Induction of corneal endothelial cells

1. A method of culturing corneal endothelial cells (CECs) in vitro comprising:

a. providing a culture of pluripotent stem cells (PSCs),

b. culturing the PSCs with a culture medium containing WNT pathway signaling inhibitor, a BMP pathway signaling inhibitor, and a TGF-β pathway signaling inhibitor for a time period sufficient to produce a culture of eye field stem cells (EFSCs), wherein the culture of EFSCs comprises 70% or more EFSCs,

c. removing EFSC medium and replacing with a culture medium that lacks the WNT pathway signaling inhibitor and promoting WNT signaling in the EFSCs by removal of EFSC media from the EFSCs to produce a culture of ocular neural crest stem cells (NCSCs), and then

d. changing the culture medium of the NCSCs to one that includes the TGF-β pathway signaling inhibitor and a ROCK signaling inhibitor suppressing TGF-β pathway signaling and ROCK signaling in the culture of NCSCs, thereby producing a culture of culturing CECs;

wherein the WNT pathway signaling inhibitor is IWP2, the BMP pathway signaling inhibitor is LDN193189, and the TGF-β pathway signaling inhibitor is SB431542.

2. The method of claim 1 , wherein the step of suppressing TGFβ and ROCK signaling is conducted in a feeder-free and serum-free defined media by applying a small molecule differentiator to the culture.

3. The method of claim 2 , wherein the small molecule differentiator comprises a ROCK inhibitor and a TGFβ inhibitor.

4. The method of claim 3 , wherein the ROCK inhibitor is H-1125 or Y27632, and wherein the TGFβ inhibitor is SB431542.

5. The method of claim 1 , wherein the CEC culture is expanded into a sheet of cells.

6. The method of claim 1 , wherein the majority of NCSCs express neural crest markers p75NTR and HNK-1.

7. The method of claim 1 , wherein the majority of EFSCs express markers PAX6 and LHX2.