Stem cell derived islet differentiation
Provided herein are methods of producing β cells and precursors thereof utilizing a Wnt signaling inhibitor or PKC activator, or both. Also provided herein are in vitro cultures comprising said cells, methods of treating a subject with a disease characterized by high blood sugar levels over a prolonged period of time by administering said cells, and devices for encapsulating said cells.
1. A method of lowering blood glucose level in a subject having diabetes, comprising administering to the subject a composition comprising a population of cells that are derived from definitive endoderm cells in vitro, wherein:
a) at least 30% of the cells in the population express NKX6.1 and ISL1;
b) at least 30% of the cells in the population express ISL1 but do not express NKX6.1;
c) less than 20% of the cells in the population express NKX6.1 but do not express ISL1; and
d) the population comprises cells that express NKX6.1 and ISL1 and exhibit glucose stimulated insulin secretion (GSIS) in vitro, and
wherein the administration of the population of cells results in reduction of blood glucose level in the subject.
2. The method of claim 1 , wherein less than 15% of the cells in the population express NKX6.1 but do not express ISL1.
3. The method of claim 1 , wherein less than 20% of the cells in the population express NXK6.1 and VMAT1.
4. The method of claim 1 , wherein less than 10% of the cells in the population express NKX6.1 and VMAT1.
5. The method of claim 1 , wherein at least 20% of the cells in the population express ARX.
6. The method of claim 1 , wherein at least 30% of the cells in the population express ARX.
7. The method of claim 1 , wherein at least 10% of the cells in the population express glucagon.
8. The method of claim 1 , wherein at least 15% of the cells in the population express glucagon.
9. The method of claim 1 , wherein at least 20% of the cells in the population express glucagon.
10. The method of claim 1 , wherein at least 40% of the cells in the population express NKX6.1 and ISL1.
11. The method of claim 1 , wherein at least 35% of the cells in the population express ISL1 but do not express NKX6.1.
12. The method of claim 1 , wherein the population of cells are in one or more cell clusters.
13. The method of claim 1 , wherein the population comprises genetically modified cells.
14. The method of claim 13 , wherein the genetically modified cells are genetically engineered to reduce an immune response.
15. The method of claim 14 , wherein the genetically engineered cells comprise a genetic disruption in beta-2-microglobulin.
16. The method of claim 14 , wherein the genetically engineered cells comprise a genetic disruption in HLA-A, HLA-B, or HLA-C.
17. The method of claim 1 , wherein the population of cells is implanted into the subject in a device.
18. The method of claim 17 , wherein the device comprises a first membrane having a first surface comprising a plurality of channels, and a second surface opposing the first surface; and a second membrane opposite and attached to the second surface of the first membrane.
19. The method of claim 18 , wherein the first membrane and the second membrane comprise polyvinylidene fluoride or polyvinylidene difluoride (PVDF), polytetrafluoroethylene (PTFE), expanded PTFE (ePTFE), polycaprolactone (PCL), polyethylene/polyethersulfone (PE/PES), polypropylene (PP), polystyrene (PS), poly(methyl methacrylate) (PMMA), poly lactic-co-glycolic acid (PLGA), poly(lactic acid) (PLLA), or any combination thereof.
20. The method of claim 1 , wherein the subject has Type I diabetes.
21. The method of claim 1 , wherein the population of cells exhibit glucose stimulated insulin secretion (GSIS) in vivo upon administration to the subject.
22. The method of claim 1 , wherein the cells that express NKX6.1 and ISL1 and exhibit glucose stimulated insulin secretion (GSIS) in vitro further mature to beta cells upon administration to the subject.
23. The method of claim 17 , wherein the device comprises apertures that allow for vascularization of the device in the subject.
24. The method of claim 1 , wherein the levels of NKX6.1 and ISL1 are determined utilizing immunocytochemistry.
25. The method of claim 1 , wherein the levels of NKX6.1 and ISL1 are determined utilizing flow cytometry.
26. The method of claim 1 , wherein the population of cells is administered to the subject intravenously.