Differentiation of human amniotic epithelial cells into corneal epithelial-like cells in vitro

AIM: To explore the feasibility that human amniotic epithelial cells (hAECs) have the potential to differentiate into corneal epithelial -like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells (S-ihCECs). METHODS: hAECs were isolated by enzyme digestion, and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA -DR. Recovered and cultured S-ihCECs, immunocytochemistry was used to detect the expression of CK3/12. The proliferation of S-ihCECs handled by different concentrations of mitomycin was detected by CCK -8. The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK -8. After filtered out the optimal conditions, we collected S-ihCECs culture media for 5 days, then prepared conditioned medium to incubate hAECs, inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs. Quantitative real time reverse transcription polymerase chain reaction (QRT-PCR) was carried out to evaluate the expression of Oct-4, NANOG, PAX6, and CK12 in the differentiation period. lmmunocytochemistry and western bloting were used to detect the expression of CK3/12. RESULTS: The culture media collected every 12h, from 20 mu g/mL mitomycin pretreatment S ihCECs could significantly promote the proliferation of hAECs. In the period of differentiation, the morphology of differentiated hAECs was obviously different compared with the control group, and the distinctive CK3/12 for corneal epithelial cells was detected. CONCLUSION: This study showed that hAECs can differentiate into corneal epithelial like cells by in vitro replication of the corneal epithelial microenvironment, using the culture media collected from S-ihCECs, and it is possible that S ihCECs culture media could be used in corneal tissue engineering.