Treatment of pluripotent cells
The present invention is provides a method for treating human pluripotent cells. In particular, the methods of the invention are directed to the treatment of human pluripotent cells, whereby the human pluripotent cells can be efficiently expanded in culture and differentiated by treating the pluripotent cells with an inhibitor of glycogen synthase kinase 3β (GSK-3B) enzyme activity.
1. A method to expand and differentiate human embryonic stem cells, comprising the steps of:
a. Culturing human embryonic stem cells, and
b. Treating the human embryonic stem cells with an inhibitor of glycogen synthase kinase 3β (GSK-3B) enzyme activity, wherein the inhibitor is 3-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione.
2. The method of claim 1 , wherein the human embryonic stem cells express at least one of the following pluripotency markers: ATP-binding cassette, subfamily G, member 2 (ABCG2), crypto, forkhead box D3 (FoxD3), Connexin43, Connexin45, POU domain transcription factor Oct4, sex-determining region Y-box2 (SOX-2), Nanog, human telomerase reverse transcriptase (hTERT), undifferentiated embryonic cell transcription factor 1 (UTF-1), zinc finger protein 42 (ZFP42), stage-specific embryonic antigen 3 (SSEA-3), stage-specific embryonic antigen 4 (SSEA-4), tumor-related antigen 1-60 (Tra1-60), and tumor-related antigen 1-81 (Tra1-81).
3. The method of claim 1 , wherein the human embryonic stem cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage.
4. The method of claim 1 , wherein the human embryonic stem cells are treated with the inhibitor of GSK-3B enzyme activity for about one to about 72 hours.
5. The method of claim 1 , wherein the human embryonic stem cells are treated with the inhibitor of GSK-3B enzyme activity for about 12 to about 48 hours.
6. The method of claim 1 , wherein the human embryonic stem cells are treated with the inhibitor of GSK-3B enzyme activity for about 48 hours.
7. The method of claim 1 , wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 100 nM to about 10004.
8. The method of claim 1 , wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 1 μM to about 10 μM.
9. The method of claim 1 , wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 10 μM.