Methods for increasing definitive endoderm production

• US 7,695,963 B2
• Filed: 09/24/2007
• Issued: 04/13/2010
• Est. Priority Date: 09/24/2007
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: CyThera, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: Baetge, Emmanuel Edward, Agulnick, Alan, D'Amour, Kevin
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US11/860,494
• Publication Number:
  US 20090093372A1
• Time in Patent Office: 932 Days
• Field of Search: 435/366
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/14 Calcium; Ca chelators; Calcitonin
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/58 Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies

Abstract

Disclosed herein are methods for increasing the production of definitive endoderm cells from pluripotent stem cells. Also disclosed herein are agents capable of increasing definitive endoderm cell production.

Claims

1. An in vitro composition comprising a TGFβ

superfamily growth factor and an antagonist of E-cadherin, said antagonist of E-cadherin bound to E-cadherin-expressing human embryonic stem cells, wherein the binding of the antagonist inhibits adhesion between said embryonic stem cells.

2. The composition of claim 1, wherein the antagonist is a polyclonal or a monoclonal E-cadherin antibody.

3. The composition of claim 1, wherein at least 10% of the human embryonic stem cells are not adhered to other human embryonic stem cells.

4. The composition of claim 1, wherein at least 50% of the human embryonic stem cells are not adhered to other human embryonic stem cells.

6. The cell culture of claim 3, wherein the calcium binding agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), 1,10 phenanthroline, diethylenetriaminepentaacetate (DTPA), hydroxyethylethylenediaminetriacetic acid (HEEDTA), diaminocyclohexanetetraacetic acid (CDTA), 1,2-Bis(2-aminophenoxy)ethane-N,N,N′

,N′

-tetraacetic acid (BAPTA), and pharmaceutically acceptable salts thereof.

7. The cell culture of claim 6, wherein at least 10% of the human embryonic stem cells are not adhered to other human embryonic stem cells.

8. The composition of claim 6, wherein at least 50% of the human embryonic stem cells are not adhered to other human embryonic stem cells.

9. The composition of claim 1, wherein said TGFβ

superfamily growth factor comprises Activin A.

10. The composition of claim 1, wherein said TGFβ

superfamily growth factor comprises Activin B.

11. The composition of claim 1, wherein said TGFβ

superfamily growth factor comprises Nodal.

12. The composition of claim 1, wherein said composition further comprises human definitive endoderm cells.

17. The composition of claim 1, wherein said TGFβ

superfamily growth factor comprises a member of the Nodal/Activin subgroup.

18. The composition of claim 1, wherein said TGFβ

superfamily growth factor comprises a member of the BMP subgroup.

5. A cell culture comprising a TGFβ

superfamily growth factor, a calcium-binding agent and E-cadherin-expressing human embryonic stem cells in a culture medium, wherein the calcium-binding agent is bound to calcium ions in the culture medium, thereby inhibiting adhesion between said embryonic stem cells.

13. The cell culture of claim 5, wherein said TGFβ

superfamily growth factor comprises Activin A.

14. The cell culture of claim 5, wherein said TGFβ

superfamily growth factor comprises Activin B.

15. The cell culture of claim 5, wherein said TGFβ

superfamily growth factor comprises Nodal.

16. The cell culture of claim 5, wherein said cell culture further comprises human definitive endoderm cells.

19. The cell culture of claim 5, wherein said TGFβ

superfamily growth factor comprises a member of the Nodal/Activin subgroup.

20. The cell culture of claim 5, wherein said TGFβ

superfamily growth factor comprises a member of the BMP subgroup.