Human limbal epithelium contains side population cells defined by expression of the ATP–binding cassette transporter ABCG2

Abstract

Abstract: : Purpose: Many types of organ–specific stem cells have recently been shown to exhibit a side population (SP) phenotype based on their ability to efflux Hoechst 33342 dye. The purpose of this study was to examine whether the limbal epithelium contains SP cells. Since the ATP–binding cassette transporter ABCG2 is the SP phenotypic determinant, ABCG2 expression in the limbal epithelium was also investigated. Methods:Human corneoscleral rims from USA eye bank eyes were used. Epithelial cells were isolated from the limbus and from the cornea, and analyzed for Hoechst 33342 dye efflux by FACS. SP and non–SP cells from limbal epithelial tissues were analyzed for ABCG2 gene expression by TaqMan real–time RT–PCR. The relative expression of the ABCG2 gene was normalized to that of GAPDH. To compare ABCG2 expression between SP and non–SP cells, the Mann–Whitney rank sum test was applied. The ABCG2 expression patterns in the limbal epithelium and the corneal epithelium were investigated by immunofluorescence using an anti–ABCG2 monoclonal antibody. Results: SP cells were detected in the limbal epithelium, but not in the corneal epithelium. The ABCG2 mRNA expression level in SP cells was significantly higher than in non–SP cells (n=4, p=0.029). Immunofluorescence revealed that ABCG2 protein is expressed in limbal epithelial basal cells, but not in corneal epithelial cells. Conclusions: Corneal epithelial stem cells have the SP phenotype defined by the expression of ABCG2. Thus, ABCG2 may serve as a useful cell surface marker in the isolation of viable corneal epithelial stem cells.

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