Methods of generating corneal cells and cell populations comprising same

Abstract

A method of generating a population of corneal epithelial cells is disclosed. The method comprises culturing human pluripotent stem cells in corneal fibroblast-conditioned medium on a solid surface comprising an extracellular matrix component thereby generating the population of corneal epithelial cells. Isolated cell populations and corneal tissues are also disclosed, as well as uses thereof.

1 - 31 . (canceled) 
     
     
         32 . A method of treating an eye disorder in a subject in need thereof, the method comprising transplanting to the subject a therapeutically effective amount of human corneal epithelial cells generated according to a method of generating a population of human corneal epithelial cells which comprises culturing human pluripotent stem cells in corneal fibroblast-conditioned medium on a solid surface comprising an extracellular matrix component thereby generating the population of human corneal epithelial cells, wherein said corneal fibroblast-conditioned medium is produced from human corneal fibroblasts which are not contaminated with limbal fibroblasts and comprises insulin, hydrocortisone and epidermal growth factor (EGF). 
     
     
         33 . The method according to  claim 32 , wherein said corneal fibroblast-conditioned medium further comprises bone morphogenetic protein-4 (BMP-4). 
     
     
         34 . The method according to  claim 32 , wherein said extracellular matrix component is selected from the group consisting of collagen IV, laminin, fibronectin and Matrigel®. 
     
     
         35 . The method according to  claim 32 , wherein said population of human corneal epithelial cells does not comprise skin cells. 
     
     
         36 . The method according to  claim 32 , wherein at least 70% of the cells of said population of human corneal epithelial cells co-express K3 and Pax6. 
     
     
         37 . The method according to  claim 32 , wherein wherein less than 10% of the cells of said population of human corneal epithelial cells express nanog and Oct4. 
     
     
         38 . A method of treating an eye disorder in a subject in need thereof, the method comprising transplanting to the subject a therapeutically effective amount of human corneal tissue generated according to a method of generating human corneal tissue which comprises:
 (a) culturing human pluripotent stem cells in corneal fibroblast-conditioned medium on a solid surface comprising an extracellular matrix component, thereby generating a population of human corneal epithelial cells, wherein said corneal fibroblast-conditioned medium is produced from human corneal fibroblasts which are not contaminated with limbal fibroblasts and comprises insulin, hydrocortisone and epidermal growth factor (EGF);   (b) dissociating the generated population of human corneal epithelial cells to generate a population of dissociated human corneal epithelial cells; and   (c) culturing said dissociated human corneal epithelial cells on a scaffold under conditions that generate human corneal tissue.   
     
     
         39 . The method according to  claim 38 , wherein said corneal fibroblast-conditioned medium further comprises bone morphogenetic protein-4 (BMP-4). 
     
     
         41 . The method according to  claim 38 , wherein said extracellular matrix component is selected from the group consisting of collagen IV, laminin, fibronectin and Matrigel®. 
     
     
         42 . The method according to  claim 38 , wherein said population of human corneal epithelial cells does not comprise skin cells. 
     
     
         43 . The method according to  claim 38 , wherein at least 70% of the cells of said population of human corneal epithelial cells co-express K3 and Pax6. 
     
     
         44 . The method according to  claim 38 , wherein wherein less than 10% of the cells of said population of human corneal epithelial cells express nanog and Oct4. 
     
     
         45 . A method of screening for an agent which enhances differentiation towards a human corneal epithelial lineage, the method comprising:
 (a) culturing human pluripotent stem cells in corneal fibroblast-conditioned medium, wherein said corneal fibroblast-conditioned medium is produced from human corneal fibroblasts which are not contaminated with limbal fibroblasts and comprises insulin, hydrocortisone and epidermal growth factor (EGF), on a solid surface comprising an extracellular matrix component in a presence of said agent; and   (b) analyzing a differentiation status of said human pluripotent stem cells, wherein an increase in differentiation compared to a differentiation in an absence of said agent is indicative of an agent which enhances differentiation towards a human corneal epithelial lineage.   
     
     
         46 . The method of  claim 45 , wherein said pluripotent stem cells comprise iPS cells. 
     
     
         47 . The method of  claim 45 , wherein said iPS cells are derived from healthy patients. 
     
     
         48 . The method of  claim 45 , wherein said iPS cells are derived from diseased patients. 
     
     
         49 . The method of  claim 45 , wherein said corneal fibroblast-conditioned medium further comprises bone morphogenetic protein-4 (BMP-4).