Experimental Study On Induced Differentiation Of Human Amniotic Epithelial Cells Into Corneal Epithelial-like Cells In Vitro

Objective To explore the feasibility that human amniotic epithelial cells(hAECs) have thepotential to differentiate into corneal epithelial-like cells under the microenvironment replicatedby spontaneously immortalized human corneal epithelial cells(S-ihCECs).Methods The fresh human amniotic tissue was delivered from36~38w caesarean, and thehAECs were obtained from two-step enzyme digestion method; The flow cytometry was carriedout to detect the expression level of CD29/90/166/73/34and HLA-DR. Recovered and culturedS-ihCECs, immunocytochemistry was used to detect the expression of CK3/12; The S-ihCECswere mitotically inactivated by adding0,10,20,30,40μg/ml mitomycin to the culture mediumand incubating these cells for2h at37°C. Removed mitomycin, then cultured S-ihCECs normally,measured absorbance at day3,5,7by using cell count kit-8(CCK8) to calculate the proliferationinhibition of each concentration; Add10,20μg/ml mitomycin to inactivate S-ihCECs, collectedculture media after culturing for12h or24h, saved it at4℃until incubated hAECs, measuredabsorbance at day1,3,5,7,9and drew the growth curve. After filtered out the optimalconditions, we collected S-ihCECs culture media for5days, then prepared induced medium toincubate hAECs, inverted phase contrast microscope and scanning electron microscope wereused to observe the change of morphology in hAECs. Quantitative Real-Time ReverseTranscription-Polymerase Chain Reaction(qRT-PCR) was carried out to evaluate the expressionof Oct-4, NANOG, PAX6, and CK12in the differentiation period. Immunocytochemistry andwestern blot were used to detect the expression of CK3/12.Results The hAECs were strong three-dimensional cells that grew in a lattice with thetriangulate, quadrangular or oval shape. hAECs expressed surface markers includingCD29/90/166/73, the expression of CD34/HLA-DR were negative. S-ihCECs initially showed afusiform appearance after attached to the culture surface, while it was a cobblestone appearancelike normal epithelial cells morphology after reached confluence; S-ihCECs could expressCK3/12.20μg/ml mitomycin could significantly inhibit the proliferation of S-ihCECs(P＜0.05),and the effect was relatively stable and durable. The group20μg/ml-12h could significantlypromote the proliferation of hAECs. After cultured with induced medium, the morphology ofhAECs was changed into much flatter, and spread out. The microcilia on differentiated hAECsbecame less and shorter. The expression of Oct-4, NANOG, PAX6were positive during thedifferentiation period. The expression of CK12was increased, then it gradually declined as timeprogressed. hAECs could express CK3/12after induced, which were confirmed by imunocytochemistry and western bloting, and the expression of CK12was earlier than CK3.Conclusion hAECs can differentiate into corneal epithelial-like cells by in vitro replication ofthe corneal epithelial microenvironment, using the culture media collected from S-ihCECs.