Generation of pancreatic endoderm from Pluripotent Stem cells using small molecules

Abstract

A method of producing pancreatic cells or pancreatic cell precursors expressing at least 5% PDX1/NKX6.1 double positive, comprising exposing definitive endoderm cells to an effective amount of one or more small molecules, to differentiate the human definitive endoderm cells into the pancreatic cells or pancreatic cell precursors. The present invention also relates to pancreatic endoderm cells produced by said methods and uses of said pancreatic endoderm cells.

1 . A method of producing pancreatic cells or pancreatic cell precursors, comprising exposing definitive endoderm cells to an effective amount of at least one BMP inhibitor, at least one kinase inhibitor, and at least one retinoic acid receptor agonist, wherein at least 5% of the resulting pancreatic cells or pancreatic cell precursors are PDX1/NKX6.1 double positive. 
     
     
         2 . The method of  claim 1 , wherein the BMP inhibitor is LDN-193189. 
     
     
         3 . The method of  claim 1 , wherein the retinoic acid receptor agonist is AM580. 
     
     
         4 . The method of  claim 1 , wherein the retinoic acid receptor agonist is a retinoic acid derivative. 
     
     
         5 . The method of  claim 1 , wherein the kinase inhibitor is an isomer of 1,9-pyrazoloanthrone with or without N-alkylation. 
     
     
         6 . The method of  claim 1 , wherein kinase inhibitor is JNK inhibitor II and the retinoic acid receptor agonist is AM580. 
     
     
         7 . The method of  claim 1 , further comprising exposing the definitive endoderm cells to bFGF. 
     
     
         8 . The method of  claim 1 , further comprising exposing the definitive endoderm cells to FGF7 or FGF10. 
     
     
         9 . The method of  claim 1 , wherein the exposing comprises a first exposure to an effective amount of LDN-193189 without bFGF and a second exposure to JNK inhibitor II, AM580, and bFGF. 
     
     
         10 . The method of  claim 2 , further comprising exposing the definitive endoderm cells to at least one compound selected from the group consisting of a wnt inhibitor and a hedgehog inhibitor prior to exposing the definitive endoderm cells to the LDN-193189. 
     
     
         11 . The method of  claim 1 , wherein said pancreatic cells or pancreatic cell precursors are at least 10% PDX1/NKX6.1 double positive. 
     
     
         12 . An isolated population of pancreatic cells or pancreatic cell precursors produced by the method of  claim 1 . 
     
     
         13 . An isolated population of pancreatic cells or pancreatic cell precursors produced by exposing definitive endoderm cells to a kinase inhibitor capable of inhibiting a target selected from the group consisting of JNK1, JNK2, JNK3, Syc, Src, GSK-3, P38, MAPK, P38 kinase, Rho kinase, MEK, Chk2, VEGFR1, VEGFR2, VEGFR3, PDGFRb, and KDR/Flk-1. 
     
     
         14 . The isolated population of pancreatic cells or pancreatic cell precursors produced by the method of  claim 13 , wherein the kinase inhibitor is JNK inhibitor II, and wherein the definitive endoderm cells are also exposed to a compound selected from the group consisting of LDN-193189, a Wnt inhibitor, a hedgehog inhibitor, and a retinoic acid receptor agonist. 
     
     
         15 . The isolated population of pancreatic cells or pancreatic cell precursors of  claim 13 , wherein the definitive endoderm cells are exposed to a retinoic acid receptor agonist. 
     
     
         16 .- 18 . (canceled)