A reconstituted telomerase-immortalized human corneal epithelium in vivo: a pilot study - PubMed (original) (raw)
A reconstituted telomerase-immortalized human corneal epithelium in vivo: a pilot study
Danielle M Robertson et al. Curr Eye Res. 2011 Aug.
Abstract
Purpose: 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.
Conflict of interest statement
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Figures
Figure 1
Double-labeling with anti-laminin (red) and DRAQ5 (blue). (A) Normal mouse cornea. (B) Presence of basement membrane following epithelial removal, note the presence of a blood vessel within the central cornea. (C) Negative control, primary antibody omitted. All images presented at identical magnification. Scale: 30 μm.
Figure 2
CMFDA CellTracker labeling of HyTK cells (green). DRAQ5 counterstaining of all nuclei (blue). (A–C) 4 hours post-transplantation. (A) Control eye confirming the absence of epithelium; (B) Labeled HyTK cells were seen evenly distributed throughout the corneal surface, scale: 80 μm; (C) Higher magnification showing an intermittent, single layer of HyTK cells, with occasional flattened cells, indicating that cells were adhering to the underlying basement membrane, scale: 40 μm. (D–F) 24 hours post-transplantation. (D) Control eye showing no evidence of re-epithelialization; (E) HyTK cells demonstrating increased flattening with concurrent multi-layering, scale: 80 μm; (F) Higher magnification confirming the presence of 2 cell layers, scale: 40 μm.
Figure 3
Double-labeling with Alexa Fluor 488 Phalloidin and PI. (A) Normal mouse corneal epithelium. Note the presence of 5–7 cell layers with columnar basal cells, wing cells, and flattened squamous surface cells. (B) HyTK cells grown for 7 days show 4–5 defined cell layers, with cuboidal basal cells and flattened squamous surface cells. (C) Negative control, primary antibody omitted. All images presented at identical magnification. Scale: 75 μm.
Figure 4
Montage of corneal surface double labeled with Alexa Fluor 488 Phalloidin and PI. (A) HyTK cells grown for 7 days; (B) Control eye, no evidence of re-epithelialization.
Figure 5
Double-labeling with cytokeratin K3 and PI. (A) After 7 days, the 4–5 cell layered reconstituted epithelium was positive for K3 in all basal and suprabasal cells. (B) Negative control, primary antibody omitted. All images presented at identical magnification. Scale: 75 μM.
Figure 6
Ganciclovir cytotoxicity assay. HyTK cells incubated for 72 hours in increasing concentrations of ganciclovir showed a significant increase in cell death at all concentrations tested with only 30% cell viability at 10 μM (p<0.001, two-way ANOVA, SNK post hoc multiple comparison test, n=3). Graph representative of three combined experiments. No loss in viability was evident in the hTCEpi cell line.
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