Direct differentiation of cardiomyocytes from embryonic stem cells

• US 9,404,085 B2
• Filed: 10/12/2012
• Issued: 08/02/2016
• Est. Priority Date: 12/22/2005
• Status: Active Grant

• Current Assignee: ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Original Assignee: ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Inventors: Davidson, Bruce Paul, Graichen, Ralph Eberhard, Zweigerdt, Robert, Xu, Xiuqin, Mummery, Christine Lindsay, Sun, William
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US13/651,151
• Publication Number:
  US 20140193909A1
• Time in Patent Office: 1,390 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/99 Serum-free medium
• CPC Class Codes: C12N 2501/02 Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/30 tumour cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0043 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5061 Muscle cells

Abstract

The present invention relates to the induction of differentiation in human embryonic stem (hES) cells to cardiomyocytes and factors such as prostaglandin I2 (PGI2), Fgf 9, Bmp6, Bmp4, Scf, Igf2, and insulin that influence the process of differentiation to cardiomyocytes. Media that is appropriate for the induction of differentiation of cardiomyocytes from hES cells is also provided wherein the media contains these factors. Genes that are upregulated in the process of cardiomyocyte differentiation are also provided.

Claims

1. A cell-free culture medium for differentiating stem cells to cardiomyocytes, comprising a prostaglandin or an analog or functional equivalent thereof.

2. The cell culture medium of claim 1, wherein the medium is serum free.

3. The cell culture medium of claim 1, wherein the medium further comprises transferrin.

4. The cell culture medium of claim 1, wherein the medium further comprises selenium.

5. The cell culture medium of claim 1, wherein the medium further comprises BMP4.

6. The cell culture medium of claim 1, wherein the medium further comprises BMP6.

7. The cell culture medium of claim 1, wherein the medium further comprises FGF9.

8. The cell culture medium of claim 1, wherein the medium further comprises SCF.

9. The cell culture medium of claim 1, wherein the medium further comprises IGF2.

10. The cell culture medium of claim 1, wherein the medium further comprises a p38 MAP kinase inhibitor.

11. The cell culture medium of claim 1, wherein the prostaglandin is PGI2.

21. The cell culture medium of claim 10, wherein the p38 MAP kinase inhibitor is SB203580.

12. A cell-free culture medium for differentiating pluripotent stem cells to cardiomyocytes, free of insulin and comprising PGI2.

13. The cell culture medium of claim 12, wherein the medium is serum free.

14. The cell culture medium of claim 12, wherein the medium further comprises transferrin.

15. The cell culture medium of claim 12, wherein the medium further comprises selenium.

16. The cell culture medium of claim 12, wherein the medium further comprises BMP4.

17. The cell culture medium of claim 12, wherein the medium further comprises BMP6.

18. The cell culture medium of claim 12, wherein the medium further comprises FGF9.

19. The cell culture medium of claim 12, wherein the medium further comprises SCF.

20. The cell culture medium of claim 12, wherein the medium further comprises IGF2.

22. A non-xenogeneic in vitro cell culture, comprising a human cardiac progenitor cell in a culture medium comprising a prostaglandin or an analog or functional equivalent thereof.

23. The cell culture of claim 22, wherein the cardiac progenitor cell beats.