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.

What is claimed is: 
     
         1 . A method for the propagation of human pluripotent stem cells comprising:
 a) Attaching a population of human pluripotent stem cells to a first volume of micro-carriers;   b) Culturing the human pluripotent stem cells on the first volume of micro-carriers in a defined medium lacking a Rho kinase inhibitor,   c) Removing the human pluripotent stem cells from the first volume of micro-carriers; and   d) Attaching the population of human 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 human 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 human pluripotent stem cells are attached to the first volume of micro-carriers, the second volume of micro-carriers, or both in medium containing a Rho kinase inhibitor. 
     
     
         6 . The method of  claim 5 , wherein the Rho kinase inhibitor is Y27632 or Glycyl-H 1152 dihydrochloride. 
     
     
         7 . The method of  claim 6 , wherein the method comprises from about 1 μM to about 10 μM of the Rho kinase inhibitor Y27632. 
     
     
         8 . The method of  claim 6 , wherein the method comprises from about 0.25 μM to about 5 μM of the Rho kinase inhibitor Glycyl-H 1152 dihydrochloride. 
     
     
         9 . The method of  claim 1 , wherein the human pluripotent stem cells are removed from the first volume of micro-carriers, the second volume of micro-carriers, or both by enzymatic treatment. 
     
     
         10 . The method of  claim 1 , wherein the first volume of micro-carriers is removed from the human pluripotent stem cells prior to attaching the cells to the second volume of micro-carriers. 
     
     
         11 . The method of  claim 1 , wherein the human pluripotent stem cells are removed from the first volume of micro-carriers and the second volume of micro-carriers by treatment with a protease. 
     
     
         12 . The method of  claim 1 , wherein the step of attaching the population of human pluripotent stem cells to the first volume of micro-carriers or the step of attaching the population of human pluripotent stem cells to a second volume of micro-carriers comprises seeding the pluripotent stem cells at about a seeding density of about 4,000 to about 30,000 cells per cm 2  of micro-carriers. 
     
     
         13 . The method of  claim 1 , wherein the method comprises agitating the micro-carriers. 
     
     
         14 . The method of  claim 1 , wherein the human pluripotent stem cells maintain pluripotentency. 
     
     
         15 . The method of  claim 12 , wherein the human pluripotent stem cells express CD9, SSEA-4, SSEA-3, TRA-1-60, and TRA-1-81. 
     
     
         16 . The method of  claim 1 , wherein the method further comprises culturing the human pluripotent stem cells on a planar substrate prior to step a). 
     
     
         17 . The method of  claim 1 , wherein the method eliminates any need for an animal component matrix. 
     
     
         18 . The method of  claim 1 , wherein the human pluripotent stem cells are propagated as single cells. 
     
     
         19 . The method of  claim 1 , wherein the human pluripotent stem cells are human embryonic stem cells.