title: Differentiation of human amniotic epithelial cells into corneal epithelial-like cells in vitro.
abstract: OBJECTIVE
  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. 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510632, Guangdong Province, China.
authors.forename: Min
authors.initials: M
authors.lastname: Yao
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Xiao-Xi
authors.initials: XX
authors.lastname: Yang
authors.forename: Xiao-Ling
authors.initials: XL
authors.lastname: Zhang
authors.forename: Qing-Shan
authors.initials: QS
authors.lastname: Ji
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhou
authors.forename: Jin-Tang
authors.initials: JT
authors.lastname: Xu
keywords: human amniotic epithelial cells; microenvironment; mytomicin; spontaneously immortalized human corneal epithelial cells; tissue engineering