DIFFERENTIATION OF PRIMATE PLURIPOTENT STEM CELLS TO CARDIOMYOCYTE-LINEAGE CELLS
The present application describes the new methods for the differentiation of primate pluripotent stem cells into cardiomyocyte-lineage cells. The methods utilize sequential culturing of the primate pluripotent stem cells in certain growth factors to produce cardiomyocyte-lineage cells. In certain embodiments of the invention, the population of cells produced by the sequential culturing is further enriched for cardiomyocyte-lineage cells so as to produce a higher percentage of those cells.
1 . A method for obtaining cardiomyocyte-lineage cells from human embryonic stem (hES) cells, comprising
a) culturing the hES cells in the presence of Activin A in the absence of a BMP; and
b) subsequently culturing the cells in the presence of a BMP.
2 . The method of claim 1 , wherein the BMP is BMP-2, BMP-4, or BMP-7.
3 . The method of claim 2 , wherein the BMP is BMP-2.
4 . The method of claim 2 , wherein the BMP is BMP-4.
5 . The method of claim 2 , wherein the BMP is BMP-7.
6 . The method of claim 1 , wherein the culturing in the presence of a BMP is done in the absence of an Activin.
7 . The method of claim 1 , wherein the hES cells are cultured in the presence of Activin A for about one day and then subsequently cultured in the presence of the BMP for about four days.
8 . The method of claim 1 , further comprising subsequent to the BMP culturing step a culturing step in which the medium does not contain Activin A or a BMP.
9 . The method of claim 8 , wherein the further culturing step is performed for at least one week.
10 . The method of claim 8 , wherein the further culturing step is performed for at least two weeks.
11 . The method of claim 8 , wherein the further culturing step is done in medium containing IGF-I or IGF-II.
12 . The method of claim 11 , wherein the further culturing step is done in medium containing IGF-I.
13 . The method of claim 1 , wherein the method does not involve a step in which embryoid bodies are formed.
14 . The method of claim 1 , wherein the method further comprises harvesting cells from the culture subsequent to the BMP culturing step and enriching the harvested cell population for cardiomyocyte-lineage cells.
15 . The method of claim 14 , wherein the harvested cell population is enriched by Percoll gradient.
16 . The method of claim 14 , wherein the enrichment involves the formation of cardiac bodies.
17 . A method of obtaining an enriched population of cardiomyocyte-lineage cells from hES cells, comprising in the following order:
a) culturing the hES cells in a serum-free medium in the presence of Activin A in the absence of a BMP for about one day;
b) subsequently culturing in a serum-free medium in the presence of a BMP in the absence of an Activin for about four days;
c) harvesting cells from the culture; and
d) enriching the harvested cell population for cardiomyocyte-lineage cells.