.

[0116] Preferably, the culturing lasts for 2-12 days.

[0117] Preferably, the culturing lasts for 8 days.

[0118] Preferably, the culturing lasts for medium exchange every 24-60 hours.

[0119] Preferably, the culturing lasts for medium exchange every 36-48 hours.

[0120] Preferably, the culturing lasts for medium exchange every 48 hours.

[0121] Method of increasing yield and purity of directed cardiomyocyte induction from pluripotent stem cells

[0122] In another aspect, the present application provides a method for improving the yield and purity of cardiomyocytes derived from pluripotent stem cells.

[0123] In one embodiment, the method comprises a step of culturing the pluripotent stem cells to form embryoid bodies;

[0124] Continuing to culture the embryoid bodies to form cardiac progenitor cells;

[0125] Continuing to culture the cardiac progenitor cells to form immature cardiomyocytes;

[0126] Continuing to culture the immature cardiomyocytes to form mature cardiomyocytes;

[0127] Continuing to culture the mature cardiomyocytes to obtain purified mature cardiomyocytes.

[0128] In one embodiment, the method comprises a step of culturing the pluripotent stem cells to form cardiac progenitor cells;

[0129] Continuing to culture the cardiac progenitor cells to form immature cardiomyocytes;

[0130] Continuing to culture the immature cardiomyocytes to form mature cardiomyocytes;

[0131] Continuing to culture the mature cardiomyocytes to obtain purified mature cardiomyocytes.

[0132] In one embodiment, the culturing of pluripotent stem cells to form embryoid bodies is performed using the aforementioned method of inducing embryoid body formation.

[0133] In one embodiment, the culturing of pluripotent stem cells to form cardiac progenitor cells is performed using the aforementioned method of inducing cardiac progenitor cells.

[0134] In one embodiment, the culturing of embryoid bodies to form cardiac progenitor cells is performed using the aforementioned method of inducing cardiac progenitor cells;

[0135] In one embodiment, the culturing of cardiac progenitor cells to form immature cardiomyocytes is performed using the aforementioned method of inducing cardiomyocytes.

[0136] In one embodiment, the culturing of immature cardiomyocytes to form mature cardiomyocytes is performed using the aforementioned method of promoting maturation of immature cardiomyocytes.

[0137] In one embodiment, the culturing of mature cardiomyocytes and ultimately purified mature cardiomyocytes is performed using the aforementioned method of purifying cardiomyocytes.

[0138] Cell

[0139] In another aspect, the present application provides an embryoid body prepared using the aforementioned method of inducing embryoid body formation.

[0140] In another aspect, the present application provides a cardiac progenitor cell prepared using the aforementioned method of inducing cardiac progenitor cells.

[0141] In another aspect, the present application provides a cardiomyocyte prepared using the aforementioned method of inducing cardiomyocytes; preferably, the cardiomyocyte is immature.

[0142] In another aspect, the present application provides a mature cardiomyocyte prepared using the aforementioned method of promoting maturation of immature cardiomyocytes.

[0143] In another aspect, the present application provides a purified mature cardiomyocyte prepared using the aforementioned method of purifying cardiomyocytes.

[0144] In another aspect, the present application provides a purified mature cardiomyocyte prepared using the aforementioned method of increasing the yield and purity of directed cardiomyocyte induction from pluripotent stem cells.

[0145] Composition or medicament

[0146] In another aspect, the present application provides a composition or medicament comprising the aforementioned cells;

[0147] The composition can further comprise other cell culture-related media, growth factors or additives;

[0148] The pharmaceutical agent also includes one or more of a pharmaceutically acceptable carrier, excipient, and pharmaceutically active agent.

[0149] Culture medium combination

[0150] In another aspect, the present application provides a medium combination for improving the yield and purity of the directed induction of cardiomyocytes from pluripotent stem cells, the medium combination comprising one or more of an EB formation medium, a cardiac progenitor cell induction and differentiation medium, a cardiomyocyte induction and differentiation medium, a cardiomyocyte maturation medium, and a cardiomyocyte purification medium.

[0151] Preferably, the medium combination comprises the aforementioned EB formation medium, cardiac progenitor cell induction and differentiation medium, cardiomyocyte induction and differentiation medium, cardiomyocyte maturation medium, and cardiomyocyte purification medium.

[0152] Preferably, the medium combination comprises the aforementioned cardiac progenitor cell induction and differentiation medium, cardiomyocyte induction and differentiation medium, cardiomyocyte maturation medium, and cardiomyocyte purification medium.

[0153] Preferably, the medium combination is the aforementioned EB formation medium, cardiac progenitor cell induction and differentiation medium, cardiomyocyte induction and differentiation medium, cardiomyocyte maturation medium, and cardiomyocyte purification medium.

[0154] Preferably, the medium combination is the aforementioned cardiac progenitor cell induction and differentiation medium, cardiomyocyte induction and differentiation medium, cardiomyocyte maturation medium, and cardiomyocyte purification medium.

[0155] Cell culture system

[0156] In another aspect, the present application also provides a cell culture system for improving the yield and purity of the directed induction of cardiomyocytes from pluripotent stem cells, the cell culture system comprising one or more of an induction of embryoid body formation unit, an induction of cardiac progenitor cell culture unit, an induction of cardiomyocyte unit, a promotion of immature cardiomyocyte maturation unit, and a purification of cardiomyocyte unit.

[0157] Preferably, the system further comprises a unit for monitoring the state of the cells and / or a unit for separating and washing the cells.

[0158] Kit

[0159] In another aspect, the present application also provides a kit for improving the yield and purity of the directed induction of cardiomyocytes from pluripotent stem cells, the kit further comprising reagents required for formulating one or more of an EB formation medium, a cardiac progenitor cell induction and differentiation medium, a cardiomyocyte induction and differentiation base medium, a cardiomyocyte maturation medium, and a cardiomyocyte purification medium.

[0160] Preferably, the kit further comprises instruments and / or devices required in the step of inducing embryoid body formation, the step of inducing cardiac progenitor cell culture, the step of inducing cardiomyocyte, the step of promoting maturation of immature cardiomyocyte, or the step of purifying cardiomyocyte.

[0161] Use

[0162] In another aspect, the present application provides use of IWR-1 and / or C59 in the preparation of a reagent for inducing differentiation of cardiac progenitor cells into cardiomyocytes; preferably, the cardiac progenitor cells are induced to differentiate from pluripotent stem cells or embryoid bodies.

[0163] Preferably, the reagent is a cardiomyocyte induction differentiation medium.

[0164] Preferably, the reagent is a combination of culture media for improving the yield and purity of the directed induction of cardiomyocytes from pluripotent stem cells.

[0165] In another aspect, the present application provides use of IWR-1 and / or C59 in improving the yield and purity of end cells differentiated from cardiac progenitor cells; preferably, the end cells are cardiomyocytes; preferably, the cardiac progenitor cells are induced to differentiate from pluripotent stem cells or embryoid bodies.

[0166] In another aspect, the present application provides use of a combination of one or more of the EB formation medium, the cardiac progenitor cell induction differentiation medium, the cardiomyocyte induction differentiation basal medium, the cardiomyocyte maturation medium, and the cardiomyocyte purification medium in the preparation of a reagent for improving the yield and purity of end cells differentiated from cardiac progenitor cells; preferably, the end cells are cardiomyocytes; preferably, the end cells are purified mature cardiomyocytes; preferably, the cardiac progenitor cells are induced to differentiate from pluripotent stem cells or embryoid bodies.

[0167] In another aspect, the present application provides use of the EB formation medium in the preparation of the aforementioned reagent for inducing embryoid body formation.

[0168] In another aspect, the present application provides use of the cardiac progenitor cell induction differentiation medium in the preparation of the aforementioned reagent for inducing cardiac progenitor cell formation.

[0169] In another aspect, the present application provides use of the cardiomyocyte induction differentiation medium in the preparation of the aforementioned reagent for inducing cardiomyocyte generation.

[0170] In another aspect, the present application provides use of the cardiomyocyte maturation medium in the preparation of the aforementioned reagent for promoting maturation of cardiomyocytes.

[0171] In another aspect, the present application provides use of a myocardial purification medium in the manufacture of a reagent for purifying cardiomyocytes as described above.

[0172] In another aspect, the present application provides use of an EB formation medium, a cardiac progenitor cell induction and differentiation medium, a cardiomyocyte induction and differentiation basal medium, a cardiomyocyte maturation medium, or a myocardial purification medium in the manufacture of a medium combination.

[0173] In another aspect, the present application provides use of a combination of one or more of an EB formation medium, a cardiac progenitor cell induction and differentiation medium, a cardiomyocyte induction and differentiation basal medium, a cardiomyocyte maturation medium, and a myocardial purification medium in the manufacture of cells for cell transplantation therapy of a disease; preferably, the disease is a cardiac disease.

[0174] In another aspect, the present application provides use of the aforementioned cells in the manufacture of a medicament for cell transplantation therapy of a disease; preferably, the disease is a cardiac disease.

[0175] General definitions:

[0176] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0177] Culture medium

[0178] The media used in the present application are commercialized media or artificially prepared.

[0179] The media that can be used in the present application include, but are not limited to, TeSR-E8, mTESR1, E8, Essential 8 TMMedium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, α-Minimal Essential Medium (α-MEM), Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Medium (IMDM), AmnioMax, Amino Max II complete Medium (Gibco, Newyork, USA), Chang's Medium, MesemCult-XF Medium (STEMCELL Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM / F12, Ham's F-12 K Medium, HepatoZYME-SFM, William's E Medium, Waymouth's Medium, or Hepatocyte Culture Medium.

[0180] The medium of the present application can further comprise cytokines, growth factors and small molecule compounds required for cell growth;

[0181] The small molecule compounds are selected from the functional groups of TGF-β signaling pathway, epigenetic modifiers, calcium ion channel agonists, and metabolic pathway modulators;

[0182] The cytokines and protein polypeptides include fibroblast growth factor 1, fibroblast growth factor 2, epidermal growth factor, platelet-derived growth factor, insulin, insulin-like growth factor 1, vascular endothelial cell growth factor, placental growth factor, leukemia inhibitory factor, stem cell factor, transferrin, and human serum albumin.

[0183] Pluripotent stem cell

[0184] Pluripotent stem cells (psc) are stem cells that have the potential to differentiate into multiple cell tissues, but lose the ability to develop into a complete body, and the developmental potential is limited. Such stem cells have the potential to differentiate into multiple cell tissues, but lose the ability to develop into a complete body. For example: embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, etc.

[0185] The pluripotent stem cells in the present application include one or more of ESC, iPSC, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, adipose stem cells, and cord blood stem cells.

[0186] Preferably, the pluripotent stem cells include ESC and / or iPSC cells.

[0187] Preferably, the pluripotent stem cells include iPSC.

[0188] Preferably, the pluripotent stem cells are iPSC.

[0189] More preferably, the pluripotent stem cells are IPSC-NFL.

[0190] The iPSC cells can be a commercial cell line or can be induced from donor cells including one or more of chorionic cells, skin (fibroblasts and keratinocytes), amniotic fluid, extraembryonic tissue (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, hepatocytes, mesenchymal stem cells, peripheral blood cells, mammary epithelial cells, adipose stem cells, umbilical cord matrix, and placenta.

[0191] Preferably, the pluripotent stem cells are cultured or passaged in an IPS basal medium containing ROCKi before being directionally induced into pluripotent stem cells.

[0192] Preferably, the concentration of ROCKi is 1-20 μM; preferably, the concentration of ROCKi is 5-15 μM; preferably, the concentration of ROCKi is 10 μM.

[0193] Preferably, the IPS basal medium is TeSR-E8 medium.

[0194] Induced pluripotent stem cell (iPS cell, iPSC, IPSC, iPSC)

[0195] Induced pluripotent stem cells (iPSCs) are somatic cells reprogrammed by the introduction of transcription factors (Oct4, Sox2, Klf4 and c-Myc) and have similar differentiation capacity as embryonic stem cells. iPSCs are an important source of cells for studying the pathogenesis of human diseases and tissue cell replacement therapy, and do not have ethical issues, so they have a very broad application prospect in the medical field. With iPSCs as the source cells, they can be expanded and induced to differentiate into specific tissue cells in vitro. iPSCs have been successfully cultured and differentiated into myocardial cells, neural cells, pancreatic cells, bone cells and other various tissue cells and different tissues.

[0196] Embryoid body (Embryoid body, EB, EB)

[0197] Embryonic stem cells (ES) or induced pluripotent stem cells (iPS) are formed under certain culture conditions in vitro, have an inner, middle and outer three germ layer structure, and have a high similarity to the early embryonic development stage of mammals in morphology. A kind of spherical structure is called embryoid body. Many differentiation systems are currently established based on the embryoid body differentiation system, such as hematopoietic stem cells, natural killer cells, neural stem cells, and cardiomyocytes. The differentiation strategy is to first prepare EB, and then use different measures to induce differentiation into target cells. The purpose of the EB route is to simulate the in vivo embryonic development process, and the cells in the EB inner, middle and outer three germ layers support each other and provide each other with a differentiation and growth microenvironment.

[0198] Cardiac progenitor cell (CPC)

[0199] Cardiac progenitor cells generally refer to multipotent stem cells with limited differentiation and proliferation capacity, which can be differentiated from CSCs and play an important role in the renewal and repair of cardiomyocytes.

[0200] Differentiation

[0201] Differentiation is the process by which non-specialized ("undifferentiated") or less specialized cells acquire the characteristics of specialized cells (e.g., nerve cells or muscle cells). Differentiated or differentiation-induced cells are cells that have a more specialized ("committed") position in a cell lineage.

[0202] Cellular transplantation therapy

[0203] Cell transplantation therapy is to transplant healthy stem cells into patients to repair or replace damaged cells or tissues, so as to achieve the purpose of cure. BRIEF DESCRIPTION OF DRAWINGS

[0204] Figure 1 A state diagram of undifferentiated IPS cells in Example 1 of the present application;

[0205] Figure 2 A state diagram of forming the embryoid body EB in Example 2 of the present application;

[0206] Figure 3 A myocardial morphology identification diagram of the cells treated with WNT inhibitors C59 and IWR-1 respectively in Example 3 of the present application, A is a morphology diagram of the myocardial cells induced by using WNT inhibitor C59, B is a morphology diagram of the myocardial cells induced by using WNT inhibitor IWR;

[0207] Figure 4 A gene identification result diagram of the cells treated with WNT inhibitors C59 and IWR-1 respectively in Example 3 of the present application, A is a result diagram of comparing the mRNA expression amount of TNNT in the negative control hIPS, the cells treated with WNT inhibitor C59 and the cells treated with WNT inhibitor IWR-1; B is a result diagram of comparing the mRNA expression amount of NKX2-5 in the negative control hIPS, the cells treated with WNT inhibitor C59 and the cells treated with WNT inhibitor IWR-1;

[0208] Figure 5 A qPCR identification result diagram of the mRNA expression of the myocardial markers of the cells treated by using Example 3 or Example 4 of the present application; A is a comparison diagram of the mRNA expression amount of TNNT; B is a comparison diagram of the mRNA expression amount of TNNT; C is a comparison diagram of the mRNA expression amount of NKX2-5; D is a comparison diagram of the mRNA expression amount of PDGFRα; E is a comparison diagram of the mRNA expression amount of SLC8A; F is a comparison diagram of the mRNA expression amount of KCNJ2;

[0209] Figure 6 A qPCR identification result diagram of the mRNA expression of the myocardial markers of the cells treated by using Example 3 or Example 4 of the present application; A is a comparison diagram of the mRNA expression amount of TNNT; B is a comparison diagram of the mRNA expression amount of TNNT; C is a comparison diagram of the mRNA expression amount of NKX2-5; D is a comparison diagram of the mRNA expression amount of PDGFRα; E is a comparison diagram of the mRNA expression amount of SLC8A; F is a comparison diagram of the mRNA expression amount of KCNJ2;

[0210] Figure 7 An immunofluorescence identification diagram of the mature autonomous beating myocardial marker cTNT in the cells prepared by the method for inducing myocardial cells optimized in Example 4 of the present application; A is a staining diagram of cTNT, B is a staining diagram of Dapi, C is a fusion staining diagram of cTNT and Dapi;

[0211] Figure 8Figure of immunofluorescence identification of mature autonomous beating myocardial marker α-actinin in cells prepared by the method for inducing myocardial cells optimized in Example 4 of the present application; A is a staining figure of α-actinin, B is a staining figure of Dapi, and C is a fusion staining figure of α-actinin and Dapi;

[0212] Figure 9 Figure of alkaline phosphatase staining results of comparative myocardial cells with or without purification culture in the method for inducing myocardial cells optimized in Example 4 of the present application; A is the morphology of non-purified cells, B is the morphology of purified cells, C is an alkaline phosphatase staining figure of non-purified cells, and D is an alkaline phosphatase staining figure of purified cells. DETAILED DESCRIPTION

[0213] The present application is further described in conjunction with the following examples, which are presented by way of illustration and not of limitation. Any modification of the examples, which do not depart from the scope of the present application, are to be considered within the scope of the present application. The following examples are presented by way of illustration and not of limitation.

[0214] General method - formulation of culture media required for the present invention

[0215] EB formation medium: PVA was added to TESR-E8 basal medium at a final concentration of 4 mg / ml;

[0216] Cardiac progenitor cell induction medium: DMEM / F-12 medium, 2 mM GlutaMAX TM Supplement, 1X B-27 TM Supplement (minus vitamin A), 25 ng / ml BMP4, 3 μM CHIR99021;

[0217] Cardiomyocyte induction medium (C59): RPMI-1640 medium, 2 mM GlutaMAX TM Supplement, 100 units / mL penicillin, 100 μg / mL streptomycin, 1X B-27 TM Supplement (minus insulin), 10 ng / ml BMP4, 2 μM C59;

[0218] Cardiomyocyte induction medium (IWR-1): RPMI-1640 medium, 2 mM GlutaMAXTM Supplement, 100 units / mL penicillin, 100 μg / mL streptomycin, IX B-27 TM Supplement (minus insulin), 10 ng / ml BMP4, 5 μM IWR-1;

[0219] Cardiomyocyte maturation medium: RPMI-1640 medium, 2 mM GlutaMAX TM Supplement, 100 units / mL penicillin, 100 μg / mL streptomycin, IX B-27 TM Supplement;

[0220] Cardiomyocyte purification medium: DMEM (without glucose) medium, 1% L-lactate, 25 μg / ml gentamicin sulfate.

[0221] Example 1, culture and passaging of IPSCs

[0222] This example takes IPSC-NFL as an example, when IPSC cells are expanded to 75-85% confluence, passaging is started. Take T25 culture dish as an example, remove the old culture medium, wash twice with room temperature PBS, then add 3 mL of 37°C preheated EDTA, place in a 37°C, 5% CO2 cell incubator for 5 min, observe under a microscope that gaps appear between individual cells. Discard the EDTA, add 3 ml of TeSR-E8 complete medium to terminate digestion, transfer to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min at room temperature. Discard the supernatant, add 1 ml of 37°C preheated TeSR-E8 medium (IPS basic medium) containing 10 μM Rocki to the cells, then resuspend by gently blowing the cells. After counting, plate on a matrigel-coated cell culture plate, for example, 2 ml of cell suspension per well in a 6-well plate, with a plating density of 2w / cm 2 .

[0223] Undifferentiated IPSCs are washed three times with DPBS, and after removing dead cells, TeSR-E8 medium is added, and the cell state is recorded under a 4X inverted microscope. The results are shown in Figure 1 , which have a typical monolayer clone-like appearance, clear edges, high nucleus-to-cytoplasm ratio, and good cell state, and can proceed to the next experiment.

[0224] Example 2, formation of IPSC embryoid bodies (EB formation culture)

[0225] When the IPSC cells are expanded to 75-85% confluence, start the digestion and passaging, for example, using a T25 flask, 0.5mM EDTA 3ml digestion for 3min, then collect the cells using a cell scraper and count, 600,000 / ml, plate in a low attachment 6-well plate, 3ml per well, use EB formation medium as the culture medium; place the cell plate in a 37°C, 5% CO2 cell incubator, shake it left and right for about 10 times (short, flat, fast), try to ensure that the cells are evenly distributed on the plate surface, then stand overnight, recorded as DAY1. After 48 hours (Day3), collect the cells into a centrifuge tube, let it settle for about 1h, replace the culture medium with EB formation medium.

[0226] After washing the EBs three times with DPBS, resuspend them using EB formation medium, take a photo under a 4X inverted microscope, record the cell state, the results are shown in Figure 2 , this method successfully induced embryoid bodies, which showed suspended growth, were very compact, and had strong three-dimensional feeling.

[0227] Example 3, comparison of results with addition of C59 or IWR-1 to cardiomyocyte induction and differentiation medium

[0228] Cell processing:

[0229] Use a six-well plate coated with Matrigel, when the IPSC cells are expanded to 75-85% confluence, pass them according to the method of Example 1, the plating density is 5w / cm 2 , and the culture medium used is TESR-E8 (IPS basic medium) + 10μM Rocki, which is recorded as DAY0.

[0230] DAY1-3 use cardiac progenitor cell induction and differentiation medium to induce and differentiate cardiac progenitor cells.

[0231] DAY4-6 use cardiomyocyte induction and differentiation medium to induce and differentiate cardiomyocytes. The culture medium used here is cardiomyocyte induction and differentiation medium containing Wnt pathway inhibitor C59 or IWR-1.

[0232] DAY7-16 use cardiomyocyte maturation medium to induce cardiomyocyte maturation.

[0233] Detection:

[0234] Cell morphology detection: under the microscope, the cell morphology is shown in Figure 3 , A is the morphology of cardiomyocytes induced using WNT inhibitor C59, the cardiomyocytes form a reticular structure and produce rhythmic beating, B is the morphology of cardiomyocytes induced using WNT inhibitor IWR, most of the cardiomyocytes present a spherical structure, and less than 10% of the cells are connected in a chengwang shape.

[0235] Marker gene: The expression amount of cell central myocyte marker gene TNNY and NKX2-5 was detected, and the specific steps of mRNA detection were as follows:

[0236] ① 200W of cells were collected, 1ml of TRIZOL was added, RNA was extracted, and the RNA concentration was determined, 1ug of RNA was reversed into cDNA, and the pre-mixing was performed according to Table 1:

[0237] Table 1. Composition table of PCR mixture

[0238]

[0239] ② Then, the reaction was performed in a Light cycler instrument according to a 3-step method, the cycle number was 45, and the reaction system was as shown in Table 2:

[0240] Table 2. PCR reaction system

[0241]

[0242] The detection results are shown in Figure 4 , the expression of the marker cannot be detected in the negative control hIPS, and the myocardial cell induction differentiation medium added with WNT inhibitor C59 or WNT inhibitor IWR can induce myocardial cells. And the expression amount of the marker in the myocardial cell induction differentiation medium treated with WNT inhibitor C59 is higher.

[0243] This example proves that the myocardial cell induction differentiation culture added with C59 or IWR can promote the formation of myocardial cells, and the effect of C59 is better.

[0244] Example 4, optimised method of inducing cardiomyocytes

[0245] Cell treatment:

[0246] The embryoid bodies formed in Example 2 were washed twice with DPBS, and then the heart progenitor cell induction differentiation medium was replaced, and the induction of heart progenitor cells was started, which is recorded as (DAY4) here.

[0247] The medium was replaced once after 48h (DAY6).

[0248] DAY7-9 started myocardial cell induction, and the medium used here was myocardial cell induction differentiation medium containing Wnt pathway inhibitor (C59 or IWR-1).

[0249] DAY10-18 induced myocardial cell maturation, and the medium used in this stage was myocardial cell maturation medium, and myocardial cell spontaneous beating can be observed at DAY14, and most of the myocardial cells can spontaneously beat (≥85%) at DAY16.

[0250] DAY 19-26, purified cardiomyocytes, the medium used in this stage is glucose-free lactic acid-containing cardiomyocyte purification medium.

[0251] The cells induced in this example (labeled as experimental group) and the cells induced by the method of Example 3 (C59 was added in the cardiomyocyte induction and differentiation medium, labeled as control group 2) were compared; specifically, the myocardial marker detection was performed: TNNT, NKX2-5, TBX5, β-MHC, PDGFRα, SLC8A, MYL3, MLC2v, KCNH2 and KCNJ2, and the method was consistent with the aforementioned mRNA detection method.

[0252] The results are shown in Figure 5 and Figure 6 , which prove that the expression of markers in the cells prepared by the optimized method is higher, thereby proving that the optimized method can improve the yield of cardiomyocytes.

[0253] Example 5, immunofluorescence identification of cells obtained from Example 4 culture

[0254] Detection:

[0255] Immunofluorescence detection was performed on the cells induced by the optimized method of inducing cardiomyocytes in Example 4. The specific steps of immunofluorescence staining are as follows: the cardiomyocytes were washed twice with PBS, 1 ml of 0.25% trypsin was added and digested for 5 min; the trypsin was discarded, and the digestion was terminated with cardiomyocyte maturation medium, and after centrifugation, the supernatant was discarded; the cells were resuspended in cardiomyocyte maturation medium, and were re-plated in a 24-well plate coated with matrigel in advance at a ratio of 1:2, and were cultured for 24-48 h; the culture medium was discarded, and PBS was washed 3 times, the first time was washed, and the last two times were immersed for 3 min each time; the cells were fixed with 4% paraformaldehyde at 4°C for 30 min, and PBS was immersed for 3 min each time; 0.3% Triton X-100 (5% BSA) was used to block the holes at room temperature for 1 h (antigens expressed on the cell membrane were omitted, 300 μl of the mixture was added to the 24-well plate to cover the bottom of the plate); primary antibody was added, and the shaking table was incubated at 4°C overnight; the primary antibody was recovered, and PBS was washed 3 times, the first time was washed, and the last two times were immersed for 5 min each time on the shaking table; secondary antibody was added, and the concentration of the secondary antibody was 1:1000, and the shaking table was incubated at room temperature for 1 h (200 μl per well; from this step, light was avoided); the secondary antibody was washed with PBS, the first time was washed, and the second time was added with 1000x dapi and shaken at room temperature for 3 min; PBS was washed twice more, and the shaking table was incubated at room temperature for 3 min; the machine was started or tin paper was used to avoid light at 4°C.

[0256] The results are shown in Figure 7 , A is the staining of cTNT, B is the staining of Dapi, and C is the fusion of cTNT and Dapi; Figure 8In the figure, A is the staining of a-actinin, B is the staining of Dapi, C is the fusion of a-actinin and Dapi; the experiment proves that the optimized method can induce the generation of cardiomyocytes.

[0257] Example 6, comparison of optimised method of inducing cardiomyocytes with and without inclusion of purification culture

[0258] Cell processing:

[0259] The cells were treated according to the method of inducing cardiomyocytes optimized in Example 4, one culture method included the step of purifying cardiomyocytes on day 19-26, and another culture method did not include the step of purifying cardiomyocytes.

[0260] Detection:

[0261] The purified and non-purified cardiomyocytes were stained by the method of staining with alkaline phosphatase, and the specific steps were as follows: 3 ml of alkaline phosphatase staining buffer, 10 μl of BCIP solution (300X), 20 μl of NBT solution (150X), 3.03 ml of BCIP / NBT staining working solution were mixed to prepare BCIP / NBT staining working solution, and the cell sample was washed with appropriate washing solution for 3-5 times, each time for 3-5 minutes. After the last washing, the washing solution was removed, and an appropriate amount of BCIP / NBT staining working solution was added to ensure sufficient coverage of the sample. Incubate at room temperature for 5-30 minutes or longer (up to 24 hours) in the dark, until the color develops to the desired depth. Remove the BCIP / NBT staining working solution, and wash with distilled water for 1-2 times to terminate the color development reaction. Fluorescence inverted microscope, 10X photographing.

[0262] The results are shown in Figure 9 Figure, A is the morphology of non-purified cells, B is the morphology of purified cells, C is the alkaline phosphatase staining of non-purified cells, and D is the alkaline phosphatase staining of purified cells.

[0263] The experimental results show that the purification step in the optimized method can improve the purity of cardiomyocytes.

Claims

1. A medium combination, characterized in that, The medium combination consists of the following media: 1) EB formation medium containing TESR-E8 basal medium and 4 mg / ml PVA; 2) Cardiac progenitor cell induction differentiation medium containing DMEM / F-12 medium, 2 mM GlutaMAX TM Supplement, 1X B-27 TM Supplement-minus vitamin A, 25 ng / ml BMP4, and 3 μM CHIR99021; 3) Cardiomyocyte induction differentiation medium containing RPMI-1640 medium, 2 mM GlutaMAX TM Supplement, 100 units / mL penicillin, 100 pg / mL streptomycin, 1X B-27 TM Supplement-minus insulin, 10 ng / ml BMP4, and 2 pM C59; 4) Cardiomyocyte maturation medium containing RPMI-1640 medium, 2 mM GlutaMAX TM Supplement, 100 units / mL penicillin, 100 μg / mL streptomycin, 1X B-27 TM Supplement; 5) Cardiomyocyte purification medium containing sugar-free DMEM medium, 1% L-lactic acid and 25 μg / ml gentamicin sulfate.

2. A method of improving yield and purity of iPSC-directed induced cardiomyocytes, characterized by, The method comprises the following steps: (1) iPSC cells are cultured using the EB formation medium of claim 1 for 3 days; (2) the EBs are cultured using the cardiac progenitor cell induction and differentiation medium of claim 1 for 3 days; (3) the cardiac progenitor cells are cultured using the cardiomyocyte induction and differentiation medium of claim 1 for 2 days; (4) the immature cardiomyocytes are cultured using the cardiomyocyte maturation medium of claim 1 for 8 days; (5) the mature cardiomyocytes are cultured using the cardiomyocyte purification medium of claim 1 for 8 days.

3. Use of the medium combination of claim 1 in the directed induction of iPSC cells to prepare cardiomyocytes.

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