Methods of enhancing stem cell differentiation into beta cells

Abstract

Disclosed herein are compositions and methods of enhancing stem cell differentiation into beta cells with use of one or more epigenetic modification compounds. The present disclosure also relates to compositions and methods of sorting and enriching the differentiated beta cells. The present disclosure also relates to compositions and methods of irradiating cell population for reducing proliferation.

1 . A method comprising:
 contacting an in vitro population of pancreatic progenitor cells or precursors thereof with an epigenetic modifying compound.   
     
     
         2 - 32 . (canceled) 
     
     
         33 . An in vitro composition comprising a cell population, wherein the cell population comprises:
 (a) at least about 20% cells expressing C-peptide and NKX6.1;   (b) at least about 60% cells expressing CHGA;   (c) at most about 20% cells expressing Cdx2; or   (d) at most about 45% cells expressing VMAT1, as measured by flow cytometry.   
     
     
         34 - 140 . (canceled) 
     
     
         141 . A method of reducing cell proliferation, comprising exposing to irradiation a cell population comprising stem cells, definitive endoderm cells, primitive gut tube cells, pancreatic progenitor cells, or endocrine cells, wherein said irradiation results in a cell population that has reduced proliferative capability as compared to a corresponding cell population that is not subject to irradiation. 
     
     
         142 - 162 . (canceled) 
     
     
         163 . An in vitro composition comprising an epigenetic modifying compound and a population of cells expressing PDX1 and NKX6.1. 
     
     
         164 . The in vitro composition of  claim 163 , wherein said epigenetic modifying compound comprises one or more of a DNA methylation inhibitor, a histone acetyltransferase inhibitor, a histone deacetylase inhibitor, a histone methyltransferase inhibitor, or a bromodomain inhibitor. 
     
     
         165 . The in vitro composition of  claim 163 , wherein said epigenetic modifying compound comprises a histone methyltransferase inhibitor. 
     
     
         166 . The in vitro composition of  claim 165 , wherein said histone methyltransferase inhibitor is selected from the group consisting of DZNep, GSK126, and EPZ6438. 
     
     
         167 . The in vitro composition of  claim 165 , wherein said histone methyltransferase inhibitor is DZNep. 
     
     
         168 . The in vitro composition of  claim 167 , wherein the composition comprises about 0.05 mM to about 50 mM, about 0.1 mM to about 10 pM, about 0.5 pM to about 5 pM, about 0.75 pM to about 2.5 pM, or about 1 pM to about 2 pM of DZNep. 
     
     
         169 . The in vitro composition of  claim 163 , wherein said epigenetic modifying compound comprises a histone deacetylase (HDAC) inhibitor. 
     
     
         170 . The in vitro composition of  claim 169 , wherein said HDAC inhibitor is selected from the group consisting of KD5170, MC1568, and TMP195. 
     
     
         171 . The in vitro composition of  claim 163 , wherein the composition further comprises a TGF-β signaling pathway inhibitor. 
     
     
         172 . The in vitro composition of  claim 171 , wherein the TGF-β signaling pathway inhibitor is selected from the group consisting of: Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124. 
     
     
         173 . The in vitro composition of  claim 171 , wherein the composition further comprises a thyroid hormone signaling pathway activator. 
     
     
         174 . The in vitro composition of  claim 173 , wherein the thyroid hormone signaling pathway activator comprises T3 or GC-1. 
     
     
         175 . The in vitro composition of  claim 173 , wherein the composition further comprises a ROCK inhibitor. 
     
     
         176 . The in vitro composition of  claim 175 , wherein the ROCK inhibitor is selected from the group consisting of: Thiazovivin, Y-27632, Fasudil/HA1077, and 14-1152. 
     
     
         177 . The in vitro composition of  claim 175 , wherein the composition further comprises a bone morphogenetic protein (BMP) signaling pathway inhibitor. 
     
     
         178 . The in vitro composition of  claim 177 , wherein the BMP signaling pathway inhibitor comprises LDN193189 or DMH-I. 
     
     
         179 . The in vitro composition of  claim 177 , wherein the composition further comprises a retinoic acid (RA) signaling pathway activator. 
     
     
         180 . The in vitro composition of  claim 179 , wherein the RA signaling pathway activator is selected from the group consisting of: retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314. 
     
     
         181 . The in vitro composition of  claim 179 , wherein the composition further comprises a sonic hedgehog (SHH) pathway inhibitor. 
     
     
         182 . The in vitro composition of  claim 181 , wherein the SHH pathway inhibitor is selected from the group consisting of: SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine. 
     
     
         183 . The in vitro composition of  claim 181 , wherein the composition further comprises a γ-secretase inhibitor. 
     
     
         184 . The in vitro composition of  claim 183 , wherein the γ-secretase inhibitor comprises XXI or DAPT. 
     
     
         185 . The in vitro composition of  claim 183 , wherein the composition further comprises a protein kinase inhibitor selected from the group consisting of staurosporine, Ro-31-8220, a bisindolylmaleimide (Bis) compound, 10′-{5″-[(methoxycarbonyl)amino]-2″-methyl}-phenylaminocarbonylstaurosporine, and a staralog. 
     
     
         186 . The in vitro composition of  claim 185 , wherein the composition further comprises a growth factor from the epidermal growth factor (EGF) family. 
     
     
         187 . The in vitro composition of  claim 186 , wherein the growth factor from the EGF family is betacellulin or EGF. 
     
     
         188 . The in vitro composition of  claim 163 , wherein the composition further comprises:
 (a) a sonic hedgehog pathway inhibitor selected from the group consisting of: SANT1, SANT2, SANT4, Cur61414, forskolin, tomatidine, AY9944, triparanol, and cyclopamine;   (b) a retinoic acid signaling pathway activator selected from the group consisting of: retinoic acid, CD1530, AM580, TTHRB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, and CD2314;   (c) a γ-secretase inhibitor comprising XXI or DAPT;   (d) a growth factor from the epidermal growth factor family comprising betacellulin or EGF;   (e) a bone morphogenetic protein signaling pathway inhibitor comprising LDN193189 or DMH-I;   (f) a TGF-β signaling pathway inhibitor selected from the group consisting of: Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, and SB-505124;   (g) a thyroid hormone signaling pathway activator comprising T3 or GC-1;   (h) a protein kinase inhibitor selected from the group consisting of: staurosporine, Ro-31-8220, a bisindolylmaleimide (Bis) compound, 10′-{5″-[(methoxycarbonyl)amino]-2″-methyl}-phenylaminocarbonylstaurosporine, and a staralog; or   (i) a ROCK inhibitor selected from the group consisting of Thiazovivin, Y-27632, Fasudil/HA1077, and 14-1152.   
     
     
         189 . The in vitro composition of  claim 167 , further comprising Thiazovivin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN193189 and staurosporine. 
     
     
         190 . The in vitro composition of  claim 189 , wherein the composition further comprises betacellulin. 
     
     
         191 . An in vitro composition comprising an epigenetic modifying compound and a population of cells expressing insulin.