{"id":{"repo_id":"utswmed","oai_identifier":"oai:utswmed-ir.tdl.org:2152.5/929"},"canonical_url":"https://search.dev.ndltd.org/etd/utswmed/oai:utswmed-ir.tdl.org:2152.5/929","repository":{"repo_id":"utswmed","name":"University of Texas Southwestern Medical Center","base_url":"https://utswmed-ir.tdl.org/server/oai/request"},"display":{"title":"Reconstitution of a Multi-layered, Differentiated Cornea by HTERT-Immortalized Corneal Epithelial Cells Transduced with Thymidine Kinase Transplanted onto Denuded Mouse Corneas","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Kalangara, Jerry P."],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Cavanagh, H. Dwight"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-10-25T17:33:30Z","date_published":"2011-10-25T17:33:30Z","updated_at":"2026-07-24T05:52:13Z","subjects":["Cornea","Corneal Transplantation","Epithelium, Corneal"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["758868616"],"render_values":[{"text":"758868616","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/2152.5/929","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Cavanagh, H. Dwight"]},{"key":"dc:creator","label":"Author","values":["Kalangara, Jerry P."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-10-25T17:33:30Z","2011-10-25"]},{"key":"dc:type","label":"Dc Type","values":["Thesis","Text"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Cornea","Corneal Transplantation","Epithelium, Corneal"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2152.5/929","758868616"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Reconstitution of a Multi-layered, Differentiated Cornea by HTERT-Immortalized Corneal Epithelial Cells Transduced with Thymidine Kinase Transplanted onto Denuded Mouse Corneas"]}]}],"canonical_facts":{"dc:contributor":["Cavanagh, H. Dwight"],"dc:creator":["Kalangara, Jerry P."],"dc:date":["2011-10-25T17:33:30Z","2011-10-25"],"dc:description":["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."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2152.5/929","758868616"],"dc:language":["en"],"dc:subject":["Cornea","Corneal Transplantation","Epithelium, Corneal"],"dc:title":["Reconstitution of a Multi-layered, Differentiated Cornea by HTERT-Immortalized Corneal Epithelial Cells Transduced with Thymidine Kinase Transplanted onto Denuded Mouse Corneas"],"dc:type":["Thesis","Text"]},"updated_at":"2026-07-24T05:52:13Z"}