title: ABCG2-dependent dye exclusion activity and clonal potential in epithelial cells continuously growing for 1 month from limbal explants.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, Dokuz Eylul University, Izmir, Turkey.
authors.forename: Ozlëm Barut
authors.initials: OB
authors.lastname: Selver
authors.forename: Alexander
authors.initials: A
authors.lastname: Barash
authors.forename: Mohaned
authors.initials: M
authors.lastname: Ahmed
authors.forename: J Mario
authors.initials: JM
authors.lastname: Wolosin
mesh_terms: D016475:3T3 Cells; D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism*; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000328:Adult; D000368:Aged; D000818:Animals; D001562:Benzimidazoles / Q000493:pharmacokinetics* / Q000633:toxicity; D001692:Biological Transport; D002102:Cadaver; D002232:Carbocyanines / Q000493:pharmacokinetics* / Q000633:toxicity; D049109:Cell Proliferation; D002469:Cell Separation; D003114:Colony-Forming Units Assay; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes / Q000493:pharmacokinetics* / Q000633:toxicity; D006801:Humans; D066298:In Vitro Techniques; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism*; D008567:Membranes, Artificial; D051379:Mice; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000378:metabolism*; D011093:Polyethylene Terephthalates; D011817:Rabbits; D013194:Staining and Labeling; D013997:Time Factors
chemical_list: C116614:ABCG2 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D008567:Membranes, Artificial; D009363:Neoplasm Proteins; D011093:Polyethylene Terephthalates; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C017807:bisbenzimide ethoxide trihydrochloride