Direct differentiation method for making cardiomyocytes from human embryonic stem cells
This invention provides a new procedure for generating cardiomyocyte lineage cells from embryonic stem cells for use in regenerative medicine. Differentiating by way of embryoid body formation or in serum is no longer required. Instead, the stem cells are plated onto a solid substrate, and differentiated in the presence of select factors and morphogens. After enrichment for cells with the appropriate phenotype, the cells are allowed to cluster into cardiac bodies™, which are remarkably homogeneous and suitable for the treatment of heart disease.
1. A direct differentiation method of obtaining cardiomyocyte lineage cells from primate pluripotent stem (pPS) cells, comprising in the following order:
a) obtaining undifferentiated pPS cells;
b) plating the undifferentiated pPS cells without forming embryoid bodies directly onto a solid surface comprising a substrate to which cardiomyocyte lineage cells adhere without forming embryoid bodies;
c) permitting the plated pPS cells to adhere onto the substrate;
d) culturing the adhered cells in the absence of serum or feeder cells, but in the presence of activin and a bone morphogenic protein to produce cardiomyocyte lineage cells;
e) harvesting the cardiomyocyte lineage cells from the culture.
2. The method of claim 1 , wherein the undifferentiated pPS cells in step a) are an established line of human embryonic stem cells.
3. The method of claim 1 , wherein the pPS cells are plated in step b) onto a substrate coated with gelatin or fibronectin.
4. The method of claim 1 , wherein the cells are adhered in step c) by culturing as undifferentiated pPS cells on said substrate for four days or more.
5. The method of claim 1 , wherein the cells are cultured in step d) with Activin A and BMP-4 for four days or more.
6. The method of claim 1 , wherein the cells are cultured after step d) in the absence of either Activin A or BMP-4 for one week or more.
7. The method of claim 1 , wherein the cells are cultured after step d) in the absence of BMP-2, but in the presence of a medium supplement containing insulin, transferrin, and selenium.
8. The method of claim 1 , further comprising separating the harvested cells into fractions according to their density; and then collecting the fractions that express MHC from an endogenous gene.
9. The method of claim 1 ; further comprising:
a) separating cells in the enriched cell population that are present as single cells from cells that are present as clusters;
b) resuspending the cells present as clusters in nutrient medium;
c) reculturing the resuspended cells in the nutrient medium; and
d) collecting and washing the recultured cells;
thereby generating cell clusters in which the clusters express a cardiomyocyte marker.
10. The method of claim 9 , wherein the single cells are separated from the clustered cells by allowing the clustered cells to settle from suspension, and cells remaining in suspension are removed.
11. The method of claim 9 , wherein the nutrient medium in which the resuspended cells are cultured contains about 20% serum or serum substitute.
12. The method of claim 9 , comprising separating, resuspending, and reculturing the cells three or more times.
13. The method of claim 9 , further comprising dispersing the cardiac bodies into a suspension of single cells and/or smaller cell clusters.
14. The method of claim 1 , further comprising culturing the cells from d) in the absence of either activin or bone morphogenic protein.