Comparative analysis of different feeder layers with 3T3 fibroblasts for culturing rabbits limbal stem cells

AIM： To explore the possibility of human umbilical cord mesenchymal stem cells（h UCMSCs）, human umbilical vein endothelial cells（h UVECs）, human dental pulp stem cells（h DPSCs） and human periodontal ligament stem cells（h PDLSCs） serving as feeder cells in co-culture systems for the cultivation of limbal stem cells.METHODS： Different feeder layers were cultured in Dulbecco＇s modified Eagle＇s medium（DMEM）/F12 and were treated with mitomycin C. Rabbits limbal stem cells（LSCs） were co-cultured on h UCMSCs, h UVECs, h DPSCs, h PDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency（CFE） assay and immunofluorescence（IPO13,CK3/12）.RESULTS： The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But h UCMSCs, h DPSCs and h PDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to h UVECs and feedercell-free culture.CONCLUSION： hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.