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Tracing the fate of limbal epithelial progenitor cells in the murine cornea.

Stem cells (Dayton, Ohio) (2014-06-27)
N Di Girolamo, S Bobba, V Raviraj, N C Delic, I Slapetova, P R Nicovich, G M Halliday, D Wakefield, R Whan, J G Lyons
ZUSAMMENFASSUNG

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 multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored 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 multicolor 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.

MATERIALIEN
Produktnummer
Marke
Produktbeschreibung

Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, ACS reagent, ≥99.5%
Sigma-Aldrich
DAPI, for nucleic acid staining
Supelco
Ethanol -Lösung, certified reference material, 2000 μg/mL in methanol
Sigma-Aldrich
Tamoxifen -citrat (Salz), ≥99%
USP
Dehydrated Alcohol, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Tris(tert-butoxy)silanol, 99.999%
Tamoxifencitrat, European Pharmacopoeia (EP) Reference Standard
Tamoxifencitrat für Performance-Test, European Pharmacopoeia (EP) Reference Standard