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J. Wolosin, M. Akinci, M. Taveras, H. Turner; Intraepithelial Lymphocytes Are a Major Component of Ocular Surface Epithelial Side Populations . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3954.
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© ARVO (1962-2015); The Authors (2016-present)
Recent work has identified cells capable of exporting Hoechst 33342 through the activity of the ABCG2 transporter (FEBS Lett 565:6; Stem Cells 23:63; J Cell Sci 118: 1715) in limbal and conjunctival epithelia (the ocular surface, or OS, epithelia). This feature causes cells to form a side population (SP) in Hoechst blue/red emission flow cytometry plots. OS epithelial SPs have also been shown to be enriched in cells displaying stem cell properties. Given that the two OS epithelia contain substantial amounts of intraepithelial lymphocytes (IELs; Curr Eye Res 13:87) and that peripheral blood lymphocytes express ABCG2 (Brit J Pharm 143:899), we have now investigated the potential inclusion of IELs within the epithelial side populations.
Single cell suspensions from rabbit and human conjunctival and limbal epithelia, generated by sequential Dispase–trypsin digestions, were used fresh or were cultured in low serum medium for 24–96 hr at 30º C with one passage after the first 6 hr. Cell suspensions were sequentially incubated with Hoechst (90 min, 37º C, 2–3 µg/ml) and FITC–conjugated anti–lymphocyte Abs (anti–CD3 for human; anti–lymphocyte antigen for rabbit) and sorted in a flow cytometer according to SP/SP– or SP–lymph+/SP–lymph– phenotypes. Our analysis further distinguished cells in terms of their size according to their forward light scatter (FSC) into cells included in the lower FSC quartile (FSCqlo) and larger, FSCqhi cells. The FSCqlo is likely to include most limbal epithelial cells (de Paiva, Stem Cells. 2005 Aug 25). Sorted cells were cytospun and stained for cytokeratin to identify epithelial cells and, for CD antigens, to identify hematopoietic cells.
In all fresh limbal and conjunctival populations SP–lymph+ cells amounted to 20–25 % of total SPs. Consistent with the known small size of peripheral lymphocytes SP–lymph+ cells were all FSCqlo, and thus, within this critical SP cell subset, lymphocytes accounted for up to 30 % (human epithelia) and 45 % (rabbit) of the cells. These results were qualitatively confirmed by the staining of sorted cells with the anti–CD Abs. Additionally, consistent with these results under 50 % of the SP FSCqlo in fresh cells were of epithelial origin. In contrast, in the SP– and SP–FSCqhi cohorts, at least 90% of the cells expressed cytokeratins. Cultured cells contained only trace amounts of lymph+ cells.
The presence of non–epithelial cells within the OS epithelia has to be considered when analyzing cell subpopulations based on the ABCG2, SP and/or size phenotypes.
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