Compositions And Methods For Self-Renewal And Differentiation In Human Embryonic Stem Cells

• US 20070281355A1
• Filed: 08/15/2005
• Published: 12/06/2007
• Est. Priority Date: 08/13/2004
• Status: Active Grant

• Current Assignee: The University of Georgia Research Foundation, Inc., Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Dalton, Stephen, Sheppard, Allan, Jones, Karen, Agulnick, Alan, D'Amour, Kevin, Baetge, E.
• Granted Patent: US 8,187,878 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 5/16 for decreasing, blocking or antagonising the activity of the thyroid hormones
• CPC Class Codes: A61P 5/18 of the parathyroid hormones
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/405 Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

The present invention provides compositions and methods for the production of differentiated mammalian cells. More particularly, the present invention provides cellular differentiation methods employing culturing the cells on a feeder layer or under feeder-free conditions in cell culture and further contacting the cells with an inhibitor of the PI3-kinase pathway for the generation of differentiated mammalian cells from pluripotent mammalian stem cells. Preferably, the differentiated cell is selected from the group consisting of a mesendodermal cell, a mesodermal cell, and an endodermal cell.

Claims

1. A method of differentiating a pluripotent mammalian cell comprising:

providing the pluripotent mammalian cell, and

contacting the pluripotent mammalian cell with an effective amount of an inhibitor of the

PI3-kinase signaling pathway and a member of the TGFβ

family to at least partially differentiate the pluripotent cell to a cell of the endoderm lineage.

2. The method of claim 1, wherein the mammalian cell is contacted with the inhibitor and the member of the TGFβ

family for greater than approximately 24 hours.

3. The method of claim 1, wherein GSK3 is activated.

4. The method of claim 1, wherein the inhibitor is selected from the group consisting of LY 294002, Rapamycin, wortmannin, lithium chloride, Akt inhibitor I, Akt inhibitor II, Akt inhibitor III, NL71-101, and mixtures of the foregoing.

5. The method of claim 1, wherein the inhibitor is LY 294002.

6. The method of claim 5, wherein LY 294002 is initially present at a concentration of approximately 1 μ

M to approximately 500 μ

M.

7. The method of claim 5, wherein LY 294002 is initially present at a concentration of approximately 20 μ

M to approximately 163 μ

M.

8. The method of claim 1, wherein the inhibitor is Rapamycin.

9. The method of claim 8, wherein Rapamycin is initially present at a concentration of approximately 0.1 nM to approximately 500 nM.

10. The method of claim 8, wherein Rapamycin is initially present at a concentration of approximately 1.5 nM to approximately 30 nM.

11. The method of claim 1, wherein the inhibitor is AktI-II.

12. The method of claim 11, wherein AktI-II is initially present at a concentration of approximately 10 μ

M to approximately 50 μ

M.

13. The method of claim 1, wherein step (b) occurs in the presence of bFGF.

14. The method of claim 13, wherein bFGF is initially present at a concentration of approximately 1 ng/ml to approximately 12 ng/ml.

15. The method of claim 1, wherein the member of the TGFβ

family is selected from the group consisting of Nodal, Activin A, Activin B, TGF-β

, BMP2, BMP4, and mixtures of the foregoing.

16. The method of claim 1, wherein the member of the TGFβ

family is Activin A.

17. The method of claim 15, wherein the member of the TGFβ

family is present in a conditioned medium.

18. The method of claim 17, wherein the conditioned medium is obtained from a feeder layer of embryonic fibroblasts.

19. The method of claim 1, wherein the mammalian pluripotent cell is selected from the group consisting of an embryonic stem cell, an ICM/epiblast cell, a primitive ectoderm cell, a primordial germ cell, and a teratocarcinoma cell.

20. The method of claim 19, wherein the mammalian pluripotent cell is a human embryonic stem cell.

21. The method of claim 1, wherein the cell is dissociated to an essentially single cell culture prior to contacting the cell with the inhibitor.

22. The method of claim 21, wherein the cell is dissociated using a protease.

23. The method of claim 22, wherein the protease is trypsin.

24. The method of claim 1, wherein the pluripotent cell is plated prior to contact with the PI3-kinase inhibitor and the member of the TGFβ

family.

25. The method of claim 1, wherein the differentiated cell is a definitive endoderm cell.

26-73. -73. (canceled)