Differentiation of human embryonic stem cells
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
A61K-038/00
A61K-035/12
C12N-005/071
출원번호
US-0183656
(2008-07-31)
등록번호
US-9096832
(2015-08-04)
발명자
/ 주소
Xu, Jean
출원인 / 주소
LifeScan, Inc.
대리인 / 주소
Gianneschi, Lois A.
인용정보
피인용 횟수 :
3인용 특허 :
72
초록▼
The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present inventio
The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.
대표청구항▼
1. A method for generating human pancreatic endocrine cells, comprising differentiating isolated pancreatic endoderm cells into the pancreatic endocrine cells by culturing the pancreatic endoderm cells in medium supplemented with glucose at a concentration from about 10 mM to about 20 mM and treatin
1. A method for generating human pancreatic endocrine cells, comprising differentiating isolated pancreatic endoderm cells into the pancreatic endocrine cells by culturing the pancreatic endoderm cells in medium supplemented with glucose at a concentration from about 10 mM to about 20 mM and treating with a factor selected from the group consisting of: a gamma secretase inhibitor, Exendin-4, and a combination of Exendin-4 and hepatocyte growth factor. 2. The method of claim 1, wherein the human pancreatic endoderm cells are differentiated from human pluripotent stem cells. 3. The method of claim 1, wherein the human pancreatic endoderm cells are derived from definitive endoderm cells. 4. The method of claim 1, wherein the human pancreatic endocrine cells are derived from human pluripotent stem cells. 5. The method of claim 2, wherein the human pluripotent stems cells are differentiated into definitive endoderm cells before being further differentiated into pancreatic endoderm cells. 6. The method of claim 1, wherein the glucose is used at a concentration of about 10 mM. 7. The method of claim 1, wherein the glucose is used at a concentration of about 20 mM. 8. The method of claim 1, wherein the human pancreatic endocrine cells are treated with the factor for about 2 days to about 30 days. 9. The method of claim 1, wherein the human pancreatic endocrine cells are treated with the factor for about 2 days to about 20 days. 10. The method of claim 1, wherein the human pancreatic endocrine cells are treated with the factor for about 2 days to about 10 days. 11. The method of claim 1, wherein the human pancreatic endocrine cells are treated with the factor for about 10 days. 12. The method of claim 1, wherein the human pancreatic endocrine cells are treated with the factor for about 4 days. 13. The method of claim 1, wherein the human pancreatic endocrine cells are treated with the factor for about 2 days. 14. The method of claim 6, wherein the human pancreatic endocrine cells are treated with glucose for about 2 days to about 30 days. 15. The method of claim 7, wherein the human pancreatic endocrine cells are treated with glucose for about 2 days to about 30 days. 16. A method for differentiating human pluripotent stem cells into human pancreatic endocrine cells, comprising the steps of: a) culturing the human pluripotent stem cells,b) differentiating the human pluripotent stem cells into human definitive endoderm cells, by treating the human pluripotent cells with activin A,c) differentiating the human definitive endoderm cells into human pancreatic endoderm cells, by treating the human definitive endoderm cells with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor, andd) differentiating the human pancreatic endoderm cells into human pancreatic endocrine cells, by culturing the human pancreatic endoderm cells in medium supplemented with glucose at a concentration from about 10 mM to about 20 mM and treating with a factor selected from the group consisting of: a gamma secretase inhibitor, Exendin-4, and a combination of Exendin-4 and hepatocyte growth factor. 17. The method of claim 16, wherein the glucose is used at a concentration of about 10 mM. 18. The method of claim 16, wherein the glucose is used at a concentration of about 20 mM. 19. The method of claim 16, wherein the human pancreatic endocrine cells are treated with the factor for about 2 days to about 30 days. 20. The method of claim 16, wherein the human pancreatic endocrine cells are treated with the factor for about 2 days to about 20 days. 21. The method of claim 16, wherein the human pancreatic endocrine cells are treated with the factor for about 2 days to about 10 days. 22. The method of claim 16, wherein the human pancreatic endocrine cells are treated with the factor for about 10 days. 23. The method of claim 16, wherein the human pancreatic endocrine cells are treated with the factor for about 4 days. 24. The method of claim 16, wherein the human pancreatic endocrine cells are treated with the factor for about 2 days. 25. The method of claim 21, wherein the human pancreatic endocrine cells are treated with glucose for about 2 days to about 30 days. 26. The method of claim 22, wherein the human pancreatic endocrine cells are treated with glucose for about 2 days to about 30 days. 27. The method of claim 16, wherein the human pluripotent stem cells are human embryonic stem cells. 28. The method of claim 27, wherein the human embryonic stem cells are derived from a cell line of the group consisting of H1 and H9. 29. The method of claim 1, wherein the human pancreatic endocrine cells are treated with a gamma secretase inhibitor. 30. The method of claim 1, wherein the human pancreatic endocrine cells are treated with a combination of Exendin-4 and hepatocyte growth factor. 31. The method of claim 1, wherein the method up-regulates expression of pancreatic endocrine markers. 32. The method of claim 1, wherein the method increases expression of one or more of NGN-3, NeuroD-1, Nkx2.2, Pax-4, insulin, or glucagon. 33. The method of claim 16, wherein the pancreatic endocrine cells are treated with a gamma secretase inhibitor. 34. The method of claim 16, wherein the pancreatic endocrine cells are treated with a combination of Exendin-4 and hepatocyte growth factor. 35. The method of claim 16, wherein the step of differentiating the human cells pancreatic endoderm cells into human pancreatic endocrine cells up-regulates expression of pancreatic endocrine markers. 36. The method of claim 1, wherein the step of differentiating the human pancreatic endoderm cells into human pancreatic endocrine cells increases expression of one or more of NGN-3, NeuroD-1, Nkx2.2, Pax-4, or glucagon. 37. The method of claim 1, wherein the method increases the fraction of pancreatic endocrine cells expressing insulin. 38. The method of claim 1, wherein the method increases the fraction of pancreatic endocrine cells expressing synaptophysin.
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