SC-beta cells and compositions and methods for generating the same
Disclosed herein are methods, compositions, kits, and agents useful for inducing β cell maturation, and isolated populations of SC-β cells for use in various applications, such as cell therapy.
1. An in vitro composition that comprises a protein kinase inhibitor, a retinoic acid signaling pathway activator, a sonic hedgehog pathway inhibitor, a TGF-beta pathway inhibitor, and a plurality of non-native human pancreatic progenitor cells that express PDX1 and NKX6.1.
2. The composition of claim 1 , wherein the protein kinase inhibitor is selected from the group consisting of: staurosporine; Ro-31-8220; a bisindolylmaleimide (Bis); 10′-{5″-[(methoxycarbonyl)amino]-2″methyl}-phenylaminocarbonylstaurosporine; a staralog; a pseudohypericin; and indirubin-3-monoxime, 5--Iodo.
3. The composition of claim 1 , wherein the composition further comprises insulin-expressing cells.
4. The composition of claim 3 , wherein the insulin-expressing cells express NKX6.1 and Chromogranin A.
5. The composition of claim 1 , wherein the protein kinase inhibitor is a protein kinase C (PKC) inhibitor.
6. The composition of claim 1 , wherein the protein kinase inhibitor comprises staurosporine or an analog thereof.
7. The composition of claim 6 , wherein the protein kinase inhibitor comprises staurosporine.
8. The composition of claim 1 , wherein:
a) the protein kinase inhibitor is selected from the group consisting of staurosporine; Ro-31-8220; a bisindolylmaleimide (Bis); 10′-{5″-[(methoxycarbonyl)amino]-2″methyl}-phenylaminocarbonylstaurosporine; a staralog; a pseudohypericin; and indirubin-3-monoxime, 5-Iodo;
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; CD 2314; DEAB; BMS 195614; ER 50891; BMS 493; CD 2665; LE 135; BMS 453; and MM 11253;
c) sonic hedgehog pathway inhibitor is selected from the group consisting of: Sant1, Sant2, Sant3, Sant4, Cur61414, forskolin, tomatidine, AY9944, and triparanol; and
d) the TGF-beta pathway inhibitor is selected from the group consisting of: ALK5 inhibitor II; A83-01; SB 431542; D 4476; GW 788388; LY 364947; LY 580276; SB 525334; SB 505124; SD 208; and GW 6604.
9. The composition of claim 1 , wherein the protein kinase inhibitor comprises staurosporine, the retinoic acid signaling pathway activator comprises retinoic acid, the sonic hedgehog pathway inhibitor comprises SANT1, and the TGF-beta pathway inhibitor comprises Alk5 inhibitor II.
10. An in vitro composition that comprises
a protein kinase inhibitor comprises staurosporine; Ro-31-8220; a bisindolylmaleimide (Bis); 10′-{5″-[(methoxycarbonyl)amino]-2″methyl}-phenylaminocarbonylstaurosporine; a staralog; a pseudohypericin; or indirubin-3-monoxime, 5-Iodo;
a TGF-beta pathway inhibitor comprising Alk5 inhibitor II;
a BMP signaling pathway inhibitor comprising LDN193189; and
a plurality of non-native human pancreatic insulin-expressing cells.
11. The composition of claim 10 , wherein the protein kinase inhibitor comprises staurosporine.
12. The composition of claim 10 , wherein the composition comprises from 10 nM to 1 μM of the protein kinase inhibitor.
13. A method of in vitro cell differentiation, comprising:
contacting an in vitro population of human pancreatic progenitor cells that express PDX1 and NKX6.1 with a retinoic acid signaling pathway activator, a sonic hedgehog pathway inhibitor, a TGF-beta pathway inhibitor, and a protein kinase inhibitor,
wherein the method results in a plurality of the pancreatic progenitor cells in the population to differentiate into insulin-expressing cells.
14. The method of claim 13 , wherein the pancreatic progenitor cells that express PDX1 and NKX6.1 are contacted for 4, 6, 8, or 10 days with the protein kinase inhibitor, the retinoic acid signaling pathway activator, and the TGF-beta pathway inhibitor.
15. The method of claim 13 , wherein the pancreatic progenitor cells that express PDX1 and NKX6.1 are contacted for 7 days with the protein kinase inhibitor.
16. The method of claim 13 , wherein the protein kinase inhibitor comprises a protein kinase C (PKC) inhibitor.
17. The method of claim 13 , wherein the protein kinase inhibitor comprises staurosporine or an analog thereof.
18. The method of claim 13 , wherein the protein kinase inhibitor comprises staurosporine.
19. The method of claim 13 , wherein the insulin-expressing cells express NKX6.1 and Chromogranin A.
20. The method of claim 13 , wherein:
a) the protein kinase inhibitor is selected from the group consisting of staurosporine;
Ro-31-8220; a bisindolylmaleimide (Bis); 10′-{5″-[(methoxycarbonyl)amino]-2″ methyl}-phenylaminocarbonylstaurosporine; a staralog; a pseudohypericin; and indirubin-3-monoxime, 5-Iodo;
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; CD 2314; DEAB; BMS 195614; ER 50891; BMS 493; CD 2665; LE 135; BMS 453; and MM 11253;
c) sonic hedgehog pathway inhibitor is selected from the group consisting of: Sant1, Sant2, Sant3, Sant4, Cur61414, forskolin, tomatidine, AY9944, and triparanol; and
d) the TGF-beta pathway inhibitor is selected from the group consisting of: ALK5 inhibitor II; A83-01; SB 431542; D 4476; GW 788388; LY 364947; LY 580276; SB 525334; SB 505124; SD 208; and GW 6604.
21. The method of claim 13 , wherein the retinoic acid signaling pathway activator comprises retinoic acid, the sonic hedgehog pathway inhibitor comprises SANT1, and the TGF-beta pathway inhibitor comprises Alk5 inhibitor II.
22. The method of claim 13 , wherein the method generates a higher percentage of cells that express Chromogranin A in a cell population resulting from the contacting with the protein kinase inhibitor as compared to a corresponding cell population generated without the contacting with the protein kinase inhibitor.
23. The method of claim 13 , wherein the method generates a higher percentage of cells that express NKX6.1 and C-peptide in a cell population resulting from the contacting with the protein kinase inhibitor as compared to a corresponding cell population generated without the contacting with the protein kinase inhibitor.
24. The method of claim 13 , wherein the pancreatic progenitor cells that express PDX1 and NKX6.1 are generated from stem cells in vitro.