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Mijeong Park, Alexander Richardson, Elvis Pandzic, Renee Whan, Erwin Lobo, Stephanie L Watson, Guy Lyons, Denis Wakefield, Nick Di Girolamo; Accelerated deployment of K14-Confetti limbal epithelial progenitor-derived clones during corneal wound-healing visualized in real-time. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2990. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Limbal epithelial stem cells (LESCs) are critically important for regenerating the cornea after wounding. However, their fate and that of their progeny during this process is poorly defined. This study aimed to map the contribution of K14+ limbal precursor cells during corneal wound-healing in a transgenic mouse model using high-resolution microscopy and sophisticated image analysis platforms.
K14CreERT2-Confetti (n=19) and WT C57BL/6 (n=47) mice were used to generate circular (2mm) corneal epithelial debridement wounds. Cell morphology, phenotype and proliferation was assessed by histology, immunofluorescence and BrdU-labelling. Intravital, light-sheet and confocal microscopy was employed to visualize the activity of K14+-Confetti LESCs and their progeny in-vivo and in an organ-culture model during injury-repair. Spatio-Temporal Image Correlation Spectroscopy (STICS) was applied to define velocity and direction of moving clones.
Wounds generally healed within 48-hrs, and during this time-frame the rate of centripetal clonal migration increased by 37.7-fold compared to the uninjured contralateral cornea (19.8 ± 3.7 μm/hr vs 0.53 ± 0.52 µm/hr respectively, p=0.015). This accelerated clonal migration was associated with a 3.5-fold increased proliferative response in the limbus compared to steady-state as indicated by BrdU-labelling index (18.5 ± 5.5 % vs 5.4 ± 1.8 % respectively, p=0.017). Despite the absence of a blood supply, neuronal signals and blinking forces, cellular dynamics recorded in ex vivo wounded corneas was similar to that observed in vivo. Moreover, movement and directionality of Confetti clones was accurately quantified by STICS, which accounted for corneal curvature, and showed that clonal migration in wounds gradually decelerated from 49.0 ± 23.4 μm/hr to 15.2 ± 4.9 μm/hr from the periphery towards the central cornea as wounds sealed.
This study provides the first visual fate-map of how K14+ LESCs partake in wound-healing, and suggests that population pressure from the limbus is the principle driving force behind the initial migratory phase responsible for rapid re-epithelialization. These findings provide clues as to the molecular events that govern LESC dynamics after wounding, and have ramification for devising better treatment modalities for patients with persistent corneal epithelial defects.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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