Differentiation Of Adipose Derived Mesenchymal Stem Cells Induced By Pax6 And Its Application

ObjectivesAdipose derived mesenchymal stem cells(ADMSCs) were multipotent adult stem cells, which have potential of multi-directional differentiation. In this research, ADMSCs differentiated into corneal epithelioid-like cells after being transfected with Pax6 gene. This attempt may hold great promise in generating corneal cell sheets for corneal epithelium repair of corneal injury. MethodsThe ADMSCs were isolated by enzymatic digestion from bilateral inguinal fat pads of healthy SD rat, which were identified using HPLC afterwards. The ADMSCs cells were designated into two groups, that is, recombinant plasmid Pax6-EGFP transfection group or blank control group. The cells in both groups were screened by G418. The colony forming units assay was initiated to compare the proliferative activity between the two groups. The Pax6-transfected cells were transplanted onto rabbit with corneal full-thickness resection, the growth of which were observed. ResultsThe ADMSCs displayed a fusiform appearance with the centripetal arrangement. The cells were positive for CD90 and CD105,but negative for CD34 and CD45. After being transfected with Pax6-EGFP plasmid, green fluorescence of green fluorescent protein(GFP) was visible under inverted microscope. The colony forming units assay demonstrated that the proliferative activity of the Pax6-EGFP-tranfected ADMSCs was lower than that in the control group. Both immunofluorescence and western blot showed that the Pax6-EGFP-tranfected ADMSCs had green fluorescence and expressed CK3-12, E-cadherin and Pax6-EGFP protein. The cells were transplanted onto New Zealand white rabbits cornea with corneal full-thickness resection. The optic detection revealed that corneal surface of the rabbits were smooth and transparent. The corneal epithelial cells were cobblestone-like cells observed in HE staining and displayed green fluorescence under the inverted microscope. ConclusionsThe ADMSCs could be induced into corneal epithelial-like cells by Pax6 gene transfection. The Pax6-EGFP c DNA-transfected ADMSCs could be successfully transplanted onto rabbit cornea with full-thickness excision of the sectorial limbus. The transplanted cells were able to replenish the corneal epithelium-like cells and repair damaged corneal surface. This research may provide a novel corneal cell sheets for corneal epithelium repair of corneal disorders.