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.

We claim: 
     
         1 . A method of enriching the proportion of islet-like cells from a cell population generated by the differentiation of human pluripotent stem cells to islet-like cells, comprising
 a) differentiating human pluripotent stem cells into a cell population comprising islet-like cells, wherein the cell population comprises cell clusters that have formed buds; and   b) separating at least some of the buds from at least some of the clusters in the cell population into at least two fractions;   
       wherein at least one of the at least two resulting fractions contains a higher proportion of islet-like cells than the cell population. 
     
     
         2 . The method of  claim 1 , wherein the islet-like cells comprise insulin-producing cells or glucagon-producing cells. 
     
     
         3 . The method of  claim 1 , wherein the human pluripotent stem cells are human embryonic stem cells. 
     
     
         4 . The method of  claim 1 , wherein the buds are separated from the clusters based on size differences between the buds and the clusters. 
     
     
         5 . The method of  claim 4 , wherein the buds are separated from the clusters by use of nylon mesh. 
     
     
         6 . The method of  claim 5 , wherein the nylon mesh is a 200 micron mesh. 
     
     
         7 . The method of  claim 5 , wherein the nylon mesh is a 70 micron mesh. 
     
     
         8 . The method of  claim 5 , wherein the nylon mesh is between 50 and 250 microns. 
     
     
         9 . The method of  claim 4 , wherein at least one of the fractions with a higher proportion of islet-like cells than the cell population is a fraction with a smaller particle size than at least one of the other fractions that result from the size separation. 
     
     
         10 . The method of  claim 9 , wherein the at least one fraction with a higher proportion of islet-like cells than the cell population has a particle size equal to or less than 200 microns. 
     
     
         11 . The method of  claim 9  wherein the at least one fraction with a higher proportion of islet-like cells than the cell population has a particle size equal to or less than 150 microns. 
     
     
         12 . The method of  claim 9 , wherein the at least one fraction with a higher proportion of islet-like cells than the cell population has a particle size equal to or less than 100 microns. 
     
     
         13 . The method of  claim 4 , wherein the cell population is fractionated into three fractions by performing two size separation steps, wherein the second separation step is performed on a fraction that results from the first separation step. 
     
     
         14 . The method of  claim 13 , wherein the first separation step is performed using a size separation value equal to or less than 50 microns and the second separation step is performed using a size separation value between 100 microns and 300 microns on the retained fraction from the first separation step; and wherein the pass-through fraction from the second separation step contains a higher proportion of islet-like cells than the cell population. 
     
     
         15 . The method of  claim 14 , wherein the first separation step is performed using a size separation value of 50 microns and the second separation step is performed using a size separation value of 150 microns, and wherein the 50 to 150 micron fraction contains higher proportion of islet-like cells than the cell population. 
     
     
         16 . The method of  claim 13 , wherein the first separation step is performed using a size separation value equal to or less than 50 microns and the second separation step is performed using a size separation value between 150 microns and 250 microns on the retained fraction from the first separation step; and wherein the pass-through fraction from the second separation step contains a higher proportion of islet-like cells than the cell population. 
     
     
         17 . The method of  claim 16 , wherein the first separation step is performed using a size separation value of 50 microns and the second separation step is performed using a size separation value of 200 microns, and wherein the 50 to 200 micron fraction contains higher proportion of islet-like cells than the cell population. 
     
     
         18 . The method of  claim 13 , wherein the first separation step is performed using a size separation value equal to or less than 20 microns and the second separation step is performed using a size separation value of 100 microns or greater on the retained fraction from the first separation step; and wherein the pass-through fraction from the second separation step contains a higher proportion of islet-like cells than the cell population. 
     
     
         19 . The method of  claim 18 , wherein the first separation step is performed using a size separation value of 20 microns and the second separation step is performed using a size separation value of 200 microns, and wherein the 20 to 200 micron fraction contains higher proportion of islet-like cells than the cell population. 
     
     
         20 . The method of  claim 1 , wherein the buds are dissociated from the clusters before the separation step b). 
     
     
         21 . The method of  claim 20 , wherein the buds are dissociated from the clusters by repeated pipetting. 
     
     
         22 . The method of  claim 20 , wherein the buds are dissociated from the clusters by enzyme treatment. 
     
     
         23 . The method of  claim 22 , wherein the enzyme is Dispase. 
     
     
         24 . The method of  claim 22 , wherein the enzyme is trypsin. 
     
     
         25 . The method of  claim 1 , wherein the buds are separated from the clusters by manually or robotically picking the buds. 
     
     
         26 . The method of  claim 1 , wherein the buds are separated from the clusters based on density differences between the buds and the clusters. 
     
     
         27 . The method of  claim 26 , wherein the buds are separated from at least some of the clusters using a Percoll gradient. 
     
     
         28 . The method of  claim 4 , wherein the buds are separated from at least some of the clusters using flow cytometry.