Di Girolamo, N., Bobba, S., Raviraj, V., Delic, N. C., Slapetova, I., Nicovich, P. R., … Lyons, J. G.
School of Medical Sciences and Biomedical Imaging Facility, University of New South Wales, Sydney, Australia; Sydney Medical School, Discipline of Dermatology and Bosch Institute, University of Sydney, Sydney, Australia.
Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolour genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbour two copies of the Brainbow 2.1 cassette, yielding up to 10 colours from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multi-coloured columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolour genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation.
… “Nikon AZ100 multi-zoom microscope illuminated with a CoolLed pE2 light source equipped with 425 nm, 470 nm, 525 nm, and 585 nm LED array modules.”…
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pE-2: A repeatable, controllable modular system with 20 different LED peaks. Instant on/off and intensity (0-100%) control.
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