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J. Wolosin, O. Barut Selver, M. Mayorano, A. Berra; The Stem Cell-Related SP Phenotype of Limbal Epithelial Cells Wanes in Low Density Culture but Expands in Biopsy Explants. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3738.
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© ARVO (1962-2015); The Authors (2016-present)
Determine the changes in the expression of the ABCG2-dependent stem cell side population (SP) and other efflux-dependent dye exclusion phenotypes in cell cultures and their relationship to clone formation index (CFI).
Limbal cells, freshly isolated by Dispase-trypsin digestion from either NDRI-supplied cadaver corneas (Hu) or from rabbits (Rb), were cultured at low density in SHEM (LD; <10,000 attaching cells/cm2). Alternatively, 2 mm-long limbal biopsies were set on permeable inserts and cultured for 4 -17 days. The ensuing explanted cells were trypsinized and re-cultured in 1 ml SHEM at LD for 16 hr. In either case, LD cultures were incubated with 4 microg Hoechst 33342 (Ho) or, alterantively, 1 microgram JC1 (a mitochondrial dye transported by ABCG2 that identifies the same population; unpublished) and 0.2 microg mitotracker deep red (MtDR, a mitochondrial dye transported by ABCC5; unpublished), trypsinized and analyzed by flow cytometry to determine dye exclusion (Ho-JC1]lo (eqiv. to SP) and MtDRlo. Rb cells were also seeded on 3T3 feeder cells to determine CFI.
After 6 to 16 hr in LD culture, freshly isolated limbal cells included small amounts of [Ho-JC1]lo (Hu: 0.2-0.8 %; Rb: 1-2 %) and MtDRlo cells (Hu: 1-3 %; Rb: 3-7 %). These ABC-dependent phenotypes rapidly decreased and were no longer detectable after 90 hr in both Hu and Rb. In contrast, cultures from explants showed strong phenotype expansion; between days 4-9, [Ho-JC1]lo cells represented 4-7 % and 7 to 18 % of the re-cultured Hu and Rb cells, respectively. MtDRlo underwent similar n-fold increases. Following confluence (>14 days) the phenotypes decreased markedly. The CFI of rabbit explants correlated with the content [Ho-JC1]lo cells in the explants.
ABC transporter-dependent phenotypes in limbal explants are preserved and expanded during explant growth. The percentile expansion suggests that the small cohort of side population cells present in the limbus are a major contributor to the explanted population.
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