XXXX -dependent dye exclusion activity and clonal potential in epithelial cells continuously growing for 1 month from limbal explants.

@entity1 @entity49320 @entity95 @entity39549 @entity39102
PURPOSE: To determine changes in @entity39102 -transport-dependent dye exclusion in outgrowths from limbal explants. METHODS: @entity1 or @entity95 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 @entity39102 -dependent efflux by flow cytometry using a newly identified substratum, JC1. @entity95 cells were sorted into JC1-excluding (JC1(low)) and main (JC1(main)) cohorts and seeded with feeder 3T3 cells to determine colony formation efficiency (CFE). RESULTS: The JC1(low) cells were all Hoechst 33342-excluding cells and vice versa, establishing the physical equivalence between JC1(low) and the side population (SP). JC1(low) cell content was reduced by three @entity39102 -specific inhibitors: FTC, @entity39549 , and @entity49320 . JC1(low) percentiles for the fresh @entity1 and @entity95 cells were 1.4% and 4.1% and CFEs for @entity95 JC1(low) and JC1(main) were 1.2% and 5.3%. In contrast, the respective JC1(low) percentiles in the first and second outgrowths were 19.5% and 27.4% and 25.8% and 32.5%, and the @entity95 JC1(low) and JC1(main) CFEs were 12.3% and 0.9%. Thus, although in FT the contribution of the JC1(low) cohort to the CFE is minimal, in the explant culture the phenotype incorporates >80% of the CFE. CONCLUSIONS: @entity39102 -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.
