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 composition comprising a 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 25% of the cells in the population express NKX6.1 but do not express ISL1;
d) less than 25% of the cells in the population express VMAT1; and
e) the population comprises at least one cell that is genetically modified.
2. The composition of claim 1 wherein less than 15% of the cells in the population express NKX6.1 but do not express 1SL1.
3. The composition of claim 1 , wherein at least 20% of the cells in the population express ARX.
4. The composition of claim 1 , wherein at least 30% of the cells in the population express ARX.
5. The composition of claim 1 , wherein at least 10% of the cells in the population express glucagon.
6. The composition of claim 1 , wherein at least 15% of the cells in the population express glucagon.
7. The composition of claim 1 , wherein at least 20% of the cells in the population express glucagon.
8. The composition of claim 7 , wherein less than 20% of the cells in the population express VMAT1.
9. The composition of claim 1 , wherein at least 40% of the cells in the population express NKX6.1 and ISL1.
10. The composition of claim 1 wherein at least 35% of the cells in the population express ISL1 but do not express NKX6.1.
11. The composition of claim 1 , wherein the population of cells are in one or more cell clusters.
12. The composition of claim 1 , wherein the population comprises cells that express NKX6.1 and ISL1 and exhibit glucose stimulated insulin secretion (GSIS) in vitro.
13. The composition population of claim 1 , wherein the at least one cell is genetically engineered to reduce an immune response.
14. The composition of claim 13 , wherein the at least one cell contains a genetic disruption in the gene encoding for beta-2-microglobulin.
15. The composition of claim 13 , wherein the at least one cell contains a genetic disruption in the gene encoding for one or more of HLA-A, HLA-B, or HLA-C.
16. The composition of claim 1 , wherein the population is in a device suitable for implantation into a human subject.
17. The composition of claim 1 , wherein the levels of NKX6.1 and ISL1 are determined utilizing immunocytochemistry.
18. The composition of claim 1 , wherein the levels of NKX6.1 and ISL1 are determined utilizing flow cytometry.
19. The composition 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.
20. The composition 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).