The Corneal epithelial stem cell marker-novel combination of high expression of p63 and a large N/C Ratio

Abstract

The cornea is a transparent avascular tissue that provides a protective barrier from the external environment and also serves as the main refractive element of the visual system. The corneal transparency relies on the normal functioning of its three tissue layers, epithelium, stroma and endothelium. Under normal circumstances, the superficial cells of the corneal epithelium undergo desquamation after terminal differentiation which is followed by replacement of lost cells from the limbus. For this system to perpetuate and maintain equilibrium, the primary source is considered to be the stem cells (SCs) residing in the limbal palisades of Vogt. Although the location of SCs is now well established, there are currently no definitive markers for identification of SCs. Therefore, a method was devised for the identification of a population of small cells with high expression of p63 combined with a large N/C ratio, using cytospin smear, doubleimmunofluorescence staining and quantitative confocal microscopy. We have demonstrated that these two parameters in combination,form a precise marker to identify SCs in the basal layer of limbus. We have further confirmed that the distinct group of small cells expressing high levels of nuclear protein p63 combined with a large N/C ratio is a precise corneal epithelial SC marker on the basis of the following findings: they are small cells (diameter 9-11μ m) with these two characteristics and are present in the limbal but not in the corneal epithelium; more specifically these cells are present in the isolated basal layer cells of limbus, but not in the isolated suprabasal/superficial cells; they are enriched by five-fold in the isolated limbal basal cells; they are positive for the SC-related markersacidic cytokeratins, K5 and negative for K3, Cx43, 14-3-3σ and Ki67; they possess slowcycling label retaining property; the isolated limbal basal layer cells have higher mRNA levels for Δ Np63a and display higher CFE; they are present in small numbers in ex vivo expanded limbal epithelium but not in that of corneal epithelium.