Differentiation of primate pluripotent stem cells to cardiomyocyte-lineage cells

• US 20070010012A1
• Filed: 06/20/2006
• Published: 01/11/2007
• Est. Priority Date: 06/22/2005
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Inventors: Hassanipour, Mohammad, Gold, Joseph
• Granted Patent: US 9,062,289 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart 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.

Claims

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.