Pluripotent Stem Cell Culture on Micro-Carriers

Abstract

The present invention is directed to methods for the growth, expansion and differentiation of pluripotent stem cells on micro-carriers.

1 . A method for the propagation of pluripotent stem cells, comprising the steps of:
 a. Attaching a population of pluripotent stem cells to a first volume of micro-carriers,   b. Culturing the pluripotent stem cells on the first volume of micro-carriers,   c. Removing the pluripotent stem cells from the first volume of micro-carriers, and   d. Attaching the population of pluripotent stem cells to a second volume of micro-carriers.   
   
   
       2 . The method of  claim 1 , wherein the steps of culturing, removing and attaching the pluripotent stem cells on micro-carriers are is repeated using subsequent volumes of micro-carriers. 
   
   
       3 . The method of  claim 1 , wherein the first volume of micro-carriers is selected from the group consisting of dextran micro-carriers and polystyrene micro-carriers. 
   
   
       4 . The method of  claim 1 , wherein the second volume of micro-carriers is selected from the group consisting of dextran micro-carriers and polystyrene micro-carriers. 
   
   
       5 . The method of  claim 1 , wherein the pluripotent stem cells are attached to the first volume of micro-carriers in medium containing a Rho kinase inhibitor. 
   
   
       6 . The method of  claim 1 , wherein the pluripotent stem cells are attached to the second volume of micro-carriers in medium containing a Rho kinase inhibitor. 
   
   
       7 . The method of  claim 1 , wherein the pluripotent stem cells are removed from the first volume of micro-carriers by enzymatic treatment. 
   
   
       8 . The method of  claim 1 , wherein the pluripotent stem cells are removed from the second volume of micro-carriers by enzymatic treatment. 
   
   
       9 . The method of  claim 1 , wherein the first volume of micro-carriers is removed from the pluripotent stem cells prior to attaching the cells to the second volume of micro-carriers. 
   
   
       10 . A method to differentiate pluripotent stem cells to insulin-expressing cells on micro-carriers comprising the steps of:
 a. Attaching the pluripotent stem cells to a volume of micro-carriers,   b. Differentiating the pluripotent stem cells to cells expressing markers characteristic of the definitive endoderm lineage,   c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage,   d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, and   e. Differentiating the cells expressing markers characteristic of the pancreatic endocrine lineage into insulin-expressing cells.