DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS

• US 20110151560A1
• Filed: 12/16/2010
• Published: 06/23/2011
• 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
• Granted Patent: US 9,150,833 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• 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 population of cells expressing markers characteristic of the pancreatic endocrine lineage that co-express NKX6.1 and insulin and wherein less than 10% of the cells in the population express glucagon.

2. The population of cells in claim 1, wherein at least 30% of the cells express NKX6.1.

3. The population of cells in claim 1, wherein at least 40% of the cells express NKX.6.1

4. The population of cells in claim 1, wherein at least 50% of the cells express NKX6.1

5. The population of cells in claim 1, wherein at least 60% of the cells express NKX6.1

6. The population of cells in claim 1, wherein at least 5% of the cells express insulin.

7. A method to generate a population of cells expressing markers characteristic of the pancreatic endocrine lineage that co-express NKX6.1 and insulin and wherein less than 10% of the cells in the population express glucagon, the method comprising the steps of:

a. Culturing pluripotent stem cells,

b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage,

c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, and

d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage that co-express NKX6.1 and insulin and minimal amounts of glucagon by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in a medium supplemented with a factor capable of inhibiting BMP, a TGFβ

receptor signaling inhibitor, and a protein kinase C activator.