Abstract
Purpose :
The mammalian cornea is a transparent, avascular tissue whose epithelium is maintained by stem cells (SC) residing in the limbus, and are thus known as limbal epithelial SC (LESC). However, controversy continues to surround the designation and destination of LESCs and their progeny. Therefore, this study was initiated to ascertain when LESCs evolve and their fate during development and aging.
Methods :
Female K14CreERT2-Confetti mice were time-mated then injected with tamoxifen (TAM) for transgene induction at embryonic (E) day 15.5. Embryos (n=22) and pups (n=4) were harvested between E16.5 and post-natal day 14 for microscopic analysis. Additionally, 1-60 wk old transgenic mice (n=28) were sacrificed at selected time-points post-TAM and their corneas imaged by confocal and intra-vital microscopy.
Results :
Individual fluorescent cells were detected in E16.5 corneas 1-day post-transgene induction which formed small colonies at E18.5. By E19.5, K14+ cells were distributed across the primordial corneal epithelium, seeding new fluorescent clones that persisted into post-natal life. Six-week old transgenic mice injected with TAM developed radial streaks derived from fluorescent K14+ progenitors, which migrated towards the central cornea. With age, fluorescent clones decreased in number from 63.6±4.2 to 26.8±9.8, while those remaining became wider (86.5±9.7μm to 281.6±51.7μm). In addition, clones deviated from their linear trajectory, converging on a central nasal-temporal demarcation zone. TUNEL staining identified increased apoptosis in this midline region.
Conclusions :
Herein, we demonstrated clonal dynamics of K14+ corneal epithelial progenitor cells during development and aging. We observed limbal segregation and loss of clones over time, possibly due to SC quiescence, functional decline, or symmetric division of SC into two differentiated daughters. Clone broadening could also be the result of symmetric division, whereby both daughters are retained in the SC pool as neighbours. Increased central apoptosis could result from eyelid frictional forces, explaining why cells deviated from their linear path to the corneal apex. This is a robust model of age-related corneal epithelial cell dynamics and may be useful to ascertain SC activity under a myriad of conditions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.