In vitro differentiation of pluripotent stem cells to pancreatic endoderm cells (PEC) and endocrine cells
A human immature endocrine cell population and methods for making an immature endocrine cell population are provided. Specifically, immature beta cells and methods for production of immature beta cells are described. Immature beta cells co-express INS and NKX6.1 and are uni-potent and thereby develop into mature beta cells when implanted in vivo. The mature beta cells in vivo are capable of producing insulin in response to glucose stimulation.
1. An in vitro cell culture comprising:
human cells, wherein at least 10% of said human cells are immature beta cells that co-express INS and NKX6.1; and
an effective amount of Y-27632 to promote survival of the immature beta cells in the cell culture.
2. The cell culture of claim 1 , wherein the cell culture is in a semipermeable encapsulation device.
3. The cell culture of claim 1 , wherein said human immature beta cells do not substantially express NGN3.
4. A cryopreserved in vitro cell culture comprising:
human cells, wherein the human cells comprise unipotent human immature beta cell suspension aggregates and wherein at least 10% of cells in the aggregates are human immature beta cells that co-express INS and NKX6.1, and
a cryopreservation agent.
5. The cell culture of claim 4 , wherein the unipotent human immature beta cell suspension aggregates are in a semipermeable encapsulation device.
6. The cell culture of claim 4 , wherein the suspension aggregates are about 50 to 200 microns in size.
7. A cryopreserved in vitro cell culture comprising:
human cells, wherein at least 10% of said human cells are immature beta cells that co-express INS and NKX6.1; and
a cryopreservation agent.
8. The cell culture of claim 7 , wherein said human immature beta cells do not substantially express NGN3.
9. The cell culture of claim 7 , wherein the immature beta cells are in a semipermeable encapsulation device.
10. The cell culture of claim 7 , wherein the immature beta cells are in a perforated encapsulation device.
11. The cell culture of claim 7 , wherein said human immature beta cells further co-expresses pancreatic-duodenal homeobox factor-1 (PDX1).
12. The cell culture of claim 7 , wherein said human immature beta cells are capable of maturing to a mature beta cells.
13. The cell culture of claim 7 , wherein said human immature beta cells further expresses MAFB.
14. The cell culture of claim 7 , further comprising Chromogranin A (CHGA)-negative cells.
15. The cell culture of claim 7 , further comprising a compound selected from the group consisting of bone morphogenetic protein, sonic hedgehog, platelet-derived growth factor, a member of the FGF superfamily, retinoic acid, retinoic acid analogs, insulin-like growth factor, a vitamin B3 derivative, and Nicotinamide.
16. The cell culture of claim 7 , further comprising a matrix.
17. A cryopreserved in vitro cell culture comprising:
human cells, wherein at least 50% of said human cells are immature beta cells that co-express INS and NKX6.1; and
a cryopreservation agent.
18. The cell culture of claim 17 , wherein the immature beta cells are in a semipermeable encapsulation device.