Method for rapidly inducing directional differentiation of embryonic stem cells into myocardial cells in vitro

Abstract

The invention relates to the technical field of cell differentiation and the technical field of myocardial tissue regenerative medicine, in particular to a method for rapidly inducing directional differentiation of embryonic stem cells into myocardial cells in vitro. A matrix sandwich method is adopted, specific small molecule compounds are added in different time periods to induce the embryonic stem cells to differentiate towards the myocardial cells, and nearly 80% of myocardial cells can be obtained on the seventh day of induced differentiation. Compared with a traditional in-vitro myocardial differentiation induction scheme, the induced differentiation method provided by the invention has the advantages that the variety of related small molecules is few, the dosage is low, the induced differentiation time can be effectively shortened from 15 days to 7 days, the induced differentiation cost is saved, and the method is suitable for large-scale popularization and application. A novel way which is more economical, simpler, more convenient, more stable and more efficient is provided for the production of the stem cell-derived myocardial cells; the cardiac muscle cells obtained by induced differentiation are relatively high in purity and relatively wide in application prospect, and are of great significance to research on drug screening, myocardial regenerative medicine and cardiac developmental biology.

Claims

1. A method for rapidly inducing embryonic stem cells to differentiate into cardiomyocytes in vitro, characterized in that: The steps include: (1) using embryonic stem cells in good growth state, and defining the day of differentiation induction as day 0 when the cell density reaches 90% or more; adding the first differentiation medium to the embryonic stem cells and culturing for 20-24 hours, which is the first day of induction culture; then, replacing the medium with the basal medium and incubating for 20-24 hours; (2) Between the second and fourth days of differentiation induction, the culture medium was replaced with a second differentiation medium and the cells were incubated; (3) During the 4th to 7th day of differentiation induction, the culture medium was replaced with basal culture medium; (4) Starting from the 7th day of differentiation induction, the culture medium was replaced with maintenance medium to obtain differentiated cardiomyocytes; The basal medium consists of RPMI-1640 and insulin-free B27 supplement; The first differentiation medium is a basal medium supplemented with CHIR99021, Activin A and BMP4; The second differentiation medium is a basal medium supplemented with Wnt-C59; The maintenance medium consisted of RPMI-1640 and B27 supplement.

2. The method according to claim 1, characterized in that The first differentiation medium is a basal medium supplemented with 4-12 μM CHIR99021, 1-2 ng / mL Activin A and 1.25-2.5 ng / mL BMP4.

3. The method according to claim 1, characterized in that The second differentiation medium is a basal medium supplemented with 2 μM Wnt-C59.

4. The method according to claim 1, characterized in that The treatment time of the second differentiation medium is 44-48 hours.

5. The method according to claim 1, characterized in that During the 2-7 days of differentiation induction, fresh culture medium was replaced every day.

6. The method according to claim 1, characterized in that During the maintenance medium culture period, fresh medium was replaced every two days.

7. The method according to claim 1, characterized in that The embryonic stem cells are human embryonic stem cells.

8. Cardiomyocytes obtained by the method according to any one of claims 1 to 7.

9. Use of the cardiomyocytes according to claim 8 in the study of the pathogenic mechanism of heart diseases and drug screening.

10. Use of the cardiomyocytes according to claim 8 in the preparation of a medicine or preparation for repairing damaged cardiomyocytes.

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