Differentiation of human embryonic stem cells into pancreatic endocrine cells

• US 10,066,210 B2
• Filed: 06/06/2013
• Issued: 09/04/2018
• Est. Priority Date: 06/08/2012
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Primary Examiner(s): Lankford, Blaine
• Application Number:
  US13/911,829
• Publication Number:
  US 20130330823A1
• Time in Patent Office: 1,916 Days
• Field of Search: None
• CPC Class Codes: C12N 2501/50 Cell markers; Cell surface determinants
• 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 differentiation of pancreatic endoderm cells to pancreatic endocrine rich clusters and to enhance insulin expression in hormone-expressing cells.

Claims

1. An in vitro cell culture comprising a culture medium supplemented with an effective amount of an Ephrin ligand to enhance insulin and NKX6.1 expression, and a population of cells comprising pancreatic endoderm cells and pancreatic endocrine precursor cells, wherein the population of cells expresses NKX6.1 and insulin, wherein the population of cells is obtained by culturing the pancreatic endoderm cells using the culture medium supplemented with the Ephrin ligand, wherein the culturing of the pancreatic endoderm cells in the medium supplemented with the Ephrin ligand enhances expression of insulin and NKX6.1 in the pancreatic endoderm cells to at least 2 times as much as the expression of insulin and NKX6.1 compared to non-treated pancreatic endoderm cells, and wherein the pancreatic endoderm cells are obtained by a stepwise in vitro differentiation of pluripotent stem cells.

2. The in vitro culture of claim 1, wherein the Ephrin ligand is Ephrin A3 or Ephrin A4.

3. The in vitro culture of claim 1, wherein the culture further comprises L-alanyl-L-glutamine.

4. The in vitro culture of claim 3, wherein the Ephrin ligand is Ephrin A3.

5. The in vitro culture of claim 3, wherein the Ephrin ligand is Ephrin A4.

6. The in vitro culture of claim 1, wherein the culture further comprises SANT-1.

7. The in vitro culture of claim 6, wherein the Ephrin ligand is Ephrin A3.

8. The in vitro culture of claim 6, wherein the Ephrin ligand is Ephrin A4.

9. The in vitro culture of claim 6, wherein the culture comprises further comprises L-alanyl-L-glutamine.