Reconstitution of a Multi-layered, Differentiated Cornea by HTERT-Immortalized Corneal Epithelial Cells Transduced with Thymidine Kinase Transplanted onto Denuded Mouse Corneas
Kalangara, Jerry P.
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OBJECTIVE: To develop an animal model for the implementation of human telomerase enzyme reverse transcriptase (hTERT)-immortalized human corneal epithelial cell line (hTCEpi) transduced with the hygromycin-thymidine kinase gene (HyTK) as a viable cell source for the reconstruction of the corneal surface. METHODS: HyTK cells were cultured in KGM-2 serum-free culture media under hygromycin B selection. Limbal stem cell deficiency (LSCD) was established in athymic nude mice (n=75) using ethylenediaminetetraacetic acid (EDTA) and mitomycin C treatment followed by mechanical debridement of corneal and limbal epithelium. Immunofluorescence (IF) using Anti-Laminin and Propidium iodide (PI) was used to assess presence of basement membrane after epithelial removal. Cultured HyTK cells were stained with Cell Tracker Green CMFDA (5-chloromethylfluorescein diacetate) and transplanted onto the right eye after epithelial removal; the left eye served as a control. Transplanted cells were evaluated at 4 hours, 1 day, and 7 days post-transplantation using laser scanning confocal microscopy (LSCM). At 4 hours and 1 day, corneas were imaged for the presence of CMFDA. At day 7, corneas were stained using antibodies to Keratin 3, Ki-67, and the fluorescent probes – PI, and Phalloidin. Cytotoxicity of ganciclovir was assessed at concentrations of 0.1, 0.5, 1.0, 5.0, and 10.0 μM using Live-Dead Assay. RESULTS: IF confirmed an intact corneal basement membrane following epithelial removal. At 4 hours and 1 day post-transplantation, CMFDA staining demonstrated that transplanted cells were dispersed throughout the corneal surface. After 7 days, HyTK cells showed stratification and IF confirmed a differentiated corneal epithelial phenotype. Incubation in ganciclovir induced a cytotoxic effect on HyTK cells in vitro and had no significant effect on the hTCEpi control cell line. This effect was significant at 0.5 µM (p < 0.05). CONCLUSIONS: These studies demonstrate the first reconstitution of a multi-layered, differentiated corneal epithelium by HyTK cells in the nude mouse model; further, proliferating HyTK cells can be killed with ganciclovir treatment in vitro, which may reduce the potential for risk of oncogenic transformation in vivo.