Differentiation and Enrichment of Islet-like Cells From Human Pluripotent Stem Cells

Abstract

Methods for differentiating human pluripotent stem cells into islet-like cells are provided. In certain embodiments, the methods utilize sequential culturing of the human pluripotent stem cells with certain factors to produce islet-like cells. In certain embodiments, the population of cells produced by the methods is further enriched for islet-like cells.

What is claimed is: 
     
         1 . A suspension of human islet-like cell clusters wherein the human islet-like cell clusters have a particle size of 200 microns or less. 
     
     
         2 . The islet-like cell clusters of  claim 1 , wherein the islet-like cell clusters have a particle size ranging from 70 microns to 200 microns. 
     
     
         3 . The islet-like cell clusters of  claim 1 , wherein the islet-like clusters express Pdx1. 
     
     
         4 . The islet-like cell clusters of  claim 1 , wherein the islet-like cell clusters express Ngn3. 
     
     
         5 . The islet-like cell clusters of  claim 1 , wherein the islet-like cell clusters express insulin. 
     
     
         6 . The islet-like cell clusters of  claim 1 , wherein the islet-like cell clusters express Pdx1, Ngn3 and insulin. 
     
     
         7 . The islet-like cell clusters of  claim 1 , wherein the insulin content of the islet-like cell clusters is at least five times the insulin content found in undifferentiated stem cells. 
     
     
         8 . A suspension of human islet-like cells comprising cell clusters that have formed buds, wherein the clusters that have formed buds have a particle size of 200 microns or less. 
     
     
         9 . The suspension of  claim 8 , wherein the clusters that have formed buds have a particle size ranging from 70 microns to 200 microns. 
     
     
         10 . The human islet-like cells of  claim 8 , wherein the human islet-like cells express Pdx1. 
     
     
         11 . The human islet-like cells of  claim 8 , wherein the human islet-like cells express Ngn3. 
     
     
         12 . The human islet-like cells of  claim 8 , wherein the human islet-like cells express insulin. 
     
     
         13 . The human islet-like cells of  claim 8 , wherein the human islet-like cells express Pdx1, Ngn3 and insulin. 
     
     
         14 . A first and a second cell population comprising: a) a first in vitro cell population comprising islet-like cells comprising cell clusters that have formed buds, and b) a second in vitro cell population comprising at least two fractions of cells, generated from the first cell population by size or density separation of at least some of the buds from at least some of the clusters,
 wherein the first cell population comprising islet-like cells is the differentiated progeny of human pluripotent stem cells.   
     
     
         15 . The first and the second population of cells of  claim 14 , wherein the first population is generated by: i) contacting human pluripotent stem cells with a medium comprising Activin A; and ii) further contacting the cells from step i) with a second medium comprising FGF-2 but lacking Activin A. 
     
     
         16 . The first and the second population of cells of  claim 14 , wherein the second population comprises a fraction with a particle size between 70 and 200 microns that is enriched in buds.