METHODS FOR INCREASING DEFINITIVE ENDODERM PRODUCTION

• US 20090093372A1
• Filed: 09/24/2007
• Published: 04/09/2009
• 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
• Granted Patent: US 7,695,963 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 definitive endoderm production comprising:

providing an agent to a cell culture comprising E-cadherin-expressing human embryonic stem cells, thereby inhibiting adhesion of the E-cadherin-expressing cells to each other; and

differentiating said E-cadherin-expressing human embryonic stem cells by contacting said cells with a medium comprising a growth factor of the Nodal/Activin subgroup of the TGFβ

superfamily of growth factors, thereby increasing the production of definitive endoderm.

2. The method of claim 1, wherein the agent binds E-cadherin.

3. The method of claim 2, wherein the agent is an antagonist of E-cadherin.

4. The method of claim 3, wherein the antagonist is a polyclonal or a monoclonal E-cadherin antibody, wherein the agent is a peptide or peptide analog.

5. The method of claim 1 wherein the agent is a peptide or a peptide analog.

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 1, wherein the agent is a calcium ion chelator.

8. The method of claim 7, 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.

9. The method of claim 1, wherein said E-cadherin-expressing human embryonic stem cells are provided with said agent in a first medium, and wherein said E-cadherin-expressing human embryonic stem cells are differentiated in a second medium.

10. The method of claim 1, wherein said first medium is different from said second medium.

11. A method for identifying an agent capable of increasing production of a cell derived from a human embryonic stem cell, said method comprising:

providing a candidate agent to a human embryonic stem cell culture;

differentiating the human embryonic stem cell in a culture medium comprising a differentiation factor known to be capable of promoting the differentiation of said human embryonic stem cells; and

determining whether the candidate agent increases the production of cells differentiated from said human embryonic stem cells by comparing the production of differentiated cells in said cell culture provided with the candidate agent to the production of differentiated cells in a human embryonic stem cell culture that has not been provided with said candidate agent but has been treated with the same differentiation factor as the cell culture provided with the candidate agent, wherein greater production of differentiated cells in the cell culture provided with the candidate agent as compared to the production of differentiated cells in the cell culture not provided with the candidate agent indicates that the candidate agent increases the production of a cell derived from a human embryonic cell.

12. The method of claim 11, wherein the differentiation factor is a growth factor of the Nodal/Activin subgroup of the TGFβ

superfamily.

13. The method of claim 11, wherein the differentiated cell is a definitive endoderm cell or derivative thereof.

14. The method of claim 11, wherein the agent is an antagonist of human E-cadherin.

15. The method of claim 11, wherein the agent is a synthetic compound.

16. The method of claim 15, wherein the agent is a natural product.

17. An in vitro composition comprising an antagonist of E-cadherin specifically bound to an E-cadherin-expressing human embryonic stem cells, wherein the binding of the antagonist inhibits adhesion between said embryonic stem cells.

18. The composition of claim 17, wherein the antagonist is a polyclonal or a monoclonal E-cadherin antibody.

19. The composition of claim 17, wherein at least 10% of the human embryonic stem cells are not adhered to other human embryonic stem cells.

20. The composition of claim 17, wherein at least 50% of the human embryonic stem cells are not adhered to other human embryonic stem cells.

22. The cell culture of claim 19, 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.

23. The cell culture of claim 22, wherein at least 10% of the human embryonic stem cells are not adhered to other human embryonic stem cells.

24. The composition of claim 22, wherein at least 50% of the human embryonic stem cells are not adhered to other human embryonic stem cells.

21. A cell culture comprising 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.

25. A method of identifying an E-cadherin agonist or antagonist comprising:

providing a peptide library of peptides derived from hESCs and an E-cadherin peptide;

screening said peptide library for peptides having high affinity binding to the E-cadherin peptide; and

selecting a member of the peptide library binding to the E-cadherin peptide wherein the affinity of the member is equivalent to or higher than that of a native homotypic E-cadherin peptide.