Reconstitution of a Multi-layered, Differentiated Cornea by HTERT-Immortalized Corneal Epithelial Cells Transduced with Thymidine Kinase Transplanted onto Denuded Mouse Corneas
| dc.contributor.advisor | Cavanagh, H. Dwight | en |
| dc.creator | Kalangara, Jerry P. | en |
| dc.date.accessioned | 2011-10-25T17:33:30Z | |
| dc.date.available | 2011-10-25T17:33:30Z | |
| dc.date.issued | 2011-10-25 | |
| dc.description.abstract | 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. | en |
| dc.identifier.oclc | 758868616 | |
| dc.identifier.uri | https://hdl.handle.net/2152.5/929 | |
| dc.language.iso | en | en |
| dc.subject | Cornea | en |
| dc.subject | Corneal Transplantation | en |
| dc.subject | Epithelium, Corneal | en |
| dc.title | Reconstitution of a Multi-layered, Differentiated Cornea by HTERT-Immortalized Corneal Epithelial Cells Transduced with Thymidine Kinase Transplanted onto Denuded Mouse Corneas | en |
| dc.type | Thesis | en |
| dc.type.material | Text | en |
| thesis.date.available | 2011-10-25 | |
| thesis.degree.department | UT Southwestern Medical School | en |
| thesis.degree.discipline | Research | en |
| thesis.degree.grantor | UT Southwestern Medical Center | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | M.D. with Distinction | en |