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. An in vitro population of cells derived from stem 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) the population comprises cells that express NKX6.1 but do not express ISL1, and less than 20% of the cells in the population express NKX6.1 but do not express ISL1; and
d) the population comprises at least one cell that is genetically modified.
2. The population of cells of claim 1 , wherein less than 15% of the cells in the population express NKX6.1 but do not express ISL1.
3. The population of cells of claim 1 , wherein less than 20% of the cells in the population express NXK6.1 and VMAT1.
4. The population of cells of claim 1 , wherein less than 10% of the cells in the population express NKX6.1 and VMAT1.
5. The population of cells of claim 1 , wherein at least 20% of the cells in the population express ARX.
6. The population of cells of claim 1 , wherein at least 30% of the cells in the population express ARX.
7. The population of cells of claim 1 , wherein at least 10% of the cells in the population express glucagon.
8. The population of cells of claim 1 , wherein at least 15% of the cells in the population express glucagon.
9. The population of cells of claim 1 , wherein at least 20% of the cells in the population express glucagon.
10. The population of cells of claim 1 , wherein at least 40% of the cells in the population express NKX6.1 and ISL1.
11. The population of cells of claim 1 , wherein at least 35% of the cells in the population express ISL1 but do not express NKX6.1.
12. The population of cells of claim 1 , wherein the population of cells are in one or more cell clusters.
13. The population of cells of claim 1 , wherein the population comprises cells that express NKX6.1 and ISL1 and exhibit glucose stimulated insulin secretion (GSIS) in vitro.
14. The population of claim 1 , wherein the at least one cell is genetically engineered to reduce an immune response.
15. The population of cells of claim 14 , wherein the at least one cell contains a genetic disruption in beta-2-microglobulin.
16. The population of cells of claim 14 , wherein the at least one cell contains a genetic disruption in HLA-A, HLA-B, or HLA-C.
17. The population of cells of claim 1 , wherein the population is in a device suitable for implantation into a human subject.
18. The population of cells of claim 1 , wherein the levels of NKX6.1 and ISL1 are determined utilizing immunocytochemistry.
19. The population of cells of claim 1 , wherein the levels of NKX6.1 and ISL1 are determined utilizing flow cytometry.
20. The population of cells of claim 1 , wherein the population is in a composition further comprising one or more of: a transforming growth factor beta (TGF-beta) signaling pathway inhibitor, a bone morphogenesis protein (BMP) inhibitor, a Rho-associated kinase (ROCK) inhibitor, a histone methylation inhibitor, a thyroid hormone signaling pathway activator, or a broad kinase inhibitor.
21. The population of cells of claim 1 , wherein the population is in a composition further comprising one or more of: an Activin-like kinase 5 (ALK5) inhibitor, LDN193189, thiazovivin, staurosporine, GC-1, or 3-deazaneplanocin A (DZNEP).
22. A population of cells, 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; and
c) the population comprises cells that express NKX6.1 but do not express ISL1, and less than 20% of the cells in the population express NKX6.1 but do not express ISL1,
wherein the population is in a device, and
wherein the device comprises:
(i) a first membrane comprising (A) a first surface comprising a plurality of channels and (B) a second surface opposing the first surface; and
(ii) a second membrane opposite and attached to the second surface of the first membrane.
23. The population of cells of claim 22 , wherein the population comprises cells that express NKX6.1 and ISL1 and exhibited glucose stimulated insulin secretion (GSIS) in vitro.
24. The population of cells of claim 23 , wherein the population comprises cells that express NKX6.1 and ISL1 and exhibit glucose stimulated insulin secretion (GSIS) in vitro and that further mature to beta cells if implanted in a patient.
25. The population of cells of claim 22 , 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.
26. The population of cells of claim 22 , wherein the device comprises apertures that allow for vascularization of the device when implanted in a subject.
27. The population of cells of claim 22 , wherein less than 10% of the cells in the population express NKX6.1 and VMAT1.
28. The population of cells of claim 22 , wherein at least 30% of the cells in the population express ARX.
29. The population of cells of claim 22 , wherein at least 20% of the cells in the population express glucagon.
30. The population of cells of claim 22 , wherein at least 40% of the cells in the population express NKX6.1 and ISL1.