A subset of human limbal epithelial cells with greater nucleus-to-cytoplasm ratio expressing high levels of p63 possesses slow-cycling property

Purpose: The purpose of this study was to evaluate the subset of limbal epithelial cells with greater nucleus-to-cytoplasm (N/C) ratio expressing high levels of p63 for their slow-cycling property, a characteristic feature of stem cells (SCs). Methods: Limbal and peripheral corneal explant cultures were pulse labeled with 5-5-bromo-2'-deoxyuridine (BrdU) for 5 days, followed by a period of 3-week chase. Cultured explants were cryosectioned and stained for BrdU. The epithelial cells in the outgrowth and those remaining on the explant were isolated and subjected to cytospin and double immunostaining for BrdU and p63, followed by identification of label-retaining cells (LRCs) and quantification of p63 expression using confocal microscopy. Results: A distinct population of small cells with large N/C ratio expressing high levels of p63 retained the BrdU label after 21-day chase. Further, this population of LRCs, negative for the differentiation marker K3, was observed in the epithelial outgrowth of limbal but not in that of peripheral cornea. LRCs were seen to migrate along the cut edge of limbal explants in culture and were also observed as clusters of small cells in the outgrowth, which contained cells with the ability to form holoclone colonies. Conclusions: These results demonstrate that the small cells with large N/C ratio and high levels of p63 have BrdU label retaining slo-cycling property, thus confirming that these 2 parameters in combination may serve as a precise marker for identification and quantification of ex vivo-expanded limbal SCs. This method would be useful to standardize the optimal culture conditions that can maintain and expand SCs for therapeutic applications.