Methods for purifying cells derived from pluripotent stem cells
The present invention is directed to methods to differentiate pluripotent stem cells. In particular, the present invention provides methods of characterization of cells differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage utilizing unique surface markers. The present invention also provides methods to enrich or sort cells expressing markers characteristic of the pancreatic endocrine lineage. The present invention also provides methods to deplete cells that may contaminate populations of cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention, thereby reducing the incidence of tumor formation in vivo following transplantation.
1. A method to produce a population of pancreatic endocrine precursor cells enriched for NeuroD, NGN3, PDX-1, and NKX6.1 comprising the steps of:
a) differentiating a population of human pluripotent stem cells into definitive endoderm cells by treating the pluripotent stem cells with a culture medium supplemented with activin A;
b) differentiating the definitive endoderm cells into a population of pancreatic endocrine precursor cells;
c) selecting pancreatic endocrine precursor cells; and
d) enriching the population of pancreatic endocrine precursor cells for NeuroD, NGN3, PDX-1, and NKX6.1 by screening the population of cells for one of the following surface markers or surface marker combinations to thereby produce an enriched population of cells that is negative for CD13 and:
1) positive for CD56,
2) positive for CD56 and CD15 lo , or
3) positive for both CD56 and CD57.
2. The method of claim 1 , wherein the population of pancreatic endocrine precursor cells is screened for cells that are positive for CD56 and negative for CD13.
3. The method of claim 1 , wherein the population of pancreatic endocrine precursor cells is screened for cells that are positive for CD56 and CD15 lo .
4. The method of claim 1 , wherein the population of pancreatic endocrine precursor cells is screened for cells that are positive for positive for both CD56 and CD57.
5. A method to produce a population of pancreatic endocrine precursor cells enriched for NeuroD, NGN3, PDX-1, and NKX6.1 comprising the steps of:
a) differentiating a population of human pluripotent stem cells into definitive endoderm cells by treating the pluripotent stem cells with a culture medium supplemented with activin A;
b) differentiating the definitive endoderm cells into a population of pancreatic endocrine precursor cells; and
c) enriching the population of pancreatic endocrine precursor cells for NeuroD, NGN3, PDX-1, and NKX6.1 by screening the population of cells for cells that are positive for CD56 and CD57, and negative for CD13.
6. A method of enriching a population of pancreatic endocrine precursor cells for NeuroD, NGN3, PDX-1, and NKX6.1 by screening the population of cells for one of the following surface markers or surface marker combinations to thereby produce an enriched population of cells that is negative for CD13 and:
1) positive for CD56,
2) positive for CD56 and CD15 lo , or
3) positive for both CD56 and CD57.
7. The method of claim 6 , wherein the population of pancreatic endocrine precursor cells is screened for cells that are positive for CD56 and negative for CD13.
8. The method of claim 6 , wherein the population of pancreatic endocrine precursor cells is screened for cells that are positive for CD56 and CD15 lo .
9. The method of claim 6 , wherein the population of pancreatic endocrine precursor cells is screened for cells that are positive for positive for both CD56 and CD57.
10. The method of claim 6 , wherein the pancreatic endocrine precursor cells are derived by in vitro differentiation of human pluripotent stem cells.
11. A method of generating pancreatic endocrine cells comprising:
obtaining a population of pancreatic endocrine precursor cells enriched for NeuroD, NGN3, PDX-1, and NKX6.1 using the method of claim 1 ; and
differentiating the pancreatic endocrine precursor cells into pancreatic endocrine cells.
12. A method of generating pancreatic endocrine cells comprising:
obtaining a population of pancreatic endocrine precursor cells enriched for NeuroD, NGN3, PDX-1, and NKX6.1 using the method of claim 6 ; and
differentiating the pancreatic endocrine precursor cells into pancreatic endocrine cells.