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O. Barut Selver, J. Wolosin; Efflux of Mitotracker Deep Red by a Multidrug Resistance Transporter Identifies a Limbal Epithelial Cell Population Related to-, and Larger Than, the Well Known Stem-Cell Side Population. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3748.
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© ARVO (1962-2015); The Authors (2016-present)
Multidrug resistance (MDR) efflux transporters of the ATP binding cassette (ABC)-type limit the accumulation of slow penetrating, supravital dyes, generating DYElo cohorts. ABC subtype G2 acts on the DNA dye Hoechst33342 (Ho) causing the formation of a Holo cohort known as a side population(SP), rich in stem cells. The objective of this study was identify MDR sub-populations in the two ocular surface epithelia through the exclusion of mitochondrial-binding dyes.
Freshly isolated limbal or conjunctival epithelial cells from cadaver corneas or abattoir rabbit were isolated by enzymatic digestion and cultured for 16 hr in 1 ml SHEM. The medium was complemented with Ho (4 microg- 90 min) and mitotracker deep red (MtDR; 0.2 microg-30 min). Dye stained cells were analyzed/sorted by flow cytometry and cultured on 3T3 feeder cells to establish clonogenic features.
Reciprocal comparisons showed that, in all four cells types studied, MtDR exclusion correlates with Ho exclusion (Figure). The lowest zone (A, MtDRlo) includes most of the Holo (SP) cells (arrows). The mid MtDR zone (B) incorporates cells that are in continuum with the Holo (zone 2) but cannot be resolved from the Hohi (3) cells in the ‘SP plot’. The inverse analysis (i.e., MtDR levels in Ho zones 1, 2 and 3) is also shown in the left panels. Both Holo and MtDRlowere abolished by the pan-MDR inhibitor reserpine. The ABCG2-specific FTC inhibitor and verapamil, though, inhibited only the Ho efflux, whereas MtDRlo was affected by MK-571, an inhibitor of ABCC-type transporters. MtDRlo cells generated robust holoclones.
MtDR efflux exclusion provides an alternative way to isolate limbal and conjunctival cells with stem/precursor features while avoiding the DNA dye intercalation involved in Ho staining. The inhibitor profile suggests involvement of ABCC5.
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