Immature side population cells in human limbal epithelium

Abstract

Abstract: : Purpose: To confirm the presence of side population(SP) cells in limbal and central cornea epithelium. Methods: Corneal tissue from eyebank eye were digested with collagenase and trypsin– EDTA into single cell suspensions. Cells were stained with Hoechst33342 dye that is preferentially expelled by SP cells. Cells were also stained with FITC labeled anti–human CD45 antibody in order to exclude cells of myeloid origin. Labelled cells were then analyzed by FACS for CD45–negative SP cells, and differentiated main population(MP) cells. Sorted cells were analyzed for FSC/SSC profiles to show the distribution of cells size. SP cells were cultured in SHEM medium containing 15% FBS. Results: The amount of CD45(–)SP cells in limbal epithelium was in the range of 0.78% (approximately 10⁴cells), whereas epithelium from the central cornea did not contain SP cells. FSC/SSC profliles showed that limbal SP cells were smaller than MP cells. Culture of SP cells for 10 days led to the proliferation of cells of corneal epithelium phenotype. Conclusions: Preferential limbal distribution of SP cells was observed in human corneas. Further genetic characterization of limbal SP cells are underway.

Keywords: cornea: basic science • flow cytometry • cornea: epithelium