High Expression of p63 Combined with a Large N/C Ratio Defines a Subset of Human Limbal Epithelial Cells: Implications on Epithelial Stem Cells

Abstract

To characterize human limbal epithelial cells based on the expression levels of nuclear protein p63 and the nucleus-to-cytoplasm (N/C) ratio. Limbal, peripheral, and central corneal epithelia were separated from the stroma by Dispase II and subsequently were treated with trypsin to obtain single-cell suspensions. Cytospin smears of the cell suspensions were double immunostained for p63 and then stained for any one of the markers (acidic cytokeratins [AE1], K5, K3, or connexin 43 [Cx43]). They were counterstained with propidium iodide. More than 100 cells from each zone were analyzed for p63 expression levels and nuclear/cellular area using quantitative confocal microscopy. A gradient of p63-positive cells was observed in corneal and limbal epithelial cells. The percentage of p63-positive cells and the level of p63 expression were significantly higher in the limbal than in the peripheral or central corneal epithelium. Two-parameter (p63 levels and N/C ratio) analysis revealed the presence of a distinct population of small cells with higher levels of p63 and a large N/C ratio in the limbal epithelium. Such limbal epithelial cells were positive for AE1 and K5 but negative for K3 and Cx43. These results suggest that this distinct group of small cells in the limbal epithelium with greater N/C ratio, expressing high levels of nuclear protein p63, probably represent corneal epithelial stem cells.