title: A reconstituted telomerase-immortalized human corneal epithelium in vivo: a pilot study.
abstract: OBJECTIVE
  Telomerase-immortalized human corneal epithelial cells have been reported to stratify and differentiate in vitro similar to native tissue. The purpose of this study was to assess the ability of a telomerase-immortalized human corneal epithelial cell line to generate a full thickness epithelium in vivo in athymic mice.
  
  
  METHODS
  Telomerized corneal epithelial cells were transduced with a retroviral vector encoding the herpes simplex thymidine kinase gene. Efficacy of the thymidine kinase suicide gene was confirmed using a live/dead assay. The epithelium was mechanically removed from athymic nude mice and remaining cells were treated with mitomycin C to prevent re-epithelialization. Telomerized corneal epithelial cells were seeded onto the denuded cornea and allowed to adhere for 4 and 24 hours. Cellular attachment was assessed using a fluorescent cell tracker. Stratification and differentiation were assessed after 7 days using phalloidin and a mouse monoclonal antibody to K3.
  
  
  RESULTS
  Telomerized corneal epithelial cells were visualized across the denuded stromal surface at 4 and 24 hours, with multi-layering evident at the latter time point. No epithelium was present in the non-treated eye. After 7 days post-transplantation cells stratified into a multilayered epithelium, with positive K3 expression in basal and suprabasal cells. Treatment with ganciclovir induced significant loss of viability in vitro.
  
  
  CONCLUSIONS
  The findings in this pilot study demonstrate that telomerized corneal epithelial cells possess the capacity to reconstitute a stratified corneal epithelium in vivo. The introduction of thymidine kinase allowed for the successful induction of cell death in proliferating cells in vitro. Collectively, these data suggest that a telomerase-immortalized corneal epithelial cell line transduced with thymidine kinase represents a potential model for studying differentiation and epithelial-niche interactions in vivo with potential applications in tissue engineering.
authors.affiliation: Department of Ophthalmology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9057, USA. Danielle.Robertson@utsouthwestern.edu
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Robertson
authors.forename: Jerry P
authors.initials: JP
authors.lastname: Kalangara
authors.forename: Rebeccah B
authors.initials: RB
authors.lastname: Baucom
authors.forename: W Matthew
authors.initials: WM
authors.lastname: Petroll
authors.forename: H Dwight
authors.initials: HD
authors.lastname: Cavanagh
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D017690:Cell Transplantation; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000601:surgery*; D003646:Debridement; D004195:Disease Models, Animal; D019573:Epithelium, Corneal / Q000201:enzymology / Q000637:transplantation*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015774:Ganciclovir / Q000633:toxicity; D041722:Genes, Transgenic, Suicide / Q000502:physiology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010865:Pilot Projects; D019098:Telomerase / Q000502:physiology*; D013937:Thymidine Kinase / Q000235:genetics
chemical_list: D013937:Thymidine Kinase; D019098:Telomerase; D015774:Ganciclovir