Differentiation of primate pluripotent stem cells to cardiomyocyte-lineage cells

• US 9,062,289 B2
• Filed: 06/20/2006
• Issued: 06/23/2015
• 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: Gold, Joseph D., Hassanipour, Mohammad
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Moloye, Titilayo
• Application Number:
  US11/471,916
• Publication Number:
  US 20070010012A1
• Time in Patent Office: 3,290 Days
• US Class Current: 1/1
• 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 a population of cells comprising α

myosin heavy chain (α

MHC) expressing cells from human embryonic stem (hES) cells by direct differentiation, comprising a) culturing the hES cells in the presence of Activin A in the absence of a BMP;

b) subsequently culturing the cells in the presence of a BMP and in the absence of Activin A for between three and five days, wherein the BMP is selected from the group consisting of BMP-2 and BMP-4 and c) subsequently culturing the cells in the presence of an insulin like growth factor (IGF) in the absence of Activin A or a BMP, wherein the IGF is selected from the group consisting of IGF-I and IGF-II, thereby producing a cell population comprising α

myosin heavy chain (α

MHC) expressing cells.

2. The method of claim 1, wherein the BMP of step b) is BMP-4.

3. 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.

4. The method of claim 1, wherein the culturing of step c) is performed for at least one week.

5. The method of claim 1, wherein the culturing of step c) is performed for at least two weeks.

6. The method of claim 1, wherein the IGF of step c) is IGF-I.

7. The method of claim 1, wherein the method does not comprise a step in which embryoid bodies are formed.

8. The method of claim 1, wherein the method further comprises harvesting cells from the culture subsequent to the IGF culturing step and enriching the harvested cell population for α

myosin heavy chain (α

MHC) expressing cells.

9. The method of claim 8, wherein the harvested cell population is enriched by a density gradient.

10. The method of claim 8, wherein the enriching the harvested cell population of α

myosin heavy chain (α

MHC) expressing cells comprises the formation of clusters of cells.

11. A method for obtaining an enriched population of α

myosin heavy chain (α

MHC) expressing cells from hES cells by direct differentiation, comprising 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 BMP-4 in the absence of Activin A for about four days;

c) subsequently culturing in a serum-free medium in the presence of IGF-I for about five days or more;

d) harvesting cells from the culture and e) enriching the harvested cell population for α

myosin heavy chain (α

MHC) expressing cells, thereby producing an enriched population of α

myosin heavy chain (α

MHC) expressing cells.

12. A method for obtaining a population of cells comprising α

myosin heavy chain (α

MHC) expressing cells from cells expressing stage specific embryonic antigen 3 (SSEA3), stage specific embryonic antigen 4 (SSEA4) and markers Tra-1-60 and Tra-1-81 by direct differentiation, comprising a) culturing the cells expressing stage specific embryonic antigen 3 (SSEA3), state stage specific embryonic antigen 4 (SSEA4) and markers Tra-1-60 and Tra-1-81 in the presence of Activin A in the absence of a BMP;

b) subsequently culturing the cells in the presence of BMP-2 or BMP-4 and the absence of Activin A for between three and five days; and

c) subsequently culturing the cells in the presence of an IGF in the absence of Activin A or a BMP wherein the IGF is selected from the group consisting of IGF-I and IGF-II, thereby producing a cell population comprising α

myosin heavy chain (α

MHC) expressing cells.

13. The method of claim 12, wherein the BMP of step b) is BMP-4.

14. The method of claim 12, wherein the cells expressing stage specific antigen embryonic antigen 3 (SSEA3), stage specific antigen embryonic antigen 4 (SSEA4) and markers Tra-1-60 and Tra-1-81 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.

15. The method of claim 12, wherein the culturing in the presence of an IGF is performed for at least one week.

16. The method of claim 12, wherein the culturing in the presence of an IGF is performed for at least two weeks.

17. The method of claim 12, wherein the IGF is IGF-I.

18. The method of claim 1, wherein the α

myosin heavy chain (α

MHC) expressing cells express cardiac troponin I.

19. The method of claim 11, wherein the α

myosin heavy chain (α

MHC) expressing cells express cardiac troponin I.

20. The method of claim 12, wherein the α

myosin heavy chain (α

MHC) expressing cells express cardiac troponin I.

21. The method of claim 1, wherein the α

myosin heavy chain (α

MHC) expressing cells beat in culture.

22. The method of claim 11, wherein the α

myosin heavy chain (α

MHC) expressing cells beat in culture.

23. The method of claim 12, wherein the α

myosin heavy chain (α

MHC) expressing cells beat in culture.