5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and Cadaver

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 (JC1(low)) and main (JC1(main)) cohorts and seeded with feeder 3T3 cells to determine colony formation efficiency (CFE).. 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.. 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.

Topics: 3T3 Cells; Adult; Aged; Animals; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzimidazoles; Biological Transport; Cadaver; Carbocyanines; Cell Proliferation; Cell Separation; Colony-Forming Units Assay; Epithelial Cells; Fluorescent Dyes; Humans; In Vitro Techniques; Limbus Corneae; Membranes, Artificial; Mice; Middle Aged; Neoplasm Proteins; Polyethylene Terephthalates; Rabbits; Staining and Labeling; Time Factors