Differentiation of human embryonic stem cells

• US 9,062,290 B2
• Filed: 11/25/2008
• Issued: 06/23/2015
• Est. Priority Date: 11/27/2007
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Rezania, Alireza
• Primary Examiner(s): Singh, Anoop
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US12/277,904
• Publication Number:
  US 20090170198A1
• Time in Patent Office: 2,401 Days
• US Class Current: 1/1
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/335 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 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• 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 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• 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
• CPC Class Codes: C12N 5/0678 : Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells and the products related to or resulting from such methods. In particular, the present invention provides an improved method for the formation of pancreatic hormone expressing cells and pancreatic hormone secreting cells. In addition, the present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer and the products related to or resulting from such methods. The present invention also provides methods to promote glucose-stimulated insulin secretion in insulin-producing cells derived from pluripotent stem cells.

Claims

1. A method for producing cells capable of glucose-stimulated insulin secretion from human pluripotent stem cells, comprising the steps of:

a) culturing the human pluripotent stem cells,

b) differentiating the human pluripotent stem cells into definitive endoderm cells by treating the human pluripotent stem cells with a medium supplemented with a TGF-β

receptor agonist,

c) differentiating the definitive endoderm cells into pancreatic endoderm cells, and

d) differentiating the pancreatic endoderm cells into pancreatic endocrine cells by treating the pancreatic endoderm cells with an inhibitor of TGF-β

R-1 kinase.

2. The method of claim 1, wherein the pancreatic endoderm cells are treated with the inhibitor of TGF-β

R-1 kinase for about one to about twelve days.

3. The method of claim 1, wherein the pancreatic endoderm cells are treated with the inhibitor of TGF-β

R-1 kinase for about five to about twelve days.

4. The method of claim 1, wherein the pancreatic endoderm cells are treated with the inhibitor of TGF-β

R-1 kinase for about twelve days.

5. The method of claim 1, wherein the inhibitor of TGF-β

R-1 kinase is used at a concentration from about 0.1 μ

M to about 100 μ

M.

6. The method of claim 1, wherein the inhibitor of TGF-β

R-1 kinase is 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine.

7. The method of claim 1, wherein the inhibitor of TGF-β

R-1 kinase is [3-(Pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole.

8. The method of claim 1, wherein the pancreatic endoderm cells are further treated with at least one factor selected from the group consisting of a factor capable of inhibiting BMP and a netrin.

9. The method of claim 1, wherein the step of differentiating the pancreatic endoderm cells into pancreatic endocrine cells is achieved by treating the pancreatic endoderm cells with a factor that inhibits the Notch signaling pathway, and the inhibitor of TGF-β

R-1 kinase.

10. The method of claim 9, wherein the cells are treated with a factor that inhibits the Notch signaling pathway, and the inhibitor of TGF-β

R-1 kinase for about 1 to about 12 days.

11. The method of claim 9, wherein the cells are treated with a factor that inhibits the Notch signaling pathway, and the inhibitor of TGF-β

R-1 kinase for about five to about twelve days.

12. The method of claim 9, wherein the cells are treated with a factor that inhibits the Notch signaling pathway, and the inhibitor of TGF-β

R-1 kinase for about twelve days.

13. The method of claim 9, wherein the factor that inhibits the Notch signaling pathway is a γ

-secretase inhibitor.

14. The method of claim 13, wherein the γ

-secretase inhibitor is L-685,458.

15. The method of claim 13, wherein L-685,458 is used at a concentration from about 0.1 μ

M to about 100 μ

M.

16. The method of claim 1, wherein the pancreatic endoderm cells are further treated with at least one factor selected from the group consisting of a factor capable of inhibiting BMP and a netrin.

17. The method of claim 1, wherein the step of differentiating the definitive endoderm cells into pancreatic endoderm cells is achieved by treating the definitive endoderm cells with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin.

18. The method of claim 17, wherein the definitive endoderm cells are treated with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin for about one to about six days.

19. The method of claim 17, wherein the definitive endoderm cells are treated with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin for about six days.

20. The method of claim 17, wherein the definitive endoderm cells are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF7, and FGF-10 for about one to about four days.

21. The method of claim 20, wherein the definitive endoderm cells are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

22. The method of claim 20, wherein the definitive endoderm cells are treated with a sonic hedgehog inhibitor, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF7, and FGF-10 for about four days.

23. The method of claim 17, wherein the definitive endoderm cells are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

24. The method of claim 23, wherein the definitive endoderm cells are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

25. The method of claim 23, wherein the definitive endoderm cells are treated with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

26. The method of claim 17, wherein the definitive endoderm cells are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, a netrin, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

27. The method of claim 26, wherein the definitive endoderm cells are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

28. The method of claim 26, wherein the definitive endoderm cells are treated with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, a netrin, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

29. The method of claim 17, wherein the factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 is FGF-7.

30. The method of claim 29, wherein the definitive endoderm cells are treated with FGF-7 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

31. The method of claim 29, wherein the definitive endoderm cells are treated with FGF-7 at a concentration of 20 ng/ml.

32. The method of claim 17, wherein the definitive endoderm cells are treated with retinoic acid at a concentration from about 1 nM to about 1 mM.

33. The method of claim 17, wherein the definitive endoderm cells are treated with retinoic acid at a concentration of 1 μ

M.

34. The method of claim 17, wherein the sonic hedgehog inhibitor is cyclopamine.

35. The method of claim 34, wherein cyclopamine is used at a concentration from about 0.1 μ

M to about 10 μ

M.

36. The method of claim 34, wherein cyclopamine is used at a concentration of 0.25 μ

M.

37. The method of claim 17, wherein the factor capable of inhibiting BMP is a BMP4 inhibitor.

38. The method of claim 37, wherein the BMP4 inhibitor is noggin.

39. The method of claim 37, wherein the definitive endoderm cells are treated with noggin at a concentration from about 500 ng/ml to about 100 μ

g/ml.

40. The method of claim 37, wherein the definitive endoderm cells are treated with noggin at a concentration of 100 ng/ml.

41. The method of claim 17, wherein the definitive endoderm cells are treated with the netrin at a concentration from about 500 ng/ml to about 100 μ

g/ml.

42. The method of claim 17, wherein the definitive endoderm cells are treated with the netrin at a concentration of 100 ng/ml.

43. The method of claim 17, wherein the netrin is selected from the group consisting of netrin 1, netrin 2, and netrin 4.