Differentiation of human embryonic stem cells

• US 9,012,218 B2
• Filed: 10/22/2009
• Issued: 04/21/2015
• Est. Priority Date: 10/31/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Primary Examiner(s): Long, Scott
• Application Number:
  US12/603,889
• Publication Number:
  US 20100112692A1
• Time in Patent Office: 2,007 Days
• US Class Current: 435/377
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/155 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 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• 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 a method to increase the expression of markers associated with the pancreatic endocrine lineage.

Claims

1. A method of increasing the expression of markers associated with pancreatic endocrine cells comprising treating human pancreatic endocrine cells with medium comprising a sufficient amount of a TGF-β

receptor agonist for about one to about five days to cause an increase in expression of markers associated with the pancreatic endocrine cells compared to pancreatic endocrine cells not treated with the TGF-β

receptor agonist, wherein the method comprises an increase in expression of PDX-1, NKX6.1, and PAX4.

2. The method of claim 1, wherein the TGF-β

receptor agonist is selected from the group consisting of activin A, activin B, activin C, peptide variants of activin A, GDF-8, GDF-11 and GDF-15.

3. The method of claim 1, wherein the pancreatic endocrine cells are enriched by obtaining a suspension of the pancreatic endocrine cells and separating the suspension of cells through a strainer;

wherein the pancreatic endocrine cells having increased expressing markers characteristic of the pancreatic endocrine are retained by the strainer.

4. The method of claim 1, wherein the method also increases expression of NKX2.2.

5. The method of claim 1, wherein the pancreatic endocrine cell is a pancreatic hormone expressing or pancreatic hormone secreting cell.

6. The method of claim 5, wherein the method also increases expression of insulin or glucagon.

7. A method of increasing the expression of markers associated with pancreatic endocrine cells in a population of pancreatic endocrine cells derived by differentiating human embryonic pluripotent stem cells, the method comprising treating the population of pancreatic endocrine cells with medium comprising a sufficient amount of a TGF-β

receptor agonist for about one to about five days, wherein the treatment increases expression of markers associated with pancreatic endocrine cells compared to pancreatic endocrine cells not treated with the TGF-β

receptor agonist, and wherein the method comprises an increase in expression of PDX-1, NKX6.1, and PAX4.

8. The method of claim 7, wherein the method also increases expression of NKX2.2.

9. The method of claim 7, wherein the pancreatic endocrine cell is a pancreatic hormone expressing or pancreatic hormone secreting cell.

10. The method of claim 9, wherein the method also increases expression of insulin or glucagon.

11. The method of claim 7, wherein the TGF-β

receptor agonist is selected from the group consisting of activin A, activin B, activin C, peptide variants of activin A, GDF-8, GDF-11 and GDF-15.

12. The method of claim 7, wherein the pancreatic endocrine cells are enriched by obtaining a suspension of pancreatic endocrine cells and separating the suspension of cells through a strainer;

wherein pancreatic endocrine cells having increased expression of markers associated with the pancreatic endocrine are retained by the strainer.