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

Invest Ophthalmol Vis Sci. 2011 Jun 17;52(7):4330-7. doi: 10.1167/iovs.10-5897.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • ABCG2 protein, human
  • ATP Binding Cassette Transporter, Subfamily B
  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • ATP-Binding Cassette Transporters
  • Benzimidazoles
  • Carbocyanines
  • Fluorescent Dyes
  • Membranes, Artificial
  • Neoplasm Proteins
  • Polyethylene Terephthalates
  • 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine
  • bisbenzimide ethoxide trihydrochloride