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Expression of p63 in Response to Phorbol-ester Stimulation in Human Limbal Epithelial Cells Expanded on Human Amniotic Membrane

1Hernandez Galindo E. E., 2Theiss C., 3Steuhl K. P., 1Meller D.,
1Universität-Gesamthochschule Essen, Zentrum für Augenheilkunde (Essen)
2Ruhr-Universität Bochum, Abteilung für Zytologie (Bochum)
3Universität-Gesamthochschule Essen, Zentrum für Augenheilkunde, Abt. für Erkrankungen des vorderen Augenabschnitts (Essen)

Purpose: To evaluate the expression of p63 protein, a gene product restricted to progenitor epithelial cells, in human limbal epithelial cells (HLE) during ex vivo expansion on preserved human amniotic membrane (AM).
Methods: Primary human limbal epithelial cells (HLE) from limbus explants were cultured with SHEM either on intact AM or plastic for 4 to 5 weeks. A set of cultures was treated with 1µg/ml phorbol 12-myristate 13-acetate (PMA) for 24 hours. A monoclonal antibody against p63 was used to study expression of p63 in HLE. Alexa Fluor 546 goat anti-mouse was applied as secondary antibody and flat mounts were analyzed by means of laser confocal microscopy.
Results: HLE cultured on AM revealed an uniform immunolabeling for p63 in central regions (83.48%) as well as peripheral regions (86.93%) of cell cultures. In contrast, HLE cultured on plastic showed a centrifugal reduction of p63 labeling from the central (87.59%) toward peripheral areas (53.42%, p<0.001). After PMA treatment, a decrease of p63 expression in HLE cultured on AM to 54.39% and 52.37% was noted (center and periphery, respectively,p<0.001), whereas the immunoreactivity to p63 of HLE cultured on plastic diminished dramatically to 28.07% in central and 22.69% (both, p<0.001) in the peripheral areas associated with marked morphological changes characterized usually by an increased cellular size with signs of desquamation.
Conclusions: AM supports p63 protein expression of HLE and maintains a higher resistance against phorbol ester indicating that characteristic signs of limbal epithelial progenitor cells might be preserved during ex vivo expansion on AM.