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 cultur
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.
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1. A method of increasing the yield of cells expressing markers characteristic of the definitive endoderm lineage by differentiating human pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, the method comprising treating the human pluripotent stem
1. A method of increasing the yield of cells expressing markers characteristic of the definitive endoderm lineage by differentiating human pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, the method comprising treating the human pluripotent stem cells with a medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one, for a period of time sufficient for the human pluripotent stem cells to differentiate into cells expressing markers characteristic of the definitive endoderm lineage, wherein the method increases the percentage of cells expressing CXCR4. 2. The method of claim 1, wherein the method increases the percentage of cells expressing CXCR4 when compared to cells treated in a medium comprising activin A. 3. The method of claim 1, wherein the medium is further lacking Wnt3A. 4. The method of claim 1, wherein the medium further comprises at least one factor selected from the group consisting of: EGF, FGF4, PDGF-A, PDGF-B, PDGF-C, PDGF-D, VEGF, muscimol, PD98059, LY294002, U0124, U0126, and sodium butyrate. 5. A method of enhancing the differentiation of pluripotent stem cells comprising culturing the pluripotent stem cells for about one to about three days in a medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one to differentiate the pluripotent cells into definitive endoderm cells. 6. The method of claim 5, wherein the method increases the percentage of cells expressing CXCR4 when compared to cells treated in a medium comprising activin A. 7. The method of claim 5, wherein the medium is further lacking Wnt3A. 8. The method of claim 5, wherein the medium further comprises at least one factor selected from the group consisting of: EGF, FGF4, PDGF-A, PDGF-B, PDGF-C, PDGF-D, VEGF, muscimol, PD98059, LY294002, U0124, U0126, and sodium butyrate. 9. The method of claim 5, wherein the medium comprises from about 0.625 μM to about 10 μM of 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜.1˜8,12-]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one. 10. The method of claim 5, wherein the method comprises culturing the pluripotent stem cells for about three days in a medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜.1˜8,12-]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one. 11. A method of increasing the yield of definitive endoderm cells by differentiating pluripotent stem cells into definitive endoderm cells comprising: culturing the pluripotent stem cells in a medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜.1˜8,12-]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one; andculturing the cells in a medium lacking activin A, and containing GDF-8 to differentiate the pluripotent stem cells into definitive endoderm cells, wherein the method increases the percentage of cells expressing CXCR4. 12. The method of claim 11, wherein the pluripotent stem cells are cultured in the medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one for about one day. 13. The method of claim 11, wherein the cells are cultured in the medium lacking activin A, and containing GDF-8 for about two days. 14. The method of claim 11, wherein the medium lacking activin A further lacks Wnt3A. 15. The method of claim 11, wherein the method increases the percentage of cells expressing CXCR4 when compared to cells treated in a medium comprising activin A. 16. The method of claim 11, wherein the medium further comprises at least one factor selected from the group consisting of: EGF, FGF4, PDGF-A, PDGF-B, PDGF-C, PDGF-D, VEGF, muscimol, PD98059, LY294002, U0124, U0126, and sodium butyrate. 17. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells. 18. A method of differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage comprising treating the pluripotent stem cells with a medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one, for a period of time sufficient for the pluripotent stem cells to differentiate into cells expressing markers characteristic of the definitive endoderm lineage. 19. The method of claim 18, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells. 20. The method of claim 18, wherein the medium further comprises at least one factor selected from the group consisting of: EGF, FGF4, PDGF-A, PDGF-B, PDGF-C, PDGF-D, VEGF, muscimol, PD98059, LY294002, U0124, U0126, and sodium butyrate. 21. The method of claim 18, wherein the cells are cultured in the medium lacking activin A, and containing GDF-8 for about one to seven days. 22. The method of claim 18, wherein the medium is further lacking Wnt3A.
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