Experimental Study of Inducing Embryonic Stem Cell to Differentiate Into Corneal Epithelium

Abstract

Abstract: : Purpose: Our project was to determine whether embryonic stem (ES) cells could be induced to differentiate into corneal epithelia with superficial corneoscleral limbal stroma. Methods: To achieve this, ES–GFP cells line D₃ were pre–induced with retinoic acid (RA). Then, the pre–induced cells were seeded on deepithelialized superficial corneoscleral slices (SCSS). At last, one group of SCSS with confluent differentiated cells in vitro were passaged for detection, another group of SCSS with confluent differentiated cells were exposed to air–liquid interface for 10 days, then implanted into subcutaneous layer of nude mice for 2 weeks for further induction in vivo. Results: No teratomas were found 2 weeks after the implantation of differentiated ES cells into nude mice. The differentiated cells showed an appearance of epithelia both in vivo and in vitro. Expression of CK3, P63 and PCNA were detected by immunohistochemical stain in the differentiated cells in both groups. Microvillis and zonula occludens were observed on the surface of the differentiated cells under electronic microscope. In control group, ES cells differentiated freely without any inducing factors. Most cells were shedding, majority of survival cells formed a dendrite–like structure as neurons, and minority appeared polymorphic. Conclusions: These results demonstrate that ES cells can differentiate into corneal epithelia on the surface of SCSS under controlled condition. Differentiated ES cells can be used as epithelial seeding cells for the reconstruction of ocular surface and corneal tissue engineering in the future.

Keywords: cornea: basic science • cornea: epithelium