Differentiation of pluripotent stem cells
The present invention is directed to methods to differentiate pluripotent stem cells. In particular, the present invention is directed to methods and compositions to differentiate pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage comprising culturing the pluripotent stem cells in medium comprising a sufficient amount of GDF-8 to cause the differentiation of the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage.
1. A method to differentiate pluripotent stem cells into definitive endoderm cells comprising culturing the pluripotent stem cells with a medium supplemented with activin A, Wnt3a, 5-chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1˜3,7˜.1˜9,13˜.1˜14,18˜] tritriaconta-3(33),4,6,9(32),10,12,14(31),15,17-nonaen-23-one and one or more of EGF and FGF4.
2. The method of claim 1 , wherein the medium is supplemented with activin A, Wnt3a, 5-chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1˜3,7˜.1˜9,13˜.1˜14,18˜] tritriaconta-3(33),4,6,9(32),10,12,14(31),15,17-nonaen-23-one and EGF.
3. The method of claim 1 , wherein the medium is supplemented with activin A, Wnt3a, 5-chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1˜3,7˜.1˜9,13˜.1-14,18˜] tritriaconta-3(33),4,6,9(32),10,12,14(31),15,17-nonaen-23-one and FGF-4.
4. The method of claim 1 , wherein the medium is supplemented with activin A, Wnt3a, 5-chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1˜3,7˜.1˜9,13˜.1˜14,18˜] tritriaconta-3(33),4,6,9(32),10,12,14(31),15,17-nonaen-23-one EGF and FGF-4.
5. The method of claim 4 , wherein the medium is further supplemented with PDGF-AB.
6. The method of claim 5 wherein the medium is further supplemented with VEGF.
7. The method of claim 4 , wherein the medium is further supplemented with GDF-8 and muscimol.
8. The method of claim 4 , wherein the medium is further supplemented with PDFG-A, VEGF, PDGF, muscimol and GDF-8.
9. The method of claim 1 , wherein the pluripotent stem cells are human pluripotent stem cells.
10. The method of claim 9 , wherein the human pluripotent stem cells are human embryonic stem cells.
11. A method to differentiate pluripotent stem cells into definitive endoderm cells comprising culturing the pluripotent stem cells with a medium supplemented with activin A and 5-chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1˜3,7˜.1˜9,13˜.1˜14,18˜] tritriaconta-3(33),4,6,9(32),10,12,14(31),15,17-nonaen-23-one.
12. The method of claim 11 , wherein the medium is further supplemented with Wnt3a.
13. The method of claim 11 , wherein the pluripotent stem cells are human pluripotent stem cells.
14. The method of claim 13 , wherein the human pluripotent stem cells are human embryonic stem cells.