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

• 作者: Jian Chen Jin-Tang Xu Min Yao Qing Zhou Qing-Shan Ji Xiao-Ling Zhang Xiao-Xi Yang
• 来源期刊: 国际眼科杂志：英文版
• 学科: 医学
• 关键词: HUMAN amniotic EPITHELIAL CELLS spontaneously immortalized HUMAN CORNEAL EPITHELIAL CELLS mytomicin MICROENVIRONMENT tissue engineering
• 年，卷（期）: 2013,（5）
• 页码范围: 564-572
• 页数: 9页
• 分类号: R329

摘要

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.