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Expression of Np63 in Response to Phorbol Ester in Human Limbal Epithelial Cells Expanded on Intact Human Amniotic Membrane

¹From the Department of Ophthalmology, University of Essen, Essen, Germany; and the ²Department of Cytology, Institute of Anatomy, University of Bochum, Bochum, Germany.

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 µg/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% ± 12.2% and 66.6% ± 16.5%, respectively; P < 0.001). After PMA treatment, Np63 expression in HLECs on plastic dramatically decreased to 20.4% ± 11.4%. However, HLECs cultured on AM showed only a moderate decrease in Np63 expression (56.4% ± 10.9%, P < 0.001) after PMA treatment. It was also observed that 72.8% ± 17.5% of the Np63-positive cells in untreated HLECs cultured on plastic coexpressed Cx43, in contrast to only 21.9% ± 3.7% of the Np63-positive cells in HLECs cultured on AM (P < 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.

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