Production of insulin producing cells

Abstract

A population of enteroendocrine cells (EEC) is obtained from a mammalian post-natal cell population, such as a population including post-natal stem cells, by treating the population with a plurality of small molecules that upregulate ChgA and promote differentiation of the cells to form the enteroendocrine cells. The upregulation of ChgA is such that the fraction of cells expressing CGA in the obtained cell population, as measured by a ChgA Immunostaining Assay, is at least about 1.5%. Small molecules that can be used to differentiate the post-natal cells into the enteroendocrine cells can include at least one of a Wnt activator, a Notch inhibitor, a Wnt inhibitor, a MEK/ERK inhibitor, a growth factor, a HDAC inhibitor, a Histone Methylation Inhibitor, a Tgf-β inhibitor, and a NeuroD1 activator. Also, the insulin expression of a population of mammalian cells is increased by treating the population with a plurality of small molecules that increase the insulin expression.

What is claimed is: 
     
         1 . A method for obtaining a population of cells that produce insulin from a population of LGR5 positive epithelial stem cells, the method comprising:
 a) contacting the population of LGR5 positive epithelial stem cells with a Notch inhibitor, a Wnt activator, and a DNA methylation inhibitor to form a first resultant cell population;   b) contacting the first resultant cell population with a Notch inhibitor, a Wnt inhibitor, a TGF-β inhibitor, and a NeuroD1 activator to form a second resultant cell population; and   c) contacting the second resultant cell population with a Notch inhibitor, a Wnt inhibitor or a GSK3β inhibitor, a MEK/ERK inhibitor and a TGF-β inhibitor, thereby forming a population of cells that produce insulin.   
     
     
         2 . The method of  claim 1 , wherein the Notch inhibitor of a) is N—[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) or a pharmaceutically acceptable salt thereof. 
     
     
         3 . The method of  claim 1 , wherein the Wnt activator of a) is R-Spondin1. 
     
     
         4 . The method of  claim 1 , wherein the DNA methylation inhibitor of a) is 5-Azacytidine or a pharmaceutically acceptable salt thereof. 
     
     
         5 . The method of  claim 1 , wherein the DNA methylation inhibitor of a) is 5-Aza-2′ deoxycytidine or a pharmaceutically acceptable salt thereof. 
     
     
         6 . The method of  claim 1 , wherein the Notch inhibitor of b) is N—[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) or a pharmaceutically acceptable salt thereof. 
     
     
         7 . The method of  claim 1 , wherein the Wnt inhibitor of b) is Wnt-059. 
     
     
         8 . The method of  claim 1 , where the TGF-β inhibitor of b) is a compound having the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         9 . The method of  claim 1 , wherein the NeuroD1 activator of b) is N-cyclopropyl-5-(2-thienyl)-3-isoxazolecarboxamide. 
     
     
         10 . The method of  claim 1 , wherein the Notch inhibitor of c) is N—[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) or a pharmaceutically acceptable salt thereof. 
     
     
         11 . The method of  claim 1 , wherein c) comprises a Wnt inhibitor, and wherein the Wnt inhibitor of c) is Wnt-059. 
     
     
         12 . The method of  claim 1 , where c) comprises a GSK3β inhibitor, and wherein the GSK3β inhibitor is a compound having the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         13 . The method of  claim 1 , wherein the MEK/ERK inhibitor of c) is N-[(2R)-2,3-Dihydroxypropoxyl]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide. 
     
     
         14 . The method of  claim 1 , wherein the TGF-β inhibitor of c) is a compound having the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         15 - 19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein a) further comprises contacting the population of LGR5 positive epithelial stem cells with a Neuro D1 activator. 
     
     
         21 . The method of  claim 20 , wherein the NeuroD1 activator of a) is N-cyclopropyl-5-(2-thienyl)-3-isoxazolecarboxamide. 
     
     
         22 . The method of  claim 1 , wherein a) further comprises contacting the population of LGR5 positive epithelial stem cells with a TGF-β inhibitor. 
     
     
         23 . The method of  claim 22 , wherein the TGF-β inhibitor of a) is a compound having the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         24 - 32 . (canceled) 
     
     
         33 . The method of  claim 1 , wherein b) further comprises contacting the first resultant cell population with a DNA methylation inhibitor. 
     
     
         34 . The method of  claim 33 , wherein the DNA methylation inhibitor of b) is 5-Azacytidine. 
     
     
         35 - 36 . (canceled) 
     
     
         37 . The method of  claim 1 , wherein c) further comprises contacting the second resultant cell population with triiodothyronine. 
     
     
         38 . The method of  claim 1 , wherein c) further comprises contacting the second resultant cell population with N-acetylcysteine. 
     
     
         39 . The method of  claim 1 , wherein the LGR5 positive epithelial stem cells are intestinal cells. 
     
     
         40 - 45 . (canceled) 
     
     
         46 . The method of  claim 1 , wherein a) occurs from day 0 to 1; b) occurs from day 1 to 3; and c) occurs from day 3 to 5. 
     
     
         47 . The method of  claim 1 , wherein in b), contacting the first resultant cell population with the Wnt inhibitor is delayed by about 12 hours. 
     
     
         48 . (canceled)