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Ozlëm Barut Selver, Alexander Barash, Mohaned Ahmed, J. Mario Wolosin; ABCG2-Dependent Dye Exclusion Activity and Clonal Potential in Epithelial Cells Continuously Growing for 1 Month from Limbal Explants. Invest. Ophthalmol. Vis. Sci. 2011;52(7):4330-4337. doi: https://doi.org/10.1167/iovs.10-5897.
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© ARVO (1962-2015); The Authors (2016-present)
To determine changes in ABCG2-transport–dependent dye exclusion in outgrowths from limbal explants.
Human or rabbit limbal strips were deposited onto inserts. Over a month, the segments were twice transferred to new inserts. Fresh tissue (FT) cells, obtained by sequential dispase–trypsin digestion and the cells growing from the explant cultures, were characterized for ABCG2-dependent efflux by flow cytometry using a newly identified substratum, JC1. Rabbit cells were sorted into JC1-excluding (JC1low) and main (JC1main) cohorts and seeded with feeder 3T3 cells to determine colony formation efficiency (CFE).
The JC1low cells were all Hoechst 33342–excluding cells and vice versa, establishing the physical equivalence between JC1low and the side population (SP). JC1low cell content was reduced by three ABCG2-specific inhibitors: FTC, Ko143, and glafenine. JC1low percentiles for the fresh human and rabbit cells were 1.4% and 4.1% and CFEs for rabbit JC1low and JC1main were 1.2% and 5.3%. In contrast, the respective JC1low percentiles in the first and second outgrowths were 19.5% and 27.4% and 25.8% and 32.5%, and the rabbit JC1low and JC1main CFEs were 12.3% and 0.9%. Thus, although in FT the contribution of the JC1low cohort to the CFE is minimal, in the explant culture the phenotype incorporates >80% of the CFE.
ABCG2-dependent dye exclusion undergoes a large expansion in explant culture and becomes associated with a high CFE. The transport increase is more pronounced at late outgrowth times, suggesting permanence of stem cells within the explant.
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