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

Purpose: To characterize human limbal epithelial cells based on the expression levels of nuclear protein p63 and the nucleus-to-cytoplasm (N/C) ratio. Methods: 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 [C×43]). 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. Results: 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 C×43. Conclusions: 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.