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Alexander Barash, Ozlem Barut Selver, Mohaned Ahmed, J Mario Wolosin; ABCG2 Transport Activity and Clonal Potential in Epithelial Cells Continuously Growing for One Month from Limbal Explants. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5133.
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© ARVO (1962-2015); The Authors (2016-present)
To determine temporal changes in ABCG2-dependent transport in cell outgrowths from limbal explants and clonogenic features of these cells.
Fresh limbal tissue cells (FLTC) were obtained by sequential dispase-trypsin digestion. Human or rabbit limbal strips were deposited onto permeable membrane inserts for explant culture. Over a 45-day period, the segments were transferred to new inserts thrice. FLTCs and the explant outgrowth cells (EOCs) that grew from each culture round were characterized for ABCG2-dependent efflux transport by flow cytometry using a newly identified substratum, the mitochondrial dye JC1, instead of Hoechst 33342, following a short reculture step common to all cells. Cells were sorted into JC1-excluding (JC1low) and non-excluding (JC1main) cohorts and seeded on feeder cells to determine colony formation efficiency (CFE).
JC1low cells were all Hoechst-excluding (‘side population’ or ‘SP’) cells and vice versa, establishing the physical equivalence between JC1low and the SP. The JC1low phenotype was abolished by FTC, Ko 143, and glafenine, all ABCG2 transport-specific inhibitors, and was not affected by inhibitors of other ABC transporters. JC1low percentiles for the fresh human and rabbit FTLCs were 1.4% and 4.1%, respectively. The CFEs for the rabbit FLTCs JC1low and JC1main were 1.2% and 5.3%, respectively, consistent with the previously described CFE SP << CFE nonSP status (Budak, J. Cell Sci. J Cell Sci. 118:1715). In the EOCs JC1low was much higher and, in fact, increased in late outgrowth rounds (human: 19.5% and 27.4%; rabbit: 25.8% and 32.5%, respectively, for 1st and 2nd round; p<0.05; n = 6). In addition, EOC JC1low CFEs >> JC1main CFEs, 9.2 and 1.4, respectively. In selected 4th and 5th-round rabbit explants, JC1low values that reached over 90%. Thus, while the contribution of JC1low (= high ABCG2 efflux cells) to CFE is minimal in FLTCs, it accounts for more than 50% of the CFE of EOCs.
During explant culture there is a large expansion of ABCG2-rich clonogenic cells. The higher concentration of these cells in late outgrowth rounds suggests that bona-fide SCs are retained within the explant.
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