ABCG2-dependent dye exclusion activity and clonal potential in epithelial cells continuously growing for 1 month from limbal explants

Purpose. To determine changes in ABCG2-transport-dependent dye exclusion in outgrowths from limbal explants. Methods. 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 (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 ABCG2-specific inhibitors: FTC, Ko143, and glafenine. JC1 ^low percentiles for the fresh human and rabbit cells were 1.4% and 4.1% and CFEs for rabbit 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 rabbit 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. 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.