Differentiation of primate pluripotent stem cells to cardiomyocyte-lineage cells

Abstract

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.

The invention claimed is: 
     
         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.