SSEA4 Is a Potential Negative Marker for the Enrichment of Human Corneal Epithelial Stem/Progenitor Cells

Abstract

To examine the expression of stage-specific embryonic antigen-4 (SSEA4) in the epithelium of the human ocular surface and characterize SSEA4(+) and SSEA4(-) limbal epithelial cells. SSEA4 expression in the human cornea and limbus was examined by RT-PCR and immunohistochemistry. SSEA4(+) and SSEA4(-) cells were then separated by using magnetic beads. The phenotypes of these two cell populations were evaluated on the basis of cell size, clonogenic assay, and expression of putative limbal stem cell (LSC) and corneal epithelial differentiation markers. SSEA4 was expressed in all layers of the corneal and anterior limbal epithelia. Discrete clusters of SSEA4(+) cells were present in the central and posterior limbal epithelia. SSEA4(+) cells accounted for an average of 40% of the total limbal epithelial cells. The SSEA4(-) population contained five times more small cells (≤11 μm in diameter) than did the SSEA4(+) population. The expression levels of the putative LSC markers ABCG2, ΔNp63α, and cytokeratin (K)14 were significantly higher in the SSEA4(-) population than in the SSEA4(+) population. The SSEA4(-) cells also expressed a significantly higher level of N-cadherin, but a lower level of the differentiation marker K12. The colony-forming efficiency in the SSEA4(-) population was 25.2% (P = 0.04) and 1.6-fold (P < 0.05) higher than in the unsorted population and the SSEA4(+) population, respectively. SSEA4 is highly expressed in differentiated corneal epithelial cells, and SSEA4(-) limbal epithelial cells contain a higher proportion of limbal stem/progenitor cells. SSEA4 could be used as a negative marker to enrich the isolation of LSCs.