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X. Ye, M. A. M. Akinci, M. A. M. Akinci, M. Taveras, J. M. Wolosin; E-Selectin Is a Cell Surface Marker for the Conjunctival and Limbal Side Population Stem Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4517.
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© ARVO (1962-2015); The Authors (2016-present)
Side population (SP) cells isolated from limbus and conjunctiva exhibit stem cell features. The purpose of this study was to identify cell surface markers expressed by these cells.
Overnight cultures of freshly isolated human conjunctival epithelial cells were stained with Hoechst, sorted into SP (~ 0.5 % of basal cells) and G0/G1 cohorts by flow cytometry (FC). The RNA of these cells was used to perform SP/nonSP comparative analysis of gene expression by microarray technology. Cytospuned SP and G1 sorted conjunctival cells and limbal and conjunctival total cultures and tissue cryosections were double stained with anti-CD62E/ E-selectin (a gene generally thought to be specific to vascular endothelial cells) and various anti-cytokeratin (KRT) Abs. FC bivariate analysis was used to determine the percentile of SP cells and cells expressing surface CD62E as well as the degree of overlap between these phenotypes. CD62E+ cells were isolated by magnetobead immunopanning and subjected to FC analysis for SP content.
The SP/G1 CD62E gene expression ratio was > 140 (aver. 4 microarray exp.). Consistent with this result, immunostaining of cytospins revealed than > 50% of the sorted SP were CD62E+ whereas the CD62E stain was observed in no more than 1% of the cytospun G1 cells. In total limbal and conjunctival cultures, after cell permeabilization and fixation, CD62E positive cells were all KRT+ and accounted for 1-2 % of the total population. Bivariate FC analysis showed that 70 % the SP cells are CD62E+ (n=2) and conversely, 35 % of the CD62+ cells are SP cells. Consistent with the later proportion, 13 % of the cells immunopanned using a CD62E Ab were SP cells. Finally, in cryosections, CD62E appeared as a faint punctuate staining in isolated basal limbal and conjunctival cells.
Limbal and conjunctival SP stem cells selectively express at high levels the endothelial cell marker CD62E/E-selectin. This unexpected expression is consistent with unique expression patterns for somatic stem cells.
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