Comparative analysis of @entity1 -derived feeder layers with 3T3 fibroblasts for the ex vivo expansion of XXXX limbal and oral epithelium.

@entity1 @entity16914 @entity19 @entity10430 @entity4206 @entity3846 @entity1358
Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for @entity1358 ( @entity1358 ). Currently utilized co-culture of stem cells with @entity19 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of @entity1 -derived feeder layers for the ex vivo expansion of epithelial (stem) cells. @entity1 limbal and oral epithelium was co-cultured with @entity19 3T3 fibroblasts, @entity1 dermal fibroblasts (DF), @entity1 mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for @entity4206 and @entity10430 , (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for @entity16914 (cornea specific marker) and @entity3846 and @entity4206 (putative stem cell markers) in all culture conditions. The level of @entity3846 and @entity4206 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p<0.01). This level was comparable to the cells cultured on DF. Expression of @entity4206 and @entity10430 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p<0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p<0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.
