Cultivation and characterization of limbal epithelial stem cells in rabbits

Purpose: In the last decades, strong evidence emerged regarding the presence of stem cells located at the corneal limbus. Our objective was to find a way to isolate and cultivate rabbit corneal stem cells in vitro, into an epithelial tissue. Materials and Methods: Two in vitro systems were developed to culture rabbit corneal stem cells: (1) limbal biopsies used as explants and cultivated on fresh denuded amniotic membrane and (2) a monolayer culture obtained by enzymatic treatment of the corneal biopsies. Genetic characterization (PCR) was performed. Specific triggers were used to induce differentiation of corneal stem cells. Results: At four weeks, 16 explant samples out of 18 cultures showed good expansion, ranging from 1 cm to 2 cm. Genetic characterization showed similar expression of genetic stem markers for corneal stem cells and placental stem cells, previously characterized (stem cell factor, Oct3/4, Vimentin, Nestin and Neurofilament). Corneal stem cells showed high Rhodamine efflux and were effective progenitors for neuronal, myocardial, osteogenic and endothelial lineage. Conclusions: In one month, it was possible to grow enough epithelial tissue with preserved proliferative state to allow transplantation on the cornea.