Methods and compositions for cell attachment and cultivation on planar substrates

Abstract

The present invention is directed to methods for the growth, expansion, and differentiation of pluripotent stem cells on planar substrates lacking an adlayer and a feeder cell layer.

What is claimed is: 
     
         1 . A method of differentiating human pluripotent stem cells into definitive endoderm cells on a porous planar substrate comprising the steps of:
 (a) adding a cell suspension of human pluripotent stem cells to the porous planar substrate;   (b) allowing the suspension of human pluripotent stem cells to attach; and   (c) differentiating the attached human pluripotent stem cells into human definitive endoderm cells by treating human pluripotent stem cells with activin A under serum free conditions,
 wherein the porous planar substrate has the following features:
 contains from about 3.1% N to about 12% N; 
 contains from at least about 12% O to at least about 55% O; 
 lacks an adlayer and a feeder cell; 
 is hydrophilic; and 
 comprises polycarbonate or mixed cellulose esters. 
 
   
     
     
         2 . The method of  claim 1 , wherein attachment of the human pluripotent stem cells to the porous planar substrate is Rho kinase independent. 
     
     
         3 . The method of  claim 1 , wherein attachment of the human pluripotent stem cells to the porous planar substrate is Rho kinase dependent. 
     
     
         4 . The method of  claim 1 , wherein the method further comprises treating the suspension of cells with a compound capable of inhibiting Rho kinase activity. 
     
     
         5 . The method of  claim 1 , wherein the attachment of the pluripotent stem cells to the substrate is enhanced by treating the suspension of cell suspension of human pluripotent stem cells with a compound capable of inhibiting Rho kinase activity prior to step (a). 
     
     
         6 . The method of  claim 5 , wherein the compound capable of inhibiting Rho kinase activity is selected from the group consisting of: Y-27632, Fasudil, H-1152 and Hydroxyfasudil. 
     
     
         7 . The method of  claim 6 , wherein the compound capable of inhibiting Rho kinase activity is Y-27632. 
     
     
         8 . The method of  claim 6 , wherein the compound capable of inhibiting Rho kinase activity is H-1152. 
     
     
         9 . The method of  claim 5 , wherein the compound capable of inhibiting Rho kinase activity is removed after the pluripotent stem cells attach to the substrate and prior to step (c). 
     
     
         10 . The method of  claim 9 , wherein the substrate comprises polycarbonate and wherein the compound capable of inhibiting Rho kinase activity is H-1152. 
     
     
         11 . The method of  claim 1 , wherein the substrate has a porosity of from about 0.45 μm to about 0.8. 
     
     
         12 . The method of  claim 1 , wherein the substrate comprises mixed cellulose esters. 
     
     
         13 . The method of  claim 12 , wherein the substrate contains about 8.8% N and about 51.9% O. 
     
     
         14 . The method of  claim 1 , wherein the substrate comprises polycarbonate and wherein the substrate contains about 3.1% N and about 14.3% O. 
     
     
         15 . The method of  claim 1 , wherein the substrate has a contact angle from about 18 degrees to about 32 degrees. 
     
     
         16 . The method of  claim 1 , wherein the human definitive endoderm cells are further differentiated into human pancreatic endoderm cells, human primitive gut tube cells, or human posterior foregut cells. 
     
     
         17 . The method of  claim 16 , wherein the human definitive endoderm cells are further differentiated into human pancreatic endocrine cells. 
     
     
         18 . The method of  claim 1 , wherein the method comprises treating human pluripotent stem cells with activin A and wnt3A under serum free conditions. 
     
     
         19 . The method of  claim 18 , wherein the method comprises treating human pluripotent stem cells with DMEM-F12 medium supplemented with activin A and wnt3A under serum free conditions.