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J. P. Kalangara, D. M. Robertson, R. B. Baucom, S. I. Ho, H. D. Cavanagh; Reconstitution of a Multi-Layered, Differentiated Cornea by HTERT-Immortalized Corneal Epithelial Cells Transduced With Thymidine Kinase Transplanted Onto Denuded Mouse Corneas. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5751. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Limbal stem cell deficiency (LSCD) prevents the regeneration of the corneal epithelium, leading to corneal opacification and blindness. Current treatments include autologous limbal stem cell transplantation, when available, or limbal allografts from cadaver tissue which demand high immunosuppressive therapy to prevent host rejection. Thus, there is a critical need to develop a transplantable artificial corneal epithelium. The purpose of this study was to assess the use of a HTERT-immortalized human corneal epithelial cell line transduced with the thymidine kinase gene (HyTK) as a source of cells for the reconstruction of the corneal surface and to evaluate the efficacy of therapeutic levels of topical gancyclovir in maintaining proliferative control over the HyTK cell line.
HyTK cells were cultured in KGM-2 serum-free culture media under hygromycin selection. LSCD was established in athymic nude mice using EDTA and Mitomycin C treatment followed by surgical excision of limbal epithelium. Immunofluorescence (IF) using Anti-Laminin and Propidium iodide (PI) was used to assess presence of basement membrane. Cultured HyTK cells were transfected with Calcein AM and transplanted onto the right eye after epithelial removal; the left eye served as a control. Transplanted cells were evaluated at 1 hour and day 7 post-transplantation using laser scanning confocal microscopy. At 1 hour, corneas were imaged for the presence of Calcein AM. At day 7, corneas were stained using antibodies to Keratins 3 and 12, ZO-1, PI, and Phalloidin. Cytotoxicity of gancyclovir was assessed at concentrations of 0.1, 0.5, 1.0, 5.0, and 10.0 µM using Live-Dead Assay.
IF confirmed an intact corneal basement membrane following epithelial removal. At 1 hour, Calcein AM 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. Gancyclovir treatment induced a cytotoxic effect on HyTK cells (p < 0.001) and had no significant effect on hTCEpi control cell line. This effect was significant at 0.5 uM.
These studies demonstrate the first reconstitution of a multi-layered, differentiated cornea by HyTK cells in the nude mouse model; furthermore, proliferating HyTK cells can be killed with topical gancyclovir treatment, which eliminates later potential risk of oncogenic transformation.
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