Differentiation of human embryonic stem cells
The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides a method to increase the expression of markers associated with the pancreatic endocrine lineage.
1. A method for differentiating pluripotent stem cells into pancreatic endocrine cells comprising the steps of:
a) Culturing the pluripotent stem cells;
b) Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage;
c) Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage;
d) Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into a population comprising pancreatic endocrine cells; and
e) Treating the population comprising pancreatic endocrine cells with a medium comprising a cyclic aniline-pyridinotriazine and a TGF-β receptor agonist to increase the number of pancreatic endocrine cells in the population,
wherein the TGF-β receptor agonist is GDF-8, GDF-11 or GDF-15.
2. The method of claim 1 , wherein the step of treating with the medium comprising a cyclic aniline-pyridinotriazine and a TGF-β receptor agonist causes an increase in expression of markers associated with the pancreatic endocrine lineage.
3. The method of claim 1 , wherein the method further comprises enriching the pancreatic endocrine cells by obtaining a suspension of the cells and separating the suspension of cells through a strainer, wherein the enriched cells are retained by the strainer.
4. The method of claim 1 , wherein the method comprises an increase in expression of PDX-1, NKX6.1 and PAX4.
5. The method of claim 1 , wherein the pancreatic endocrine cell is a pancreatic hormone expressing or pancreatic hormone secreting cells.
6. A method for differentiating pluripotent stem cells into pancreatic endocrine cells comprising:
a) Differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage;
b) Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage;
c) Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into a population comprising pancreatic endocrine cells; and
d) Treating the population comprising pancreatic endocrine cells with a medium comprising a cyclic aniline-pyridinotriazine and a TGF-β receptor agonist to increase the number of pancreatic endocrine cells in the population,
wherein the TGF-β receptor agonist is GDF-8, GDF-11 or GDF-15.
7. The method of claim 6 , wherein the step of treating with the medium comprising a cyclic aniline-pyridinotriazine and a TGF-β receptor agonist causes an increase in expression of markers associated with the pancreatic endocrine lineage.
8. The method of claim 6 , wherein the method further comprises enriching the pancreatic endocrine cells by obtaining a suspension of the cells and separating the suspension of cells through a strainer, wherein the enriched cells are retained by the strainer.
9. The method of claim 6 , wherein the method comprises an increase in expression of PDX-1, NKX6.1 and PAX4.
10. The method of claim 6 , wherein the pancreatic endocrine cell is a pancreatic hormone expressing or pancreatic hormone secreting cells.
11. The method of claim 1 , wherein step e) comprises treating the pancreatic endocrine cells with the medium comprising a cyclic aniline-pyridinotriazine and a TGF-β receptor agonist for about three to about five days.
12. The method of claim 6 , wherein step d) comprises treating the pancreatic endocrine cells with the medium comprising a cyclic aniline-pyridinotriazine and a TGF-β receptor agonist for about three to about five days.
13. The method of claim 1 , wherein the step of treating increases expression of PDX-1, NKX6.1 and PAX4.
14. The method of claim 6 , wherein the step of treating increases expression of PDX-1, NKX6.1 and PAX4.
15. A method for differentiating pluripotent stem cells into pancreatic endocrine cells comprising:
a) Differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage;
b) Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage;
c) Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into a population comprising pancreatic endocrine cells; and
d) Treating the population comprising pancreatic endocrine cells with a medium comprising a cyclic aniline-pyridinotriazine and GDF-8, GDF-11 or GDF-15 to increase expression of PDX-1, NKX6.1 and PAX4 in the population.
16. The method of claim 15 , wherein the step of treating also increases NKX2.2, insulin and glucagon expression in the population.
17. The method of claim 15 , wherein the method further comprises enriching the pancreatic endocrine cells by obtaining a suspension of the cells and separating the suspension of cells through a strainer, wherein the enriched cells are retained by the strainer.