Use of noggin, an ALK5 inhibitor and a protein kinase c activator to produce endocrine cells

• US 10,704,025 B2
• Filed: 03/13/2017
• Issued: 07/07/2020
• Est. Priority Date: 12/23/2009
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Xu, Jean
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US15/457,956
• Publication Number:
  US 20170183630A1
• Time in Patent Office: 1,212 Days
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• 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 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• 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 2501/70 Enzymes
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• 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 into insulin producing cells. In particular, the present invention provides a method to produce cells expressing markers characteristic of the pancreatic endocrine lineage that co-express NKX6.1 and insulin and minimal amounts of glucagon.

Claims

1. A method of producing a population of NKX6.1 and insulin co-expressing human pancreatic endocrine cells comprising culturing human pancreatic endoderm cells in a medium supplemented with noggin, an inhibitor of ALK5, and a protein kinase C activator to obtain a population of NKX6.1 and insulin co-expressing human pancreatic endocrine cells, wherein less than 10% of the cells in the population express glucagon.

2. The method of claim 1, wherein the method comprises culturing for about 6 to about 12 days.

3. The method of claim 1, wherein the inhibitor of ALK5 is ALK5 inhibitor II.

4. The method of claim 1, wherein the protein kinase C activator is (2S, 5S)-(E, E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam.

5. The method of claim 1, wherein at least 30% of the pancreatic endocrine cells express NKX6.1.

6. The method of claim 1, wherein at least 40% of the pancreatic endocrine cells express NKX6.1.

7. The method of claim 1, wherein at least 50% of the pancreatic endocrine cells express NKX6.1.

8. The method of claim 1, wherein at least 60% of the pancreatic endocrine cells express NKX6.1.

9. The method of claim 1, wherein at least 5% of the pancreatic endocrine cells express insulin.

10. A method of producing human pancreatic endocrine cells comprising culturing human pancreatic endoderm cells in a medium supplemented with noggin, an inhibitor of ALK5, and a protein kinase C activator and obtaining human pancreatic endocrine cells.

11. The method of claim 10, wherein less than 10% of the pancreatic endocrine cells in the population express glucagon.

12. The method of claim 10, wherein the method comprises culturing for about 6 to about 12 days.

13. The method of claim 10, wherein the inhibitor of ALK5 is ALK5 inhibitor II.

14. The method of claim 10, wherein the protein kinase C activator is (2S, 5S)-(E, E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam.

15. The method of claim 10, wherein at least 30% of the pancreatic endocrine cells express NKX6.1.

16. The method of claim 10, wherein at least 40% of the pancreatic endocrine cells express NKX6.1.

17. The method of claim 10, wherein at least 50% of the pancreatic endocrine cells express NKX6.1.

18. The method of claim 10, wherein at least 60% of the pancreatic endocrine cells express NKX6.1.

19. The method of claim 10, wherein at least 5% of the pancreatic endocrine cells express insulin.