Epithelial cell characteristics of cultured human limbal explants

Aim: To determine the immunohistochemical characteristics of putative corneal epithelial stem cells remaining on limbal explants maintained in culture. Methods: Human limbal explant cultures were generated from 25 residual corneoscleral donor rims following penetrating keratoplasty. Serial sections of these explants were studied using immunohistochemical techniques with a panel of antibodies, on day 0 and 1, 2, and 3 weeks. Results: The number of epithelial cells expressing cytokeratin 19 and vimentin increased with duration in culture, while the number of cells expressing cytokeratin 3 decreased. Connexin 43 expression was lost by 1 week in culture. p63 was expressed by cells that had migrated around the explant and the number of p63 positive cells decreased with longer duration in culture. The explants were initially negative for Ki67, but the epithelial cells were positive at 1 week, and expression of Ki67 was progressively lost with increasing duration in culture. The initial uniform staining of the epithelium for epidermal growth factor receptor and a enolase remained unchanged at 3 weeks. Conclusions: There is an expansion of less differentiated ( cytokeratin 3 negative and CK19/vimentin positive) epithelial cells on corneoscleral explants maintained in culture for 3 weeks. The pattern of expression of p63 noted in this study does not support the suggestion that it is a marker of limbal stem cells. The decline in p63 and Ki67 expression among the epithelial cells of the cultured explant button implies that as the epithelial sheet outgrowing from the explant button reaches confluence, the proliferative status of the cells remaining on the explant button declines. These findings are of clinical relevance as explants of limbal tissue are used in limbal stem cell transplantation. There is no information available to date on the fate of epithelial cells on such explants. This study provides some insight into this and suggests that an expansion of the stem cell pool or its progeny may occur in limbal explants.