Abstract

PURPOSE. To evaluate the effect of phorbol 12-myristate 13-acetate (PMA) on the expression of ⌬Np63 in human limbal epithelial cells (HLECs) during ex vivo expansion on amniotic membrane (AM). METHODS. Primary HLECs were cultured either on AM or plastic surfaces and were treated with 1 g/mL PMA for 24 hours. Expression of ⌬Np63 and the differentiation-associated gap junctional protein connexin 43 (Cx43) were studied by laser scanning microscopy. RESULTS. The labeling index (LI) of ⌬Np63 was higher in HLECs cultured on AM than in HLECs grown on plastic (81.4% Ϯ 12.2% and 66.6% Ϯ 16.5%, respectively; P Ͻ 0.001). After PMA treatment, ⌬Np63 expression in HLECs on plastic dramatically decreased to 20.4% Ϯ 11.4%. However, HLECs cultured on AM showed only a moderate decrease in ⌬Np63 expression (56.4% Ϯ 10.9%, P Ͻ 0.001) after PMA treatment. It was also observed that 72.8% Ϯ 17.5% of the ⌬Np63-positive cells in untreated HLECs cultured on plastic coexpressed Cx43, in contrast to only 21.9% Ϯ 3.7% of the ⌬Np63-positive cells in HLECs cultured on AM (P Ͻ 0.001). The latter indicates that growth over AM preserves limbal phenotype, whereas growth over plastic surface induces or allows transition toward corneal peripheral phenotype. CONCLUSIONS. ⌬Np63 protein is typically detected in human corneal epithelial cells with high proliferative capacity including, limbal epithelial stem cells (SCs) and probably also transient amplifying cells (TACs). AM supports ⌬Np63 protein expression in HLECs and maintains a higher resistance against phorbol ester-induced differentiation, indicating that characteristic signs of limbal epithelial progenitor cells may be preserved during ex vivo expansion on AM. (Invest Ophthalmol Vis Sci.