Sc-beta cells and compositions and methods for generating the same

Abstract

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 .- 30 . (canceled) 
     
     
         31 . An in vitro composition that comprises a gamma-secretase inhibitor, a plurality of non-native pancreatic human progenitor cells that express PDX1 and NKX6.1, and one or more of a protein kinase inhibitor, a retinoic acid signaling pathway activator, a growth factor from the EGF family, and a sonic hedgehog pathway inhibitor. 
     
     
         32 . The composition of  claim 31 , wherein the composition further comprises insulin-expressing cells. 
     
     
         33 . The composition of  claim 32 , wherein the insulin-expressing cells express NKX6.1 and Chromogranin A. 
     
     
         34 . The composition of  claim 31 , wherein the composition comprises a protein kinase inhibitor, wherein the protein kinase inhibitor is selected from the group consisting of staurosporine, Ro-31-8220, a bisindolylmaleimide (Bis) compound, 10′-{5″-[(methoxycarbonyl)amino]-2″metlryl}-phenylaminocarbonylstaurosporine, and a staralog. 
     
     
         35 . The composition of  claim 34 , wherein the protein kinase inhibitor is staurosporine. 
     
     
         36 . The composition of  claim 31 , wherein the composition comprises a retinoic acid pathway signaling activator, 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, CD 2314, DEAB, BMS 195614, ER 50891, BMS 493, CD 2665, LE 135, BMS 453, and MM 11253. 
     
     
         37 . The composition of  claim 36 , wherein the retinoic acid pathway signaling activator is retinoic acid. 
     
     
         38 . The composition of  claim 31 , wherein the composition comprises a growth factor from the EGF family, wherein the growth factor from the EGF family is selected from the group consisting of EGF and betacellulin. 
     
     
         39 . The composition of  claim 38 , wherein the growth factor from the EGF family is betacellulin. 
     
     
         40 . The composition of  claim 31 , wherein the composition comprises a sonic hedgehog pathway inhibitor, wherein the sonic hedgehog pathway inhibitor is selected from the group consisting of Sant1, Sant2, Sant3, Sant4, Cur61414, forskolin, tomatidine, AY9944, and triparanol. 
     
     
         41 . The composition of  claim 40 , wherein the sonic hedgehog pathway inhibitor is Sant-1. 
     
     
         42 . The composition of  claim 31 , wherein the composition comprises a TGF-beta pathway inhibitor, wherein 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. 
     
     
         43 . The composition of  claim 42 , wherein the TGF-beta pathway inhibitor is ALK5 inhibitor II. 
     
     
         44 . The composition of  claim 31 , wherein the gamma-secretase inhibitor is selected from the group consisting of XXI and DAPT. 
     
     
         45 . The composition of  claim 31 , wherein the gamma-secretase inhibitor is XXI. 
     
     
         46 . The composition of  claim 31 , wherein the gamma-secretase inhibitor is selected from the group consisting of XXI and DAPT; and wherein the composition comprises one or more of:
 a) a protein kinase inhibitor, wherein the protein kinase inhibitor is selected from the group consisting of staurosporine, Ro-31-8220, a bisindolylmaleimide (Bis) compound, 10′-{5″-[(methoxycarbonyl)amino]-2″metlryl}-phenylaminocarbonylstaurosporine, and a staralog;   b) a retinoic acid pathway signaling activator, 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, CD 2314, DEAB, BMS 195614, ER 50891, BMS 493, CD 2665, LE 135, BMS 453, and MM 11253;   c) a growth factor from the EGF family, wherein the growth factor from the EGF family is selected from the group consisting of EGF and betacellulin; and/or   d) a sonic hedgehog pathway inhibitor, wherein the sonic hedgehog pathway inhibitor is selected from the group consisting of Sant1, Sant2, Sant3, Sant4, Cur61414, forskolin, tomatidine, AY9944, and triparanol.   
     
     
         47 . The composition of  claim 46 , wherein the composition comprises:
 a) a gamma-secretase inhibitor selected from the group consisting of XXI and DAPT;   b) a protein kinase inhibitor, wherein the protein kinase inhibitor is selected from the group consisting of staurosporine, Ro-31-8220, a bisindolylmaleimide (Bis) compound, 10′-{5″-[(methoxycarbonyl)amino]-2″metlryl}-phenylaminocarbonylstaurosporine, and a staralog;   c) a retinoic acid pathway signaling activator, 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, CD 2314, DEAB, BMS 195614, ER 50891, BMS 493, CD 2665, LE 135, BMS 453, and MM 11253; and   d) a sonic hedgehog pathway inhibitor, wherein the sonic hedgehog pathway inhibitor is selected from the group consisting of Sant1, Sant2, Sant3, Sant4, Cur61414, forskolin, tomatidine, AY9944, and triparanol.   
     
     
         48 . The composition of  claim 47 , wherein the composition comprises: XXI, staurosporine, retinoic acid, and Sant-1. 
     
     
         49 . The composition of  claim 48 , wherein the composition further comprises ALK5 inhibitor II. 
     
     
         50 . The composition of  claim 49 , wherein the composition further comprises betacellulin. 
     
     
         51 . The composition of  claim 31 , wherein the pancreatic progenitor cells are genetically modified cells or progenies thereof.