Differentiation of human embryonic stem cells

• US 9,150,833 B2
• Filed: 12/16/2010
• Issued: 10/06/2015
• 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): Singh, Anoop
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US12/970,330
• Publication Number:
  US 20110151560A1
• Time in Patent Office: 1,755 Days
• Field of Search: 435/366, 435/377
• US Class Current: 1/1
• 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 to generate a population of pancreatic endocrine cells that co-express NKX6.1 and insulin, the method comprising the steps of:

a) culturing human pluripotent stem cells;

b) differentiating the human pluripotent stem cells of step a) into definitive endoderm cells by culturing the human pluripotent stem cells in medium comprising a TGF-β

receptor agonist;

c) differentiating the definitive endoderm cells of step b) into pancreatic endoderm cells by culturing the definitive endoderm cells in the presence of an FGF family member and retinoic acid; and

d) differentiating the pancreatic endoderm cells of step c) into pancreatic endocrine cells by culturing the pancreatic endoderm cells in a medium supplemented with noggin, an inhibitor of ALK5 and a protein kinase C activator, wherein the pancreatic endocrine cells co-express NKX6.1 and insulin and wherein less than 10% of the cells in the population express glucagon.

2. The method of claim 1, wherein the inhibitor of ALK5 in step d) is ALK5 inhibitor II.

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

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

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

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

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

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

9. A method to generate a population of pancreatic endocrine cells that co-express NKX6.1 and insulin, the method comprising the steps of:

a) culturing human pluripotent stem cells;

b) differentiating the human pluripotent stem cells of step a) into definitive endoderm cells by culturing the human pluripotent stem cells in medium comprising a TGF-β

receptor agonist;

c) differentiating the definitive endoderm cells of step b) into pancreatic endoderm cells by culturing the definitive endoderm cells in the presence of an FGF family member and retinoic acid; and

d) differentiating the pancreatic endoderm cells of step c) into pancreatic endocrine cells by culturing the pancreatic endoderm cells in a medium supplemented with noggin, a TGF-β

receptor signaling inhibitor and (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam, wherein the pancreatic endocrine cells co-express NKX6.1 and insulin and wherein less than 10% of the cells in the population express glucagon.

10. The method of claim 9, wherein at least 30% of the cells express NKX6.1.

11. The method of claim 9, wherein at least 40% of the cells express NKX6.1.

12. The method of claim 9, wherein at least 50% of the cells express NKX6.1.

13. The method of claim 9, wherein at least 60% of the cells express NKX6.1.

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