Methods for differentiating and purifying pancreatic endocrine cells

Abstract

The present invention relates to compositions and methods comprising cell surface markers for pluripotent-derived cells, in particular, pancreatic endoderm-type cells, derived from pluripotent stem cells.

What is claimed is: 
     
         1 . A method of enriching for pancreatic endocrine cells expressing NKX6.1 and insulin comprising:
 a) exposing an in vitro cell population comprising pancreatic endocrine cells expressing NKX6.1 and insulin having been ex-vivo differentiated from pluripotent stem cells to a ligand that binds CD49a, wherein said CD49a is the only positive cell surface marker used in the method of enriching;   b) selecting cells which bind said ligand that binds CD49a;   c) culturing said cells which bind said ligand that binds CD49a, thereby enriching for pancreatic endocrine cells expressing NKX6.1 and insulin.   
     
     
         2 . A method of enriching for pancreatic endocrine cells expressing NKX6.1 and insulin comprising:
 a) exposing an in vitro cell population comprising pancreatic endocrine cells expressing NKX6.1 and insulin having been ex-vivo differentiated from pluripotent stem cells to a ligand that binds a negative cell surface marker that is not present on said pancreatic endocrine cells expressing NKX6.1 and insulin;   b) selecting cells which do not bind said ligand that binds said negative cell surface marker;   c) exposing said cells that do not bind said ligand that binds said negative cell surface marker to a ligand that binds CD49a;   d) selecting cells which bind said ligand that binds CD49a, thereby enriching for pancreatic endocrine cells expressing NKX6.1 and insulin.   
     
     
         3 . The method of  claim 2 , wherein said negative cell surface marker is selected from the group consisting of CD66C, CD73, CD340, CD44 and CD49f. 
     
     
         4 . The method of  claim 1 , wherein said ligand is an antibody or binding fragment thereof. 
     
     
         5 . The method of  claim 2 , wherein said ligand is an antibody or binding fragment thereof. 
     
     
         6 . The method of  claim 1 , further comprising ex vivo differentiating pancreatic endocrine cells from human pluripotent stem cells prior to step (a). 
     
     
         7 . The method of  claim 2 , further comprising ex vivo differentiating pancreatic endocrine cells from human pluripotent stem cells prior to step (a). 
     
     
         8 . The method of  claim 2 , further comprising culturing said cells which bind said ligand that binds CD49a. 
     
     
         9 . The method of  claim 1 , wherein said culturing is performed under conditions which allow forming aggregates of pancreatic endocrine cells expressing NKX6.1 and insulin. 
     
     
         10 . The method of  claim 9 , wherein said conditions comprise 1-10 mM glucose and/or EDTA. 
     
     
         11 . The method of  claim 8 , wherein said culturing is performed under conditions which allow forming aggregates of pancreatic endocrine cells expressing NKX6.1 and insulin. 
     
     
         12 . The method of  claim 11 , wherein said conditions comprise 1-10 mM glucose and/or EDTA. 
     
     
         13 . A method of treating diabetes in a subject, the method comprising transplantation of a therapeutically effective amount of the aggregates of pancreatic endocrine cells expressing NKX6.1 and insulin or  claim 10  into the subject, thereby treating diabetes. 
     
     
         14 . A method of treating diabetes in a subject, the method comprising transplantation of a therapeutically effective amount of the aggregates of pancreatic endocrine cells expressing NKX6.1 and insulin or  claim 12  into the subject, thereby treating diabetes.