Pancreatic endocrine cells and methods thereof

• US 10,456,424 B2
• Filed: 07/26/2017
• Issued: 10/29/2019
• Est. Priority Date: 07/31/2007
• 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:
  US15/660,885
• Publication Number:
  US 20170354690A1
• Time in Patent Office: 825 Days
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 7/12 Antidiuretics, e.g. drugs for diabetes insipidus ADH A61P5/10
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• 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 2501/21 Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/999 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

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.

Claims

1. A method for generating human pancreatic endocrine cells, comprising differentiating pancreatic endoderm cells into pancreatic endocrine cells by culturing the pancreatic endoderm cells with a medium supplemented with Exendin-4, then removing the medium supplemented with Exendin-4 and subsequently culturing the cells in medium supplemented with hepatocyte growth factor (HGF), Exendin-1 and insulin growth factor (IGF)-1.

2. The method of claim 1, wherein the pancreatic endoderm cells are differentiated from human pluripotent stem cells.

3. The method of claim 1, wherein the pancreatic endoderm cells are derived from definitive endoderm cells.

4. The method of claim 1, wherein the pancreatic endoderm cells are treated with a gamma secretase inhibitor, or Exendin-4, or Exendin-4 and HGF for about 2 days to about 30 days.

5. The method of claim 1, wherein the method comprises treating the pancreatic endoderm cells with a medium supplemented with a gamma secretase inhibitor.

6. The method of claim 1, wherein the method comprises treating the pancreatic endoderm cells with a medium supplemented with Exendin-4.

7. The method of claim 1, wherein the method comprises treating the pancreatic endoderm cells with a medium supplemented with Exendin-4 and HGF.

8. A method for differentiating pluripotent stem cells into pancreatic endocrine cells, comprising the steps of:

a. Differentiating human pluripotent stem cells into definitive endoderm cells by treating the pluripotent cells with activin A;

b. Differentiating the definitive endoderm cells into pancreatic endoderm cells by treating the definitive endoderm cells with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor (FGF); and

c. Differentiating the pancreatic endoderm cells into pancreatic endocrine cells by treating the pancreatic endoderm cells with Exendin-4, then removing the medium supplemented with Exendin-4 and subsequently culturing the cells in medium supplemented with hepatocyte growth factor (HGF), Exendin-1 and insulin growth factor (IGF)-1.

9. The method of claim 8, wherein the method comprises treating the pluripotent cells with activin A and a GSK-3B inhibitor.

10. The method of claim 8, wherein the FGF is FGF-2 or FGF-4.

11. The method of claim 8, wherein the method comprises treating the definitive endoderm cells with FGF-2 and retinoic acid or FGF-4 and retinoic acid.

12. The method of claim 8, wherein the pancreatic endoderm cells are treated with a gamma secretase inhibitor, or Exendin-4, or 4 and HGF for about 2 days to about 30 days.

13. The method of claim 11, wherein the method comprises treating the pancreatic endoderm cells with a medium supplemented with a gamma secretase inhibitor.

14. The method of claim 11, wherein the method comprises treating the pancreatic endoderm cells with a medium supplemented with Exendin-4.

15. The method of claim 11, wherein the method comprises treating the pancreatic endoderm cells with a medium supplemented with Exendin-4 and HGF.