Differentiation of human embryonic stem cells into single hormonal insulin positive cells

• US 10,358,628 B2
• Filed: 08/20/2015
• Issued: 07/23/2019
• Est. Priority Date: 12/22/2011
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US14/831,115
• Publication Number:
  US 20150353895A1
• Time in Patent Office: 1,433 Days
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/38 Vitamins
• 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 2501/15 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 2501/19 Growth and differentiation factors [GDF]
• 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 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• 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 the differentiation of pluripotent stem cells. In particular, the present invention provides methods to produce a population of cells, wherein greater than 10% of the cells in the population express markers characteristic of single hormonal pancreatic beta cells.

Claims

1. An in vitro culture comprising a differentiated population of human pancreatic endoderm cells in which greater than 10% of the cells are single hormonal insulin positive cells and a population of human pancreatic foregut cells in a differentiation medium, wherein the differentiation medium differentiates pancreatic foregut cells into the differentiated population of pancreatic endoderm cells in which greater than 10% of the cells are single hormonal insulin positive cells and wherein said differentiation medium comprises one of the following:

a retinoid, ascorbic acid, and a shh-inhibitor,

a retinoid, ascorbic acid and SANT-1,

a retinoid, ascorbic acid, a shh-inhibitor and a TGF-β

ligand,

a retinoid, ascorbic acid, a shh-inhibitor and ALK5 inhibitor,

a retinoid, ascorbic acid, SANT-1 and a TGF-β

ligand,

a retinoid, ascorbic acid, a shh-inhibitor, ALK5 inhibitor, and a BMP inhibitor,

a retinoid, ascorbic acid, SANT-1, a TGF-β

ligand and a BMP inhibitor;

or

a retinoid, ascorbic acid, a shh-inhibitor, a TGF-β

ligand, and LDN-19318,

wherein the presence of ascorbic acid in the differentiation medium increases the number of insulin positive cells and decreases the number of glucagon positive cells and somatostatin positive cells compared to differentiation medium without ascorbic acid.

2. The in vitro culture of claim 1, wherein the human pancreatic foregut cells are obtained by stepwise differentiation of human embryonic stem cells cultured in a medium supplemented with a TGF-β

ligand.

3. The in vitro culture of claim 2, wherein the medium in said stepwise differentiation further comprises a WNT activator.

4. The in vitro culture of claim 1, wherein the human pancreatic foregut cells are obtained by stepwise differentiation of human definitive endoderm cells cultured in a medium supplemented with an FGF ligand.

5. The method of claim 1, wherein the differentiation medium further comprises a PKC activator.

6. The method of claim 1, wherein the differentiation medium comprises the retinoid, ascorbic acid, and the shh-inhibitor, and further comprises a PKC activator.

7. The in vitro culture of claim 4, wherein the medium in said stepwise differentiation further comprises ascorbic acid.

8. The in vitro culture of claim 2, wherein the human pancreatic foregut cells are obtained by stepwise differentiation of human pluripotent stem cells cultured in a medium supplemented with a TGF-β

ligand.

9. The in vitro culture of claim 2, wherein the medium in said stepwise differentiation further comprises GDF-8.

10. The in vitro culture of claim 9, wherein the medium in said stepwise differentiation further comprises GSK3β

inhibitor.

11. The in vitro culture of claim 10, wherein the medium in said stepwise differentiation further comprises MCX compound.

25. The in vitro culture of claim 1, wherein greater than 30% of the differentiated population of pancreatic endoderm cells are PDX-1+, NKX6.1+, SOX2−

, and CDX2−

.

28. The in vitro culture of claim 1, wherein the differentiation medium comprises the retinoid, ascorbic acid, and the shh-inhibitor.

29. The in vitro culture of claim 1, wherein the differentiation medium comprises the retinoid, ascorbic acid and SANT-1.

30. The in vitro culture of claim 1, wherein the differentiation medium comprises the retinoid, ascorbic acid, the shh-inhibitor and the TGF-β

ligand.

31. The in vitro culture of claim 1, wherein the differentiation medium comprises the retinoid, ascorbic acid, the shh-inhibitor and the ALK5 inhibitor.

32. The in vitro culture of claim 1, wherein the differentiation medium comprises the retinoid, ascorbic acid, SANT-1 and the TGF-β

ligand.

33. The in vitro culture of claim 1, wherein the differentiation medium comprises the retinoid, ascorbic acid, the shh-inhibitor, the ALK5 inhibitor, and the BMP inhibitor.

34. The in vitro culture of claim 1, wherein the differentiation medium comprises the retinoid, ascorbic acid, SANT-1, the TGF-β

ligand and the BMP inhibitor.

35. The in vitro culture of claim 1, wherein the differentiation medium comprises the retinoid, ascorbic acid, the shh-inhibitor, the TGF-β

ligand, and LDN-19318.

12. An in vitro culture comprising human pancreatic foregut cells in a differentiation medium supplemented with one of the following:

a retinoid, ascorbic acid, and a shh-inhibitor;

a retinoid, ascorbic acid and SANT-1;

a retinoid, ascorbic acid, a shh-inhibitor, and a TGF-β

ligand;

a retinoid, ascorbic acid, a shh-inhibitor and ALK5 inhibitor;

a retinoid, ascorbic acid, SANT-1 and a TGF-β

ligand;

a retinoid, ascorbic acid, a shh-inhibitor, ALK5 inhibitor, and a BMP inhibitor;

a retinoid, ascorbic acid, SANT-1, a TGF-β

ligand, and a BMP inhibitor;

or

wherein the differentiation medium differentiates the human pancreatic foregut cells into a differentiated population of human pancreatic endoderm cells in which greater than 10% of the cells are single hormonal insulin positive cells, and wherein the presence of the ascorbic acid in the differentiation medium increases the number of insulin positive cells and decreases the number of glucagon positive cells and somatostatin positive cells compared to differentiation medium without ascorbic acid.

13. The in vitro culture of claim 12, wherein the pancreatic foregut cells are obtained from stepwise differentiation of human pluripotent stem cells.

14. The in vitro culture of claim 13, wherein the stepwise differentiation comprises culturing human pluripotent stem cells in medium supplemented with GDF-8.

15. The in vitro culture of claim 14, wherein the medium in said stepwise differentiation further comprises a GSK3β

inhibitor.

16. The in vitro culture of claim 15, wherein the medium in said stepwise differentiation further comprises MCX compound.

26. The in vitro culture of claim 12, wherein greater than 30% of the differentiated population of pancreatic endoderm cells are PDX-1+, NKX6.1+, SOX2−

, and CDX2−

.

17. An in vitro culture of a differentiated population of human pancreatic endoderm cells in which greater than 10% of the cells are single hormonal insulin positive cells and a population of human pancreatic foregut cells in a differentiation medium supplemented with a retinoid, ascorbic acid and SANT-1, wherein the differentiation medium differentiates pancreatic foregut cells into the differentiated population of pancreatic endoderm cells in which greater than 10% of the cells are single hormonal insulin positive cells, and wherein the presence of the ascorbic acid in the differentiation medium increases the number of insulin positive cells and decreases the number of glucagon positive cells and somatostatin positive cells compared to differentiation medium without ascorbic acid.

18. The in vitro culture of pancreatic endoderm cells of claim 17, wherein the pancreatic foregut cells are obtained from stepwise differentiation of human pluripotent stem cells.

19. The in vitro culture of claim 18, wherein the stepwise differentiation comprises culturing human embryonic stem cells in medium supplemented with GDF-8.

20. The in vitro culture of claim 19, wherein the medium in said stepwise differentiation further comprises a GSK3β

inhibitor.

21. The in vitro culture of claim 20, wherein the medium in said stepwise differentiation further comprises MCX compound.

22. The in vitro culture of claim 17, wherein the differentiation medium is further supplemented with a TGF-β

ligand, a BMP inhibitor or both a TGF-β

ligand and a BMP inhibitor.

23. The in vitro culture of claim 22, wherein the TGF-β

ligand is ALK5 inhibitor.

24. The in vitro culture of claim 22, wherein the BMP inhibitor is LDN-193189.

27. The in vitro culture of claim 17, wherein greater than 30% of the differentiated population of pancreatic endoderm cells are PDX-1+, NKX6.1+, SOX2−

, and CDX2−

.