Greater growth potential of p63-positive epithelial cell clusters maintained in human limbal epithelial sheets

PURPOSE. To determine the growth potential of p63-positive cell clusters maintained in human limbal epithelial sheets. METHODS. Intact human limbal epithelial sheets were isolated from corneoscleral rims and cultured with or without being rendered into single cells by trypsinization on either plastic or 3T3 fibroblast feeder layers. Clonal growth on 3T3 fibroblast feeder layers was compared between monolayers in sheets or single cells and between areas with or without laser-microdissected, p63-enriched cell clusters. Immunostaining, immunoblot analysis, and reverse transcription-polymerase chain reaction of such differentiation markers as keratin (K)-3 and -12 and such progenitor markers as p63, ABCG2, and MDR-1 were also compared. RESULTS. Clusters of small p63-positive cells were enriched in limbal palisades of dispase-isolated epithelial sheets immediately or after brief cultivation on plastic in SHEM. Clonal growth of p63-rich cell clusters was higher than areas without clusters (P < 0.001). Clonal growth of epithelial monolayers derived from sheets was also higher than that derived from single cells (P < 0.01). Although expression of ABCG2 and MDR-1 transcripts was similar, cells from epithelial sheets expressed higher protein levels of p63 and lower protein levels of K3 and -12 than single cells, whether they were cultured on plastic or as 3T3 fibroblast feeder layers. CONCLUSIONS. Limbal palisades contain clusters of p63-rich progenitor cells. Maintenance of such cluster architecture during ex vivo expansion yields higher growth potential than being dispersed into single cells.