Cardiomyocytes and methods of producing and purifying cardiomyocytes
The invention provides methods for producing a culture of cardiomyocytes and cultures of cardiomyocytes. Exemplary methods of producing and cultures of cardiomyocytes include a population of cells including cells having spontaneous and periodic electrical activity, and/or including nodal, sino-atrial or pacemaker cells; immature cardiomyocytes (cardiomyoblasts); mature contractile cardiomyocytes; or a mixed population of two or more of such cells.
1. A method for producing a culture of cardiomyocytes, comprising:
A) Providing a culture of stem cells that are at least 90% confluent or the cells have overgrown to form multiple layers of cells, or proliferating stem cells until the cells are at least 90% confluent or the cells have overgrown to form multiple layers of cells;
B) Inducing formation of mesoderm by contacting the overgrown stem cells with a bone morphogenic protein (BMP) receptor ligand and an fibroblast growth factor (FGF) receptor ligand for a period of about 2 to 15 days; and
C) promoting cardiomyocyte formation by contacting mesoderm cells with neural cells, or a neural cell conditioned culture supernatant, for a period of about 1 to 21 days, thereby producing a culture of cardiomyocytes.
2. The method of claim 1 , wherein the cardiomyocytes comprise a population of cells comprising cells having spontaneous and periodic electrical activity.
3. The method of claim 2 , wherein the cells having spontaneous and periodic electrical activity comprise nodal, sino-atrial or pacemaker cells; immature cardiomyocytes (cardiomyoblasts); mature contractile cardiomyocytes; or a mixed population thereof.
4. The method of claim 1 , wherein the stem cells of step A) comprise a cell monolayer with 100% or more confluency.
5. The method of claim 1 , wherein the stem cells of step A) form a multilayer culture.
6. The method of claim 1 , wherein the stem cells of step A) have a density of about 150,000 to about 250,000 cells/cm 2.
7. The method of claim 1 , wherein step B) further comprises addition of an acid.
8. The method of claim 7 , wherein the acid comprises lactic, citric, fumaric, ascorbic, folic, malic, succinic, oxalacetic, or ketoglutaric acid.
9. The method of claim 1 , wherein step B) further comprises subjecting the mesoderm cells to hypoxia.
10. The method of claim 1 , wherein the stem cells, mesoderm cells or cardiomyocytes are subjected to an oxygen concentration of about 0.5 to 2.0%.
11. The method of claim 1 , wherein step B) further comprises addition of an agent that inhibits cell death or apoptosis.
12. The method of claim 11 , wherein the agent comprises selenium, lithium; ascorbic acid or ascorbate or superoxide dismutase (SOD).
13. The method of claim 12 , wherein the selenium comprises sodium selenite.
14. The method of claim 12 , wherein the selenium is at a concentration of 1 to 20 ng/ml, or 0.5 to 50 ng/ml.
15. The method of claim 12 , wherein the lithium comprises lithium carbonate.
16. The method of claim 12 , wherein the lithium is at a concentration of 1 to 20 ng/ml, or 0.5-50 ng/ml.
17. The method of claim 12 , wherein the ascorbic acid or ascorbate comprises sodium or calcium ascorbate.
18. The method of claim 12 , wherein the ascorbic acid or ascorbate is at a concentration of 0.1 to 20 ug/ml, or 1 ug/ml.
19. The method of claim 12 , wherein the superoxide dismutase (SOD) is at a concentration of 1 to 100 u/ml, or 1 to 10 u/ml.