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CN113337458A - Method for improving yield and purity of pluripotent stem cells directionally induced cardiac muscle cells - Google Patents

Method for improving yield and purity of pluripotent stem cells directionally induced cardiac muscle cells Download PDF

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CN113337458A
CN113337458A CN202110612164.XA CN202110612164A CN113337458A CN 113337458 A CN113337458 A CN 113337458A CN 202110612164 A CN202110612164 A CN 202110612164A CN 113337458 A CN113337458 A CN 113337458A
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吴理达
顾雨春
刘润琪
朱杰瑞
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Chengnuo Regenerative Medical Technology Zhuhai Hengqin New Area Co ltd
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Abstract

The invention provides a method for improving yield and purity of pluripotent stem cells directionally induced cardiac muscle cells, which is characterized by comprising one or more of the following steps: EB forming culture; inducing differentiation culture of the cardiac progenitor cells; inducing and differentiating the cardiac muscle cells; myocardial cell maturation culture and myocardial purification culture. On the other hand, the invention also provides a culture medium, a culture medium combination, a cell culture system and a kit for improving the yield and purity of the pluripotent stem cells in the directional induction of the myocardial cells.

Description

Method for improving yield and purity of pluripotent stem cells directionally induced cardiac muscle cells
Technical Field
The invention relates to the technical field of biology, in particular to a method for improving yield and purity of pluripotent stem cells directionally induced cardiac muscle cells.
Background
Cardiovascular disease remains a leading cause of death worldwide. In humans and mammals, cardiomyocytes have the ability to divide and proliferate prenatally, and this ability decreases rapidly after birth. Adult cardiomyocytes have little ability to divide and proliferate. In the case of cardiac tissue necrosis diseases such as myocardial infarction, since adult cardiomyocytes have lost the ability to proliferate and divide and the necrotic tissue cannot be repaired by regeneration of cardiomyocytes, the cardiac function deterioration caused by such diseases is irreversible. Although the myocardial contractility can be increased by the use of drugs to improve the pumping capacity of the heart, the aggravation of the cardiac load may rather worsen the condition.
Replacement of necrotic cells by normal cardiomyocyte transplantation is one of the fundamental treatments for such heart diseases. Cell transplantation therapy aimed at the repair of injured cardiac tissue stem cells may greatly improve the current state of cardiac therapy. 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 the essential cells that form the heart, opening up a new era of cardiovascular research, drug discovery, toxicology testing, and cardiac therapy. Obtaining large quantities of highly purified hPSCs-derived cardiomyocytes is critical for basic cardiac research as well as clinical transformation applications. Differentiation of hPSCs into cardiac vessels, particularly Cardiomyocytes (CMS), Smooth Muscle Cells (SMCs), and Endothelial Cells (ECs), has currently made significant progress.
The reported methods for differentiating cardiac muscle using stem cells mainly have the following defects: the efficiency of inducing the differentiation of the myocardial cells is not high, and the obtained myocardial cells are mixed cell populations of pacemaker cells, atrial myocytes and ventricular myocytes, and the accurate directional differentiation cannot be realized.
Induced pluripotent stem cells differ from one hPSCs cell to another in terms of yield and purity of the resulting tissue cells. The tumorigenic potential of the remaining undifferentiated cells exacerbates the safety issue of using hPSC-derived cardiovascular cells. In order to solve the problems, a high-yield, high-efficiency and high-purity myocardial cell induction method is formed by regulating the morphology before cell induction, a key development signal path and a respiration mode after induction is finished.
Experiments prove that the method provided by the invention can induce the pluripotent stem cells to differentiate into the myocardial cells, and the culture medium used by the method not only can enable the pluripotent stem cells to be rapidly and efficiently differentiated into the myocardial cells, but also can be used for producing the myocardial cells in a large scale, has stable quality and high safety, and provides a large amount of cell sources for tissue engineering, drug research and development and cell treatment. The invention has great application value.
Disclosure of Invention
The invention provides a method, a culture medium combination, a cell culture system and a kit for improving yield and purity of pluripotent stem cells in directionally induced cardiac muscle cells.
Culture medium
EB formation Medium
In one aspect, the invention provides an EB formation medium.
Preferably, the EB forming medium is an IPS basal medium to which the organic compound PVA and/or the organic compound MC is added.
Preferably, the IPS basal medium is selected from one or more of: TeSR-E8, mTESR1, E8 and Essential 8TMMedium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Minimal Essential Medium (BME), F-10, F-12, alpha-minimum Essential Medium (alpha-MEM), G-minimum Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMPM (IMDM), Iscove's Modified Eagle's Medium, AmOne or more of nioMax, a novel second generation amniotic fluid Medium (Amino Max ii complex Medium, Gibco, Newyork, USA), Chang's Medium, mee mult-XF Medium (stem cell Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM/F12, Ham's F-12K Medium, hepatozym-SFM, William's electron, Waymouth's Medium, or Hepatocyte Culture Medium;
preferably, the IPS basal medium comprises one or more of TeSR-E8, mTESR1, or E8.
Preferably, the IPS basal medium comprises TeSR-E8.
Most preferably, the IPS basal medium is TeSR-E8.
Preferably, the concentration of the organic compound PVA is 1-8 mg/ml; preferably, the concentration of the organic compound PVA is 2-5mg/ml, in particular 2mg/ml, 3mg/ml, 4mg/ml, 5 mg/ml; most preferably, the concentration of the organic compound PVA is 4 mg/ml.
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% to 0.5%.
In one embodiment, the IPS basal medium culture is a commercial medium; preferably, the TeSR-E8, mTESR1 or E8 are commercially available media from Stemcell.
Culture medium for inducing differentiation of cardiac progenitor cells
In another aspect, the invention provides a medium for inducing differentiation of cardiac progenitor cells.
Preferably, the cardiac progenitor cell differentiation-inducing medium is prepared by adding a cytokine and/or a GSK-3 inhibitor to a cardiac progenitor cell differentiation-inducing basal medium.
Preferably, the cytokines include TGF- β, activin (activin), Bone Morphogenic Protein (BMP), Growth Differentiation Factor (GDF); preferably, the cytokine comprises a bone morphogenic protein; preferably, the cytokine comprises bone morphogenic protein 4(BMP 4); preferably, the cytokine is bone morphogenetic protein 4(BMP 4).
Preferably, the concentration of BMP4 is 10-40 ng/ml; preferably, the concentration of BMP4 is 20-30ng/ml, specifically, 20ng/ml, 21ng/ml, 22ng/ml, 23ng/ml, 24ng/ml, 25ng/ml, 26ng/ml, 27ng/ml, 28ng/ml, 29ng/ml, 30 ng/ml; preferably, the concentration of BMP4 is 25 ng/ml.
Preferably, the GSK-3 inhibitor is the GSK-3 inhibitor CHIR 99021; preferably, the concentration of CHIR99021 is 1-8. mu.M; preferably, 3 μ M to 5 μ M; specifically, 3. mu.M, 4. mu.M, 5. mu.M.
In one embodiment, the primary component in the basic medium for inducing differentiation of cardiac progenitor cells is the first cell culture medium and/or glutamine.
Preferably, the first cell culture medium is selected from one or more of: TeSR-E8, mTESR1, E8 and Essential 8TMMedium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Minimal Essential Medium (BME, Basal Medium Eagle), F-10, F-12, alpha-minimum Essential Medium (alpha-MEM), G-minimum Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Eagle's Medium), AmnioMax, novel second generation amniotic fluid Medium (Amino Max II complex Medium, Gibco, Newyork, USA), Chang's Medium, Mesemcult-XF Medium (STEMCELL Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM/F12, Ham's F-12K Medium, HeatoZYME-SFM, William's EMedium, Waymouth's Medium or HeatoCyte Culture Medium.
Preferably, the first cell culture medium is DMEM/F-12 medium.
Preferably, said glutamine is a substitute therefor; preferably, the substitute is GlutaMAXTMSupplement。
In one embodiment, the basic medium for inducing differentiation of cardiac progenitor cells further comprises one or more of thioglycerol, 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-alpha tocopherol, DL-alpha-tocopheryl acetate, oleic acid, pipecolic acid, and biotin.
Preferably, the basic culture medium for inducing differentiation of cardiac progenitor cells further comprises thioglycerol, 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-alpha tocopherol acetate, oleic acid, pipecolic acid and biotin.
Culture medium for inducing differentiation of cardiac muscle cells
In another aspect, the invention provides a culture medium for inducing differentiation of cardiomyocytes; preferably, the cardiomyocyte differentiation induction culture medium is a cardiomyocyte differentiation induction culture basic medium added with one or more of cytokines, wnt pathway inhibitors and additives.
Preferably, the cytokines include TGF- β, activin (activin), Bone Morphogenic Protein (BMP), Growth Differentiation Factor (GDF); preferably, the cytokine comprises a bone morphogenic protein; preferably, the cytokine comprises bone morphogenic protein 4(BMP 4); preferably, the cytokine is bone morphogenetic protein 4(BMP 4).
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, Hematiin, Specnuezhenide, Prodigiosin, Ginkgetin, XAV939, KY02111, and C59.
Preferably, the Wnt pathway inhibitor comprises C59 and/or IWR-1.
Preferably, the Wnt pathway inhibitor is C59 and/or IWR-1
More preferably, the Wnt pathway inhibitor is C59.
Preferably, the concentration of BMP4 is 0-20 ng/ml; preferably, the concentration of BMP4 is 5-15ng/ml, in particular 5ng/ml, 6ng/ml, 7ng/ml, 8ng/ml, 9ng/ml, 10ng/ml, 11ng/ml, 12ng/ml, 13ng/ml, 14ng/ml, 15 ng/ml; preferably, the concentration of BMP4 is 10 ng/ml.
In one embodiment, the concentration of C59 is 0.1 to 10 μ M; preferably, the concentration of C59 is 0.5-8 μ M; preferably, the concentration of C59 is 1-5 μ M; specifically, the content of the compound comprises 1 mu M, 2 mu M, 3 mu M, 4 mu M and 5 mu M; preferably, the concentration of C59 is 1.5-2.5 μ M; specifically, it includes 1.6. mu.M, 1.7. mu.M, 1.8. mu.M, 1.9. mu.M, 2. mu.M, 2.1. mu.M, 2.2. mu.M, 2.3. mu.M, 2.4. mu.M, 2.5. mu.M; preferably, the concentration of C59 is 2 μ M.
In one embodiment, the concentration of said IWR-1 is 0.1 to 10. mu.M; preferably, the concentration of said IWR-1 is 0.5-8. mu.M; preferably, the concentration of said IWR-1 is 3-8. mu.M; specifically, the preparation method comprises 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M and 8. mu.M; preferably, the concentration of said IWR-1 is 4.5-5.5. mu.M; specifically, it includes 4.6. mu.M, 4.7. mu.M, 4.8. mu.M, 4.9. mu.M, 5M, 5.1. mu.M, 5.2. mu.M, 5.3. mu.M, 5.4. mu.M, 5.5. mu.M; preferably, the concentration of said IWR-1 is 5. mu.M.
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-alpha tocopherol, DL-alpha-tocopheryl acetate, oleic acid, pipecolic acid and biotin.
In one embodiment, the main component of the basic medium for inducing differentiation of cardiomyocytes is the second cell culture medium and/or glutamine.
Preferably, the basic culture medium for inducing the differentiation of the myocardial cells further comprises penicillin (Penicilin), streptomycin (streptomycin), actinomycin D, ampicillin, carbenicillin, streptomycin sulfate, polymyxin B sulfate, neomycin sulfate, kanamycin sulfate and gentamicin sulfate.
Preferably, the basic medium for inducing differentiation of the cardiomyocytes comprises two antibiotics.
Preferably, the basic medium for inducing differentiation of the myocardial cells comprises Penicilin and streptomycin.
Preferably, the second cell culture medium is selected from one or more of: TeSR-E8, mTESR1, E8 and Essential 8TMMedium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Minimal Essential Medium (BME, Basal Medium Eagle), F-10, F-12, alpha-minimum Essential Medium (alpha-MEM), G-minimum Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Eagle's Medium), AmnioMax, novel second generation amniotic fluid Medium (Amino Max II complex Medium, Gibco, Newyork, USA), Chang's Medium, Mesemcult-XF Medium (STEMCELL Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM/F12, Ham's F-12K Medium, HeatoZYME-SFM, William's EMedium, Waymouth's Medium or HeatoCyte Culture Medium.
Preferably, the cell culture medium is RPMI-1640.
Preferably, said glutamine is a substitute therefor; preferably, the substitute is GlutaMAXTMSupplement。
Myocardial cell maturation medium
In another aspect, the invention provides a cardiomyocyte maturation medium.
Preferably, the cardiomyocyte maturation medium is identical in composition to the basic medium for inducing differentiation of cardiomyocytes.
In one embodiment, the cardiomyocyte maturation medium further comprises one or more of the following: 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, pipecolic acid, biotin, and human insulin.
Myocardial purification culture medium
In another aspect, the invention provides a myocardial purification medium.
Preferably, the myocardium purification medium is a third cell culture medium to which L-lactic acid and/or gentamicin sulfate is added.
Preferably, the third cell culture medium is selected from one or more of: TeSR-E8, mTESR1, E8 and Essential 8TMMedium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Minimal Essential Medium (BME, Basal Medium Eagle), F-10, F-12, alpha-minimum Essential Medium (alpha-MEM), G-minimum Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Eagle's Medium), AmnioMax, novel second-generation amniotic fluid Medium (Amino Max II complex Medium, Gibco, Newyork, USA), Chang's Medium, Mesemcult-XF Medium (STEMCELL Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM/F12, Ham's F-12K Medium, HeatoZYME-SFM, William's EMedium, Waymouth's Medium or HeatoCyte Culture Medium; preferably, the third cell culture medium is DMEM medium.
Preferably, the DMEM medium is a medium containing less than 1000mg/L glucose or a medium without glucose.
Preferably, the volume percentage of the L-lactic acid is 0.5% to 5%, specifically 0.5%, 1%, 2%, 3%, 4%, 5%; preferably, the volume percentage of L-lactic acid is 1%.
Preferably, the concentration of the gentamicin sulfate in the culture medium is 15-35 mug/ml, and preferably, the concentration of the gentamicin sulfate in the culture medium is 20-30 mug/ml, specifically, 20 mug/ml, 21 mug/ml, 22 mug/ml, 23 mug/ml, 24 mug/ml, 25 mug/ml, 26 mug/ml, 27 mug/ml, 28 mug/ml, 29 mug/ml and 30 mug/ml; preferably, the concentration of gentamicin sulfate in the culture medium is 25. mu.g/ml.
Method
Method for inducing formation of embryoid bodies
In another aspect, the present invention provides a method of inducing embryoid body formation, the method comprising the step of inducing pluripotent stem cells to form embryoid bodies.
Preferably, the method comprises the step of culturing the pluripotent stem cells using an EB formation medium;
preferably, the culture time is 1-5 days;
preferably, the length of the culture is 2-4 days;
more preferably, the incubation period is 3 days.
Preferably, the pluripotent stem cells comprise one or more of ESC, iPSC, cellular hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, adipose stem cells, umbilical cord blood stem cells.
Preferably, the pluripotent stem cells comprise ESCs and/or ipscs.
Preferably, the pluripotent stem cells are ESCs and/or iPSCs.
More preferably, the pluripotent stem cell is IPSC-NFL.
In one embodiment, the method of inducing embryoid body formation further comprises the step of measuring the degree of polymerization of the cell before and/or after inducing embryoid body formation.
In one embodiment, the induced embryoid body formation begins passaging after the pluripotent stem cells expand to 50-95% of the degree of polymerization.
Preferably, the induced embryoid body formation begins with passage after expansion of the pluripotent stem cells to a degree of polymerization of 65-90%.
Preferably, the induced embryoid body formation begins with passage after expansion of the pluripotent stem cells to a degree of polymerization of 75-85%.
Method for inducing cardiac progenitors
In another aspect, the invention 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.
Preferably, the method comprises the step of culturing the embryoid bodies or pluripotent stem cells using a cardiac progenitor cell-inducing differentiation medium.
Preferably, the incubation period is 1-5 days.
Preferably, the incubation period is 2-4 days.
Preferably, the incubation period is 3 days.
Preferably, the culture is run with a change of broth every 24-60 hours; preferably, the culture is run with fluid changes every 36-48 hours; preferably, the culture is performed with the liquid changed every 40-60 hours; preferably, the incubation is performed with a change every 48 hours.
Preferably, the embryoid body is prepared by the method for inducing embryoid body formation.
Preferably, the pluripotent stem cells comprise one or more of ESC, iPSC, cellular hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, adipose stem cells, umbilical cord blood stem cells.
Preferably, the pluripotent stem cells comprise ESCs and/or ipscs.
Preferably, the pluripotent stem cells are ESCs and/or iPSCs.
More preferably, the pluripotent stem cell is IPSC-NFL.
Method for inducing cardiac muscle cells
In another aspect, the invention provides a method of inducing cardiomyocytes, the method comprising the step of inducing cardiac progenitor cells to form cardiomyocytes; preferably, the cardiomyocytes are immature.
Preferably, the cardiac progenitor cells are cultured by the method for inducing cardiac progenitor cells as described above.
Preferably, the method comprises the step of culturing cardiac progenitor cells using a cardiomyocyte-inducing differentiation medium.
Preferably, the method is culturing cardiac progenitor cells using a cardiomyocyte-inducing differentiation medium.
Preferably, the culturing lasts for 1-5 days.
Preferably, the culturing lasts for 2-3 days.
Preferably, the culturing lasts for 2-3 days.
Preferably, the incubation is performed with a change of solution every 24-60 hours.
Preferably, the incubation is performed with a change of solution every 36-48 hours.
Preferably, the incubation is performed with a change of broth every 48 hours.
Method for promoting maturation of immature cardiomyocytes
In another aspect, the present invention provides a method for promoting maturation of immature cardiomyocytes cultured by the method for inducing cardiomyocytes described above.
Preferably, the method comprises the step of culturing immature cardiomyocytes using a cardiomyocyte maturation medium.
Preferably, the incubation period is 1-15 days.
Preferably, the incubation period is 2-12 days.
Preferably, the incubation period is 7-10 days.
Preferably, the incubation period is 8-9 days.
Preferably, the incubation is performed with a change of broth every 12-60 hours.
Preferably, the solution is changed every 36-60 hours on days 1-6 of the culture.
Preferably, the medium is changed every 48 hours on days 1-6 of the culture.
Preferably, the solution is changed every 6-36 hours on day 7-10 of the culture.
Preferably, the medium is changed every 24 hours on day 7-10 of the culture.
Preferably, the myocardial cell maturation refers to the gradual formation of a cell network by the cells, followed by the beginning of beating, and finally the formation of a myocardial fiber network.
Preferably, the cardiomyocyte maturation is further confirmed by means of immunological or molecular detection of cardiomyocyte formation; preferably, the molecular assay is the assay for one or more of TNNT, NKX2-5, TBX5, β -MHC, PDGFR α, SLC8A, MYL3, MLC2v, KCNH2, and KCNJ 2; preferably, the immunoassay is the detection of cTNT and/or a-actin.
Method for purifying cardiomyocytes
In another aspect, the invention provides a method of purifying a cardiomyocyte, which is a mature cardiomyocyte; preferably, the mature cardiomyocytes are cultured by the method for promoting maturation of immature cardiomyocytes described above.
Preferably, the method comprises the step of culturing mature cardiomyocytes using a myocardial purification medium.
Preferably, the culturing lasts for 1-15 days.
Preferably, the culturing lasts for 2-12 days.
Preferably, the culturing lasts for 8 days.
Preferably, the incubation is continued with a change of solution every 24-60 hours.
Preferably, the incubation is continued with a change of solution every 36-48 hours.
Preferably, the incubation is continued with a change of solution every 48 hours.
Method for improving yield and purity of pluripotent stem cells directionally induced cardiac muscle cells
In another aspect, the invention provides a method for improving yield and purity of pluripotent stem cells in directionally induced cardiomyocytes.
In one embodiment, the method comprises the steps of culturing the pluripotent stem cells to form embryoid bodies;
continuing to culture the embryoid bodies to form cardiac progenitor cells;
continuing to culture said cardiac progenitor cells to form immature cardiomyocytes;
continuing to culture said immature cardiomyocytes to form mature cardiomyocytes;
and continuing to culture the mature myocardial cells to finally obtain the purified mature myocardial cells.
In one embodiment, the method comprises the steps of culturing pluripotent stem cells to form cardiac progenitor cells;
continuing to culture said cardiac progenitor cells to form immature cardiomyocytes;
continuing to culture said immature cardiomyocytes to form mature cardiomyocytes;
and continuing to culture the mature myocardial cells to finally obtain the purified mature myocardial cells.
In one embodiment, the culturing of pluripotent stem cells to form embryoid-like bodies is performed using the method for inducing embryoid-like body formation described above.
In one embodiment, the culturing of pluripotent stem cells to form cardiac progenitor cells is performed using the method for inducing cardiac progenitor cells described above.
In one embodiment, the culturing of the embryoid bodies to form cardiac progenitor cells is performed using the method of inducing cardiac progenitor cells described above;
in one embodiment, culturing the cardiac progenitor cells to form immature cardiomyocytes is performed using the method of inducing cardiomyocytes described above.
In one embodiment, culturing the immature cardiomyocytes to form mature cardiomyocytes is performed using the aforementioned method of promoting maturation of immature cardiomyocytes.
In one embodiment, the culturing of mature cardiomyocytes and the resulting purification of mature cardiomyocytes is performed using the method described above for purifying cardiomyocytes.
Cells
In another aspect, the present invention provides an embryoid body prepared by the method for inducing embryoid body formation.
In another aspect, the present invention provides a cardiac progenitor cell produced using the aforementioned method of inducing cardiac progenitor cells.
In another aspect, the present invention provides a cardiomyocyte prepared using the aforementioned method for inducing cardiomyocytes; preferably, the cardiomyocytes are immature.
In another aspect, the present invention provides a mature cardiomyocyte prepared using the aforementioned method for promoting maturation of an immature cardiomyocyte.
In another aspect, the present invention provides a purified mature cardiomyocyte produced using the aforementioned method of purifying cardiomyocytes.
In another aspect, the present invention provides a purified mature cardiomyocyte prepared by the method for improving the yield and purity of pluripotent stem cells.
Composition or medicament
In another aspect, the invention provides a composition or medicament comprising the aforementioned cells;
the composition further comprises other cell culture related media, growth factors or additives;
the medicament further comprises one or more of a pharmaceutically acceptable carrier, an excipient, and a pharmaceutically active agent.
Culture medium combination
In another aspect, the present invention provides a culture medium composition for improving yield and purity of pluripotent stem cells in directionally induced cardiomyocytes, wherein the culture medium composition comprises one or more of an EB formation medium, a cardiac progenitor cell-induced differentiation medium, a cardiomyocyte maturation medium, and a cardiomyocyte purification medium.
Preferably, the culture medium combination comprises the aforementioned EB formation medium, cardiac progenitor cell-induced differentiation medium, cardiomyocyte maturation medium, and myocardium purification medium.
Preferably, the culture medium combination comprises the aforementioned cardiac progenitor cell differentiation induction culture medium, cardiac muscle cell maturation culture medium and cardiac muscle purification culture medium.
Preferably, the culture medium combination is the aforementioned EB formation medium, cardiac progenitor cell-induced differentiation medium, cardiomyocyte maturation medium, and myocardium purification medium.
Preferably, the culture medium combination is the aforementioned cardiac progenitor cell-induced differentiation medium, cardiac myocyte maturation medium and cardiac muscle purification medium.
Cell culture system
In another aspect, the present invention further provides a cell culture system for improving yield and purity of pluripotent stem cells in directionally induced cardiomyocytes, wherein the cell culture system comprises one or more of an induction embryoid body formation unit, an induction cardiac progenitor cell culture unit, an induction cardiomyocyte unit, an immature cardiomyocyte maturation promotion unit and a purification cardiomyocyte unit.
Preferably, the system further comprises a unit for monitoring the state of the cells and/or a unit for performing separate washing of the cells.
Reagent kit
On the other hand, the invention also provides a kit for improving the yield and purity of the pluripotent stem cells by directionally inducing the cardiac muscle cells, and the kit also comprises one or more reagents required by preparing an EB (Epstein-Barr) forming culture medium, a cardiac progenitor cell induced differentiation culture medium, a cardiac muscle cell induced differentiation basal culture medium, a cardiac muscle cell maturation culture medium and a cardiac muscle purification culture medium;
preferably, the kit further comprises instruments and/or devices required for the step of inducing embryoid body formation, the step of inducing cardiac progenitor cells to culture, the step of inducing cardiomyocytes, the step of promoting maturation of immature cardiomyocytes or the step of purifying cardiomyocytes.
Applications of
In another aspect, the present invention provides the use of IWR-1 and/or C59 for the preparation of an agent 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.
Preferably, the agent is a cardiomyocyte differentiation-inducing medium.
Preferably, the agent is a combination of media that increases the yield and purity of pluripotent stem cells directed to induce cardiomyocytes.
In another aspect, the present invention provides the use of IWR-1 and/or C59 for increasing the yield and purity of end-cells induced differentiation of cardiac progenitors; preferably, the terminal cell is a cardiomyocyte; preferably, the cardiac progenitor cells are induced to differentiate from pluripotent stem cells or embryoid bodies.
In another aspect, the present invention provides the use of a combination of one or more of an EB formation medium, a cardiac progenitor cell differentiation induction medium, a cardiomyocyte differentiation induction basal medium, a cardiomyocyte maturation medium, and a cardiomyocyte purification medium in the preparation of a reagent for increasing the yield and purity of terminal cells derived from cardiac progenitor cells by induction differentiation; preferably, the terminal cell is a cardiomyocyte; preferably, the terminal cell is a purified mature cardiomyocyte; preferably, the cardiac progenitor cells are induced to differentiate from pluripotent stem cells or embryoid bodies.
In another aspect, the invention provides the use of an EB forming medium in the preparation of a reagent for inducing embryoid body formation as described above.
In another aspect, the present invention provides the use of a medium for inducing differentiation of cardiac progenitor cells in the preparation of the aforementioned agent for inducing formation of cardiac progenitor cells.
In another aspect, the invention provides the use of a cardiomyocyte-inducing differentiation medium in the preparation of the aforementioned agent for inducing cardiomyogenesis.
In another aspect, the invention provides the use of a cardiomyocyte maturation medium in the preparation of the aforementioned agent for promoting cardiomyocyte maturation.
In another aspect, the invention provides the use of a myocardial purification medium in the preparation of a reagent for purifying cardiomyocytes as described above.
In another aspect, the invention provides the use of an EB formation medium, a cardiac progenitor cell-induced differentiation medium, a cardiomyocyte-induced differentiation basal medium, a cardiomyocyte maturation medium, or a cardiomyocyte purification medium in the preparation of a culture medium combination.
In another aspect, the present invention provides the use of a combination of one or more of an EB formation medium, a cardiac progenitor cell-induced differentiation medium, a cardiomyocyte-induced differentiation basal medium, a cardiomyocyte maturation medium, and a cardiomyocyte purification medium for the preparation of cells for cell transplantation therapy of disease; preferably, the disease is a heart disease.
In another aspect, the invention provides the use of the aforementioned cells in the preparation of a medicament for the treatment of a disease by cell transplantation; preferably, the disease is a heart disease.
General definition:
unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Culture medium
The medium used in the present invention is a commercial medium or a human-prepared medium.
Media that can be used in the present application include, but are not limited to, TeSR-E8, mTESR1, E8, Essential 8TMMedium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Minimal Essential Medium (BME, Basal Medium Eagle), F-10, F-12, alpha-minimum Essential Medium (alpha-MEM), G-minimum Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Eagle's Medium), AmnioMax, novel second generation amniotic fluid Medium (Amino Max II complex Medium, Gibco, Newyork, USA), Chang's Medium, Mesemcult-XF Medium (STEMCELL Technologies, Vancouver, Canada), RPMI 1640, Ham's F12, DMEM/F12, Ham's F-12K Medium, HeatoZYME-SFM, William's EMedium, Waymouth's Medium or HeatoCyte Culture Medium.
The culture medium of the present invention may further comprise cytokines, growth factors, and small molecule compounds required for cell growth;
the small molecule compound is selected from the functional groups of TGF-beta signal path, appearance modifier, calcium channel stimulant, metabolic path regulator and the like;
the cytokines and protein polypeptides include fibroblast growth factor 1, fibroblast growth factor 2, epidermal growth factor, platelet derived growth factor, insulin-like growth factor 1, vascular endothelial growth factor, placental growth factor, leukemia inhibitory factor, stem cell factor, transferrin, and human serum albumin.
Pluripotent stem cells
Pluripotent stem cells (pscs) have the potential to differentiate into various cell tissues, but lose the ability to develop into complete individuals, and the development potential is limited. Such stem cells have the potential to differentiate into multiple cell tissues, but lose the ability to develop an intact individual. For example: embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, and the like.
The pluripotent stem cells comprise one or more of ESC, iPSC, cellular hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, adipose stem cells and umbilical cord blood stem cells.
Preferably, the pluripotent stem cells comprise ESC and/or iPSC cells.
Preferably, the pluripotent stem cells comprise ipscs.
Preferably, the pluripotent stem cells are ipscs.
More preferably, the pluripotent stem cell is IPSC-NFL.
The iPSC cells can be commercial cell lines or can be induced from donor cells including one or more of villus 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.
Preferably, prior to the directed induction of pluripotent stem cells, the pluripotent stem cells are cultured or passaged in IPS basal medium containing a ROCKi.
Preferably, the concentration of said ROCKi is 1-20 μ M; preferably, the concentration of said ROCKi is 5-15 μ M; preferably, the concentration of the ROCKi is 10 μ M.
Preferably, the IPS basal medium is TeSR-E8 medium.
Induced pluripotent stem cells (iPS cells, iPSCs, IPSC, iPSC)
Induced pluripotent stem cell (induced pluripotent stem cells) somatic cells are introduced into cells which are reprogrammed by transcription factors (Oct4, Sox2, Klf4 and c-Myc) and have differentiation capacity similar to that of embryonic stem cells, so that the induced pluripotent stem cell (induced pluripotent stem cell) becomes an important cell source for researching human disease pathogenesis and tissue cell replacement therapy, and has no ethical problem, and the application prospect in the field of medical treatment is very wide. iPSCs are used as source cells, can be amplified in vitro and induced to differentiate into specific tissue cells, and various somatic cells and different tissues such as cardiac muscle, nerve, pancreas, bone and the like are successfully cultured and differentiated by applying the iPSCs.
Embryoid bodies (Embryoid bodies, EB, EBs)
The embryonic stem cell (ES) or induced pluripotent stem cell (iPS) is a spherical structure which is formed under a certain in vitro culture condition, has internal, intermediate and external three-germ-layer structures, and has high similarity with the early embryonic development stage of mammals in morphology, and is called an embryoid body. At present, many differentiation systems are established based on embryoid body differentiation systems, such as hematopoietic stem cells, natural killer cells, neural stem cells, cardiac muscle cells and the like, and the differentiation strategies are to prepare EB first and then to directionally induce and differentiate into target cells by different measures. Through the EB path, the aim is to simulate the development process of an in vivo embryo, and cells of the internal, the external and the middle three germ layers in the EB support each other and provide a microenvironment for differentiation and growth mutually.
Cardiac Progenitors (CPC)
Cardiac progenitors generally refer to multipotent stem cells with defined differentiation and proliferation capabilities, which can be differentiated from CSCs, and also play an important role in cardiomyocyte renewal and repair.
Differentiation
Differentiation is a process by which non-specialized ("uncommitted") or less specialized cells acquire the characteristics of a specialized cell (e.g., a neural cell or a muscle cell). Differentiated or differentiation-induced cells are cells that have a more specialized ("committed") location in the cell lineage.
Cell transplantation therapy
Cell transplantation therapy is the transplantation of healthy stem cells into a patient's body to repair or replace damaged cells or tissues, thereby achieving the goal of healing.
Drawings
FIG. 1 is a diagram showing the state of undifferentiated IPS cells in example 1 of the present invention;
FIG. 2 is a diagram showing a state of forming an embryoid body EB in example 2 of the present invention;
FIG. 3 is a chart showing the morphological identification of cardiomyocytes induced by WNT inhibitor C59 and the morphological identification of cardiomyocytes induced by WNT inhibitor IWR-1, respectively, in example 3 of the present invention;
FIG. 4 is a graph showing the results of gene identification of cells in which WNT inhibitor was used with C59 and IWR-1, respectively, in example 3 of the present invention, and A is a graph showing the results of comparing the mRNA expression level of TNNT in negative control hIPS, cells in which WNT inhibitor was used with C59, and cells in which WNT inhibitor was used with IWR-1; b is a graph comparing the results of the expression of NKX2-5 mRNA in negative control hIPS, WNT inhibitor cells cultured with C59, and WNT inhibitor cells cultured with IWR-1;
FIG. 5 is a graph of the qPCR identification of myocardial marker mRNA expression in cells treated according to example 3 or example 4 of the present invention; a is a graph comparing the expression level of mRNA of TNNT; b is a graph comparing the expression level of mRNA of TNNT; c is a graph comparing the expression level of NKX2-5 mRNA; d is a graph comparing the expression level of PDGFR alpha mRNA; e is a comparison graph of the mRNA expression level of SLC 8A; f is a graph comparing the expression level of KCNJ2 mRNA;
FIG. 6 is a graph of the qPCR identification of myocardial marker mRNA expression in cells treated according to example 3 or example 4 of the present invention; a is a graph comparing the expression level of mRNA of TBX 5; b is a graph comparing the expression level of mRNA in beta-MHC; c is a graph comparing the mRNA expression levels of MYL 3; d is a graph comparing the expression level of mRNA of MLC2 gamma;
FIG. 7 is a graph of immunofluorescence identifying mature autonomously pulsating myocardial marker cTNT in cells prepared by the optimized method for inducing cardiomyocytes in example 4 of the present invention; a is a staining pattern for cTNT, B is a staining pattern for Dapi, and C is a fusion staining pattern of cTNT and Dapi;
FIG. 8 is a graph of immunofluorescence identification of mature autonomously pulsating cardiac marker α -actin in cells obtained by the method for inducing cardiomyocytes optimized in example 4 of the present invention; a is a staining pattern for alpha-actin, B is a staining pattern for Dapi, and C is a fusion staining pattern for alpha-actin and Dapi;
FIG. 9 is a graph showing the results of alkaline phosphatase staining of comparative cardiomyocytes with or without purified culture in the method for inducing cardiomyocytes optimized in example 4 of the present invention; a is the morphology of unpurified cells, B is the morphology of purified cells, C is the alkaline phosphatase staining pattern of unpurified cells, and D is the alkaline phosphatase staining pattern of purified cells.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
General procedure-preparation of the culture Medium required for the invention
EB formation medium: PVA was added to a minimal medium of TESR-E8 to a final concentration of 4 mg/ml;
heart progenitor cell differentiation induction medium: DMEM/F-12 Medium, 2mM GlutaMAXTM Supplement，1X B-27TM Supplement(minus vitamin A)，25ng/ml BMP4，3μM CHIR99021；
Cardiomyocyte-inducing differentiation medium (C59): RPMI-1640 Medium, 2mM GlutaMAXTM Supplement，100units/mL penicillin，100μg/mL streptomycin，1X B-27TM Supplement(minus insulin)，10ng/ml BMP4，2μM C59；
Cardiomyocyte-inducing differentiation medium (IWR-1): RPMI-1640 Medium, 2mM GlutaMAXTMSupplement，100units/mL penicillin，100μg/mL streptomycin，1X B-27TMSupplement(minus insulin)，10ng/ml BMP4，5μM IWR-1；
Cardiomyocyte maturation medium: RPMI-1640 Medium, 2mM GlutaMAXTM Supplement，100units/mL penicillin，100μg/mL streptomycin，1X B-27TM Supplement；
Myocardium purification medium: DMEM (without glucose), 1% L-lactic acid, 25. mu.g/ml gentamicin sulphate.
Example 1 culture and passaging of IPSC
This example illustrates the case of IPSC-NFL, and passage is initiated when IPSC cells expand to 75-85% DP. Using T25 petri dishes as an example, old media was aspirated, washed twice with room temperature PBS, followed by 3mL of EDTA pre-warmed to 37 ℃ and placed at 37 ℃ in 5% CO2The cell culture box (2) is used for 5min, and gaps among single cells are observed under a microscope. EDTA was discarded, and 3ml of TeSR-E8 complete medium was added to stop the digestion, which was transferred to a 15ml centrifuge tube and centrifuged at 1000rpm for 5min at room temperature. The supernatant was discarded, and the cells were gently pipetted with 1ml of 37 ℃ pre-warmed TeSR-E8 medium (IPS basal medium) supplemented with 10. mu.M Rocki and then resuspended. After counting, the plates were plated on matrigel-coated cell culture plates, for example, in 6-well plates, with 2ml of cell suspension per well and a plating density of 2w/cm2。
Undifferentiated IPSC is washed for three times by using DPBS, dead cells are removed, a TeSR-E8 culture medium is added, a picture is taken under a 4X inverted microscope, the cell state is recorded, and the result is shown in figure 1, the cell has a typical single-layer clone sample, the edge is clear, the cell nucleus-mass ratio is high, the cell state is good, and the next experiment can be carried out.
Example 2 formation of IPSC embryoid bodies (EB formation culture)
Digestion passage was started when the IPSC cells expanded to 75-85% DP, as exemplified by a T25 flask, 0.5mM EDTA3ml for 3min, followed by cell scraping to collect the cells for counting, 60 ten thousand/ml, plating onto low affinity 6-well plates, 3ml per well, and forming the medium using EB; the cell plate was placed at 37 ℃ in 5% CO2The cell culture box was shaken about 10 times (short, flat, fast) from front to back and from left to right to ensure uniform distribution of cells on the culture plate surface, and then left overnight (DAY 1). After 48 hours (Day3), the cells were collected in a centrifuge tube, allowed to settle naturally for about 1 hour, and the medium was changed to EB-forming medium.
After the EB is washed three times by using DPBS, the EB is used for forming a culture medium for heavy suspension, the picture is taken under a 4X inverted microscope, the cell state is recorded, and the result is shown in figure 2.
Example 3 comparison of results of addition of C59 or IWR-1 to cardiomyocyte-inducing differentiation medium
Cell treatment:
using Matrigel-plated six-well plates, passaging was performed according to the method of example 1 when IPSC cells were expanded to 75-85% degree of polymerization, and the plating density was 5w/cm2The medium used was TESR-E8(IPS basal medium) + 10. mu.M Rocki, here designated DAY 0.
DAY1-3 induced differentiation of cardiac progenitor cells using cardiac progenitor cell-inducing differentiation medium.
DAY4-6 induced differentiation of cardiomyocytes using cardiomyocyte-inducing differentiation medium. The medium used here was a cardiomyocyte differentiation-inducing medium containing Wnt pathway inhibitor C59 or IWR-1, respectively.
DAY7-16 induced cardiomyocyte maturation using cardiomyocyte maturation media.
And (3) detection:
and (3) cell morphology detection: cell morphology under microscope as shown in figure 3, a is the morphology of cardiomyocytes induced by WNT inhibitor C59, which form a network and produce rhythmic beating, B is the morphology of cardiomyocytes induced by WNT inhibitor IWR, which mostly appear as spherical structures, with fewer than 10% of the cells connected as chengwang.
Marker genes: detecting the expression levels of the marker genes TNNY and NKX2-5 of the cardiac muscle cells in the cells, wherein the specific steps of mRNA detection are as follows:
collecting 200W cardiac muscle cells, adding 1ml TRIZOL, extracting RNA, determining RNA concentration, inverting 1. mu.g of RNA into cDNA, and premixing according to the following table 1:
TABLE 1 composition of PCR mixture
Figure BDA0003096288460000191
Secondly, putting the mixture into a Light cycler instrument to perform reaction according to a 3-step method, wherein the number of the cycles is 45, and the reaction system is shown in the table 2:
TABLE 2 PCR reaction System
Figure BDA0003096288460000192
As shown in FIG. 4, the expression of marker could not be detected in negative control hIPS, and cardiomyocytes were induced by using cardiomyocyte differentiation-inducing medium supplemented with WNT inhibitor C59 or WNT inhibitor IWR. And the expression level of the marker is higher in the cells treated by the culture medium for inducing differentiation of the myocardial cells added with the WNT inhibitor C59.
This example demonstrates that both cardiomyocytes induced differentiation cultures with the addition of C59 or IWR promote cardiomyocyte formation and that C59 is more effective.
Example 4 optimized methods for inducing cardiomyocytes
Cell treatment:
after the embryoid bodies formed in example 2 were washed twice with DPBS, the cardiac progenitor induction differentiation medium was replaced to start induction of cardiac progenitors, which is herein designated (DAY 4).
After 48h (DAY6), the medium was changed once.
DAY7-9 began cardiomyocyte induction, and the medium used here was cardiomyocyte-inducing differentiation medium containing Wnt pathway inhibitor (C59 or IWR-1).
DAY10-18 induced cardiomyocyte maturation, the culture medium used at this stage was a cardiomyocyte maturation medium, DAY14 observed cardiomyocyte spontaneous beating, and DAY16 most cardiomyocytes were able to beat autonomously (85% or more).
DAY19-26 purified cardiomyocytes, the culture medium used at this stage was glucose-free, lactic acid-containing, purified myocardium medium.
Comparing the cells induced in this example (labeled as experimental group) with those induced in example 3 (myocardial cell differentiation medium supplemented with C59, labeled as control group 2); specifically, the detection was a cardiomyocyte marker detection: TNNT, NKX2-5, TBX5, beta-MHC, PDGFR alpha, SLC8A, MYL3, MLC2v, KCNH2 and KCNJ2, in a manner consistent with the mRNA detection method described above.
As a result, as shown in FIGS. 5 and 6, it was confirmed that the expression level of the marker was higher in the cells prepared by the present optimization method, and thus it was confirmed that the present optimization method can improve the yield of cardiomyocytes.
Example 5 immunofluorescence identification example 4 cells cultured
And (3) detection:
immunofluorescence assays were performed on cells induced by the method of inducing cardiomyocytes optimized in example 4. The specific steps of immunofluorescence staining are as follows: washing cardiac muscle cells twice with PBS, adding 1ml of 0.25% pancreatin, and digesting for 5 min; discarding pancreatin, terminating digestion with myocardial cell maturation culture medium, centrifuging, and discarding supernatant; cardiomyocyte maturation medium basic suspension of cells in a 1: 2, replating the mixture in a 24-well plate coated by matrigel in advance, and culturing for 24-48 h; discarding the culture solution, washing with PBS for 3 times, washing for the first time, and then washing for two times, each time for 3 min; fixing cells with 4% paraformaldehyde at 4 deg.C for 30min, and washing with PBS for 3 times, each for 3 min; 0.3% Triton X-100 (prepared by 5% BSA) for blocking the perforation for 1h at room temperature (antigen expressed on cell membrane omits the step, 300. mu.l of mixed solution is added into a 24-well plate, and the plate bottom is covered); adding primary antibody, shaking overnight at 4 deg.C; recovering primary antibody, washing with PBS for 3 times, washing for the first time, and then washing for 5min with shaking table for two times; adding secondary antibody with the concentration of 1:1000, shaking for 1h at room temperature (200 μ l per well; keeping away from light all the time from this step); washing the secondary antibody with PBS, washing for the first time, and adding 1000x dapi for the second time and shaking for 3min at room temperature; washing PBS twice, and shaking at room temperature for 3 min; storing on a machine or tin foil at 4 deg.C in dark.
The results are shown in FIG. 7, where A is the staining for cTNT, B is the staining for Dapi, and C is the fusion of cTNT and Dapi; in FIG. 8, A is a staining for α -actin, B is a staining for Dapi, and C is a fusion of α -actin and Dapi; the experiment proves that the optimization method can induce and generate the myocardial cells.
Example 6 optimized comparison of methods of inducing cardiomyocytes with or without purified culture
Cell treatment:
cells were treated according to the method for inducing cardiomyocytes optimized in example 4, one culture method included the step of purification of cardiomyocytes for DAY19-26 of DAY DAY, and another culture method included no step of purification of cardiomyocytes.
And (3) detection:
purified and unpurified cardiomyocytes were stained with alkaline phosphatase staining as follows: 3ml of alkaline phosphatase color development buffer solution, 10. mu.l of BCIP solution (300X), 20. mu.l of NBT solution (150X) and 3.03ml of BCIP/NBT staining working solution are mixed uniformly to prepare BCIP/NBT staining working solution, and a cell sample is washed 3-5 times with proper washing solution for 3-5 minutes each time. And after the last washing is finished, removing the washing solution, and adding a proper amount of BCIP/NBT dyeing working solution to ensure that the sample can be fully covered. Incubate at room temperature in the dark for 5-30 minutes or longer (up to 24 hours) until the color is developed to the desired shade. And removing the BCIP/NBT dyeing working solution, and washing with distilled water for 1-2 times to terminate the color reaction. Fluorescence inverted microscope, 10X photograph.
As shown in FIG. 9, A is the morphology of unpurified cells, B is the morphology of purified cells, C is the alkaline phosphatase staining pattern of unpurified cells, and D is the alkaline phosphatase staining pattern of purified cells.
Experimental results show that the purification step in the optimized method can improve the purity of the myocardial cells.

Claims (10)

1. A culture medium comprising any one of:
1) EB formation medium; preferably, the EB forming medium is an IPS basal medium added with organic compounds PVA and/or MC;
preferably, the IPS basal medium is TeSR-E8;
preferably, the concentration of the organic compound PVA is 1-8 mg/ml; most preferably, the PVA concentration is 4 mg/ml;
preferably, the concentration of the organic compound MC is 0.1% -1%; most preferably, the MC concentration is 0.3% -0.5%;
2) a cardiac progenitor cell induced differentiation medium; preferably, the cardiac progenitor cell differentiation induction medium is prepared by adding a cytokine and/or a GSK-3 inhibitor into a cardiac progenitor cell differentiation induction basic medium;
preferably, the cytokine is BMP 4; preferably, the concentration of BMP4 is 25 ng/ml;
preferably, the GSK-3 inhibitor is CHIR 99021; preferably, the concentration of CHIR99021 is 3. mu.M-5. mu.M;
preferably, the main component in the basic medium for inducing differentiation of cardiac progenitor cells is the first cell culture medium and/or glutamine;
preferably, the first cell culture medium is DMEM/F-12 medium;
preferably, said glutamine is a substitute therefor; preferably, the substitute is GlutaMAXTMSupplement；
Preferably, the basic culture medium for inducing differentiation of the cardiac progenitor cells further comprises thioglycerol, 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-alpha tocopherol acetate, oleic acid, pipecolic acid and biotin;
3) a myocardial cell induced differentiation culture medium; preferably, the myocardial cell differentiation induction culture medium is a myocardial cell differentiation induction base culture medium added with one or more of cytokines, wnt pathway inhibitors and additives;
preferably, the cytokine is BMP 4; preferably, the concentration of BMP4 is 10 ng/ml;
preferably, the Wnt pathway inhibitor comprises C59 and/or IWR-1; more preferably, the Wnt pathway inhibitor is C59 and/or IWR-1;
preferably, the concentration of C59 is 0.1-10 μ M; preferably, the concentration of C59 is 2 μ M;
preferably, the concentration of said IWR-1 is 0.1-10. mu.M; preferably, the concentration of said IWR-1 is 5. mu.M;
preferably, the additive is retinol, catalase, reduced glutathione, superoxide dismutase, transferrin, T3, L-carnitine, ethanolamine, galactose, putrescine, sodium selenite, corticosterone, linoleic acid, linolenic acid, progesterone, DL-alpha tocopherol acetate, oleic acid, pipecolic acid, and biotin;
preferably, the main component of the basic medium for inducing differentiation of the myocardial cells is the second cell culture medium and/or glutamine;
preferably, the basic culture medium for inducing the differentiation of the myocardial cells further comprises Penicilin and streptomycin;
preferably, the second cell culture is RPMI-1640;
preferably, said glutamine is a substitute therefor; preferably, the substitute is GlutaMAXTMSupplement；
4) A cardiomyocyte maturation medium; preferably, the composition of the myocardial cell maturation medium is consistent with that of a myocardial cell differentiation induction basic medium;
preferably, the cardiomyocyte maturation medium further comprises the following components: 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, pipecolic acid, biotin, and human insulin;
5) a myocardium purification medium; the myocardial purification culture medium is obtained by adding L-lactic acid and/or gentamicin sulfate to a third cell culture medium;
preferably, the third cell culture medium is DMEM medium;
preferably, the DMEM medium is a medium containing less than 1000mg/L glucose or a medium without glucose;
preferably, the volume percentage of the L-lactic acid is 1-5%; preferably, the volume percentage is 1%;
preferably, the concentration of the gentamicin sulfate in the culture medium is 15-35 mug/ml; preferably, the concentration of gentamicin sulfate in the medium is 25. mu.g/ml.
2. A method of inducing embryoid body formation, the method comprising inducing a pluripotent stem cell to form an embryoid body;
preferably, the method cultures pluripotent stem cells using the EB formation medium of claim 1; preferably, the culture time is 2-4 days; more preferably, the length of the incubation is 3 days;
preferably, the pluripotent stem cells comprise one or more of ESCs, ipscs, cellular hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, adipose stem cells, umbilical cord stem cells; preferably, the pluripotent stem cells are ESCs and/or ipscs; more preferably, the pluripotent stem cell is IPSC-NFL.
3. A method of inducing cardiac progenitors comprising inducing embryoid bodies or pluripotent stem cells to form cardiac progenitors;
preferably, the method comprises culturing an embryoid body or pluripotent stem cell using the cardiac progenitor induced differentiation medium of claim 1;
preferably, the culture time is 2-4 days;
preferably, the culture is run with a change of broth every 24-60 hours; preferably, the incubation is performed with a change every 48 hours;
preferably, the embryoid body is prepared by the method of claim 2;
preferably, the pluripotent stem cells comprise one or more of ESCs, ipscs, cellular hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, adipose stem cells, umbilical cord stem cells; preferably, the pluripotent stem cells are ESCs and/or ipscs; more preferably, the pluripotent stem cell is IPSC-NFL.
4. A method of inducing cardiomyocytes, comprising inducing cardiac progenitor cells to form cardiomyocytes; preferably, the cardiomyocytes are immature; preferably, said cardiac progenitor cells are cultured by the method of claim 3;
preferably, the method comprises culturing cardiac progenitor cells using the cardiomyocyte-inducing differentiation medium of claim 1;
preferably, the culturing lasts for 2-3 days;
preferably, the culture is performed by replacing the culture solution every 24-60 hours; preferably, the liquid change is performed every 48 hours.
5. A method of promoting maturation of immature cardiomyocytes, wherein the immature cardiomyocytes are cultured according to the method of claim 4;
preferably, the method comprises culturing immature cardiomyocytes using the cardiomyocyte maturation medium of claim 1; preferably, the culture time is 7-10 days;
preferably, the medium is changed every 48 hours on days 1-6 and every 24 hours on days 7-10 of the culture.
6. A method of purifying a cardiomyocyte, wherein the cardiomyocyte is a mature cardiomyocyte; preferably, the mature cardiomyocytes are cultured by the method of claim 5;
preferably, the method comprises culturing mature cardiomyocytes using the myocardial purification medium of claim 1;
preferably, the culturing lasts for 1-15 days; preferably, the culturing lasts for 8 days;
preferably, the culture is continued with the change of the solution every 24 to 60 hours; preferably, the incubation is continued with a change of solution every 48 hours.
7. A method for improving yield and purity of pluripotent stem cells directionally induced cardiac muscle cells, wherein the method is selected from any one of the following:
1) the method comprises culturing the pluripotent stem cells to form embryoid bodies;
continuing to culture the embryoid bodies to form cardiac progenitor cells;
continuing to culture said cardiac progenitor cells to form immature cardiomyocytes;
continuing to culture said immature cardiomyocytes to form mature cardiomyocytes;
continuing to culture the mature cardiomyocytes to finally obtain purified mature cardiomyocytes;
2) the method comprises culturing pluripotent stem cells to form cardiac progenitor cells;
continuing to culture said cardiac progenitor cells to form immature cardiomyocytes;
continuing to culture said immature cardiomyocytes to form mature cardiomyocytes;
continuing to culture the mature cardiomyocytes to finally obtain purified mature cardiomyocytes;
preferably, the method of claim 2 is used to culture pluripotent stem cells to form embryoid bodies;
preferably, said culturing embryoid bodies to form cardiac progenitor cells is performed using the method of claim 3;
preferably, said culturing pluripotent stem cells to form cardiac progenitor cells is performed using the method of claim 3;
preferably, said culturing cardiac progenitor cells to form immature cardiomyocytes is performed using the method of claim 4;
preferably, said culturing immature cardiomyocytes to form mature cardiomyocytes is performed using the method of claim 5;
preferably, the culturing of mature cardiomyocytes and the resulting purification of mature cardiomyocytes is performed using the method of claim 6.
8. A cell selected from any one of the following:
1) an embryoid body produced by the method of claim 2;
2) a cardiac progenitor cell made by the method of claim 3;
3) an immature cardiomyocyte produced by the method of claim 4;
4) a mature cardiomyocyte produced by the method of claim 5;
5) a purified mature cardiomyocyte produced by the method of claim 6;
6) a purified mature cardiomyocyte produced by the method of claim 7.
9. A culture medium composition for improving the yield and purity of pluripotent stem cells directionally inducing cardiomyocytes, wherein the culture medium composition comprises one or more of the culture media of claim 1;
preferably, the medium combination comprises the EB formation medium, cardiac progenitor cell-induced differentiation medium, cardiomyocyte maturation medium, and cardiomyocyte purification medium of claim 1;
preferably, the culture medium combination comprises the cardiac progenitor differentiation-inducing culture medium, the cardiac myocyte maturation culture medium and the cardiac muscle purification culture medium of claim 1.
10. An application, characterized in that the application comprises any one of the following:
1) use of IWR-1 and/or C59 in the preparation of an agent 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;
preferably, the agent is a cardiomyocyte-inducing differentiation medium;
preferably, the cardiomyocyte differentiation-inducing medium is the cardiomyocyte differentiation-inducing medium according to claim 1;
preferably, the agent is the culture medium combination for improving the yield and purity of the pluripotent stem cells for directionally inducing the cardiac muscle cells as claimed in claim 9;
2) use of IWR-1 and/or C59 for increasing the yield and purity of end-stage cells induced by differentiation of cardiac progenitors; preferably, the terminal cell is a cardiomyocyte; preferably, the cardiac progenitor cells are induced to differentiate from pluripotent stem cells or embryoid bodies;
3) use of the medium of claim 1 for the preparation of an agent for increasing the yield and purity of terminal cells induced from differentiation of cardiac progenitors; preferably, the terminal cell is a cardiomyocyte; preferably, the terminal cell is a purified mature cardiomyocyte; preferably, the cardiac progenitor cells are induced to differentiate from pluripotent stem cells or embryoid bodies;
4) use of a culture medium combination according to claim 9 for the preparation of an agent for increasing the yield and purity of end-cells induced differentiation of cardiac progenitors; preferably, the terminal cell is a cardiomyocyte; preferably, the terminal cell is a purified mature cardiomyocyte; preferably, the cardiac progenitor cells are induced to differentiate from pluripotent stem cells or embryoid bodies;
5) use of the EB formation medium of claim 1 in the preparation of an agent for inducing embryoid body formation;
6) use of a cardiac progenitor differentiation-inducing medium according to claim 1 for the preparation of an agent for inducing the formation of cardiac progenitors;
7) use of the medium for inducing differentiation of cardiomyocytes according to claim 1 for the preparation of an agent for inducing cardiomyogenesis;
8) use of a cardiomyocyte maturation medium according to claim 1 in the preparation of an agent that promotes cardiomyocyte maturation;
9) use of the myocardial purification medium of claim 1 in the preparation of a reagent for purifying cardiomyocytes;
10) use of the culture medium of claim 1 for the preparation of the combination of culture media of claim 9;
11) use of the medium of claim 1 for the preparation of cells for the treatment of diseases by cell transplantation; preferably, the disease is a heart disease;
12) use of the cell of claim 8 in the preparation of a medicament for the treatment of a disease by cell transplantation; preferably, the disease is a heart disease;
13) use of a culture medium combination according to claim 9 for the preparation of cells for the treatment of diseases by cell transplantation; preferably, the disease is a heart disease.
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