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 Oct4,NANOG,PAX6,and CK12 in the differentiation period.Immunocytochemistry and western bloting were used to detect the expression of CK3/12.RESULTS:The culture media collected every 12h,from 20μg/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. 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 analyze the expression of CD29 / 90/166/73/34 and HLA-DR. Revepted and cultured S-ihCECs, immunocytochemistry was used to detect the expression of CK3 / 12. The proliferation of S ihCEC 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 transcr PCR-PCR was performed to evaluate the expression of Oct4, NANOG, PAX6, and CK12 in the differentiation period. Immunocytochemistry and western blotting were used to detect the expression of CK3 / 12.RESULTS: The culture media collected every 12h, from 20 μg / 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 that S-ihCECs culture media could be used in corneal tissue engineering.