Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof
Disclosed herein are methods for generating SC-β cells using chemically defined, completely serum free media, and isolated populations of SC-β cells for use in various applications, such as cell therapy.
1. A serum free in vitro composition that comprises a ROCK inhibitor, a sonic hedgehog pathway inhibitor, and a plurality of pancreatic progenitor cells that express PDX1 and CDX2, wherein the composition does not include a BMP inhibitor.
2. The composition of claim 1 , wherein the plurality of pancreatic progenitor cells do not express NKX6.1.
3. The composition of claim 1 , wherein the composition further comprises cells that express PDX1 and NKX6.1.
4. The composition of claim 1 , wherein the ROCK inhibitor is a small molecule inhibitor.
5. The composition of claim 1 , wherein the ROCK inhibitor is selected from the group consisting of: AS 1892802, GSK 429286, RKI-1147 dihydrochloride, SB772077B dihydrochloride, SR 3677 dihydrochloride, Rho Kinase Inhibitor, Rho Kinase Inhibitor II, Rho Kinase Inhibitor III, thiazovivin, Y-27632, Fasudil hydrochloride, and H-1152 dihydrochloride.
6. The composition of claim 1 , wherein the ROCK inhibitor is thiazovivin.
7. The composition of claim 1 , wherein the ROCK inhibitor is Y-27632.
8. The composition of claim 1 , wherein the composition comprises from 0.1 μM to 110 μM of the ROCK inhibitor.
9. The composition of claim 1 , wherein the composition further comprises one or more of a retinoic acid signaling pathway activator, and a fibroblast growth factor.
10. The composition of claim 9 , wherein the composition comprises the retinoic acid signaling pathway activator, and the fibroblast growth factor.
11. The composition of claim 1 , wherein the composition further comprises a retinoic acid signaling pathway activator.
12. The composition of claim 11 , wherein the retinoic acid signaling pathway activator is selected from the group consisting of: retinoic acid, CD 1530, AM 580, TTNPB, CD 437, Ch 55, BMS 961, AC 261066, AC 55649, AM 80, BMS 753, tazarotene, adapalene, and CD 2314.
13. The composition of claim 12 , wherein the composition comprises retinoic acid.
14. The composition of claim 1 , wherein the sonic hedgehog pathway inhibitor is selected from the group consisting of: SANT1, SANT2, SANT3, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine.
15. The composition of claim 14 , wherein the composition comprises SANT1.
16. The composition of claim 1 , wherein the composition further comprises a fibroblast growth factor.
17. The composition of claim 16 , wherein the fibroblast growth factor is selected from the group consisting of: keratinocyte growth factor, FGF2, FGF8B, FGF10, and FGF21.
18. The composition of claim 17 , wherein the fibroblast growth factor is keratinocyte growth factor.
19. The composition of claim 10 , wherein:
a) the ROCK inhibitor is selected from the group consisting of: AS 1892802, GSK 429286, RKI-1147 dihydrochloride, SB772077B dihydrochloride, SR 3677 dihydrochloride, Rho Kinase Inhibitor, Rho Kinase Inhibitor II, Rho Kinase Inhibitor III, thiazovivin, Y-27632, Fasudil hydrochloride, and H-1152 dihydrochloride;
b) the retinoic acid signaling pathway activator is selected from the group consisting of: retinoic acid, CD 1530, AM 580, TTNPB, CD 437, Ch 55, BMS 961, AC 261066, AC 55649, AM 80, BMS 753, tazarotene, adapalene, and CD 2314;
c) the sonic hedgehog pathway inhibitor is selected from the group consisting of: SANT1, SANT2, SANT3, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine; and
d) the fibroblast growth factor is selected from the group consisting of: keratinocyte growth factor, FGF2, FGF8B, FGF10, and FGF21.
20. The composition of claim 1 , wherein the composition comprises thiazovivin, KGF, SANT1, and retinoic acid.
21. The composition of claim 1 , wherein the composition does not include a PKC activator.
22. The composition of claim 20 , wherein the composition does not include a PKC activator.