Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells

• US 20130330823A1
• Filed: 06/06/2013
• Published: 12/12/2013
• Est. Priority Date: 06/08/2012
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Granted Patent: US 10,066,210 B2
• Time in Patent Office: Days
• US Class Current: 435/375
• CPC Class Codes: C12N 2501/50 Cell markers; Cell surface determinants
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides methods to promote differentiation of pancreatic endoderm cells to pancreatic endocrine rich clusters and to enhance insulin expression in hormone-expressing cells.

Claims

1. A method of inducing insulin expression in hormone-expressing cells comprising culturing pancreatic endoderm cells with an Ephrin ligand.

6. The method of any one of claims 1 to 5, wherein the Ephrin ligand is Ephrin A3 or Ephrin A4

7. The method of claim 6, wherein the pancreatic endoderm cells are obtained by a stepwise differentiation of pluripotent stem cells.

8. The method of claim 7, wherein the pluripotent stem cells are human embryonic pluripotent stem cells.

9. Insulin and NKX6.1-expressing cells prepared by the method of claim 1.

2. The method of claim 2, wherein culturing the pancreatic endoderm cells with the Ephrin ligand also enhances expression of NKX6.1.

3. The method of claim 2, wherein culturing the pancreatic endoderm cells with an Ephrin ligand enhances expression of insulin and NKX6.1 in the pancreatic endoderm cells when compared with the expression of insulin and NKX6.1 in non-treated pancreatic endoderm cells.

4. The method of claim 3, wherein the pancreatic endoderm cells do not substantially express CDX2 or SOX2.

5. The method of claim 4, wherein the pancreatic endoderm cells express approximately about less than 10% CDX2 or SOX2.

10. A population of cells prepared by culturing pancreatic endoderm cells with an Ephrin ligand, wherein the population of cells expresses enhanced insulin and NKX6.1 as compare to pancreatic endoderm cells not treated with an Ephrin ligand.

11. The population of cells of claim 10, wherein the Ephrin ligand is Ephrin A3 or Ephrin A4.

12. A method of enhancing expression of somatostain in hormone-expressing cells, comprising culturing pancreatic endoderm cells in medium comprising Activin A or Activin C.

13. The method of claim 12, wherein expression of insulin, glucagon, and ghrelin is suppressed.

14. The method of claim 12, wherein the population of pancreatic endoderm cells treated with Activin A or Activin C expresses more somatostatin as a population of pancreatic endoderm cells non-treated with Activin A or Activin C.

15. The method of claim 14, wherein the expression of insulin is suppressed in the population of pancreatic endoderm cells treated with Activin A or Activin C as compared to the expression of insulin in a population of pancreatic endoderm cells non-treated with Activin A or Activin C.

16. The method of claim 12, wherein the expression of glucagon in the population of pancreatic endoderm cells treated with Activin A or Activin C is suppressed as compared to the expression of glucagon in a population of pancreatic endoderm cells non-treated with Activin A or Activin C.

17. The method of claim 12, wherein the expression of ghrelin is suppressed in the population of pancreatic endoderm cells treated with Activin A or Activin C as compared to the expression of ghrelin in a population of pancreatic endoderm cells non-treated with Activin A or Activin C.

18. The method of any one of claims 12 to 18, wherein the pancreatic endoderm cells do not substantially express CDX2 or SOX2.

19. The method of claim 18, wherein the pancreatic endoderm cells treated with Activin A or Activin C are obtained by a stepwise differentiation of pluripotent cells.

20. The method of claim 19, wherein the pluripotent cells where the pancreatic endoderm cells are derived from are human embryonic pluripotent cells.

21. A method of enhancing expression of NKX6.1 by treating pancreatic endoderm cells in medium comprising semaphorin 3a or Epigen.

22. The method of claim 21, wherein the population of pancreatic endoderm cells treated with medium comprising semaphorin 3a or Epigen expresses an enhanced amount of NKX6.1 as compared to pancreatic endoderm cells non-treated with medium comprising semaphorin 3a or Epigen.

23. The method of claim 21, wherein the level of expression of insulin, glucagon, and gherlin is not affected in pancreatic endoderm cells treated with medium comprising semaphorin 3a or Epigen as compared to pancreatic endoderm cells not treated with medium comprising semaphorin 3a or Epigen.

24. A method for inducing formation of endocrine clusters, comprising culturing pancreatic endocrine cells with a sphingosine-1 receptor agonist.

25. The method of claim 24, wherein the sphingosine-1 receptor agonist used for treating pancreatic endocrine cells is sphingosine-1-phosphate (SIP).

26. The method of claim 24, wherein the pancreatic endocrine cells are obtained by a stepwise differentiation of pluripotent stem cells.

27. The method of claim 26, wherein the pluripotent stem cells are human embryonic pluripotent stem cells.