Methods for increasing definitive endoderm production

• US 7,993,916 B2
• Filed: 04/12/2010
• Issued: 08/09/2011
• Est. Priority Date: 09/24/2007
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Baetge, Emmanuel Edward, Agulnick, Alan, D'Amour, Kevin
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US12/758,726
• Publication Number:
  US 20100197014A1
• Time in Patent Office: 484 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 method for increasing production of human definitive endoderm cells comprising:

providing an inhibitor of E-cadherin-mediated cellular adhesion to human pluripotent stem cells, thereby inhibiting adhesion between said human pluripotent stem cells; and

differentiating said human pluripotent stem cells by contacting said cells with a medium comprising at least one TGFβ

superfamily growth factor, thereby increasing the production of human definitive endoderm cells.

2. The method of claim 1, wherein the inhibitor of E-cadherin-mediated cellular adhesion is an antagonist of E-cadherin.

3. The method of claim 2, wherein the antagonist of E-cadherin binds E-cadherin.

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

5. The method of claim 2, wherein the antagonist of E-cadherin is a peptide.

6. The method of claim 5, wherein the peptide is an E-cadherin peptide corresponding to an E-cadherin extracellular domain at amino acid residues 600-700 of human E-cadherin (SEQ ID NO:

1).

7. The method of claim 2, wherein the antagonist of E-cadherin is a peptide analog.

8. The method of claim 1, wherein the inhibitor of E-cadherin-mediated cellular adhesion comprises a calcium ion chelator.

9. The method of claim 8, wherein the calcium ion chelator 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.

10. The method of claim 1, wherein said human pluripotent stem cells are provided with said inhibitor of E-cadherin-mediated cellular adhesion in a first medium, and wherein said pluripotent human stem cells are differentiated in a second medium.

11. The method of claim 1, wherein said human pluripotent stem cells comprise embryonic stem cells.

12. The method of claim 11, wherein said human embryonic stem cells are grown on a feeder layer prior to differentiation.

13. The method claim 1, wherein said human pluripotent stem cells are maintained in a culture vessel prior to differentiation.

14. The method of claim 13, wherein said culture vessel further comprises fibroblasts.

15. The method of claim 1, wherein said TGFβ

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

16. The method of claim 1, wherein said TGFβ

superfamily growth factor comprises a member of the BMP subgroup.