DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS

• US 20100015100A1
• Filed: 07/31/2008
• Published: 01/21/2010
• Est. Priority Date: 07/31/2007
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Xu, Jean
• Granted Patent: US 9,096,832 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 7/12 Antidiuretics, e.g. drugs for diabetes insipidus ADH A61P5/10
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• 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 2501/21 Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

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.

Claims

1. A method for generating cells expressing markers characteristic of the pancreatic endocrine lineage, comprising differentiating cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF, in medium supplemented with glucose at a concentration from about 10 mM to about 20 mM.

2. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are differentiated from pluripotent stem cells.

3. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are derived from cells expressing markers characteristic of the definitive endoderm lineage.

4. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are derived from pluripotent stem cells.

5. The method of claim 2, wherein the pluripotent stems cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage before being further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage.

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 cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 2days to about 30 days.

9. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 2 days to about 20 days.

10. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 2 days to about 10 days.

11. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 10 days.

12. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 4 days.

13. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 2 days.

14. The method of claim 6, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with glucose for about 2 days to about 30 days.

15. The method of claim 7, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with glucose for about 2 days to about 30 days.

27. A method for treating a patient having diabetes by transplanting the cells of claim 1 into the patient.

16. A method for differentiating pluripotent stem cells into cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:

a. Culturing the pluripotent stem cells,

b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, by treating the pluripotent cells with activin A,

c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor, and

d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF, in medium supplemented with glucose at a concentration from about 10 mM to about 20 mM.

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 cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 2 days to about 30 days.

20. The method of claim 16, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 2 days to about 20 days.

21. The method of claim 16, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 2 days to about 10 days.

22. The method of claim 16, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 10 days.

23. The method of claim 16, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 4 days.

24. The method of claim 16, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin 4 and HGF for about 2 days.

25. The method of claim 21, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with glucose for about 2 days to about 30 days.

26. The method of claim 22, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with glucose for about 2 days to about 30 days.

28. A method for treating a patient having diabetes by transplanting the cells of claim 16 into the patient.

29. The method of claim 16, wherein the pluripotent stem cells are embryonic stem cells.

30. The method of claim 29, wherein the embryonic stem cells are human embryonic stem cells.

31. The method of claim 30, wherein the human embryonic stem cells are derived from a cell line of the group consisting of H1 and H9.