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EE Hernandez, C Theiss, KP Steuhl, D Meller; p63 Expression in Response to Phorbol Ester Stimulation in Human Limbal Epithelial Cells Expanded on Intact Human Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1628.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate the expression of p63 protein, a gene product restricted to progenitor epithelial cells, in human limbal epithelial cells (HLEC) during ex vivo expansion on preserved human amniotic membrane (AM). Methods: Primary human limbal epithelial cells (HLEC) from corneal 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) containing medium for 24 hours and afterwards switched to PMA-free medium for 3 days. Then, all cell cultures were terminated and fixed in 4% paraformaldehyde. Monoclonal antibody against p63 was used to study expression of p63 in HLEC. Alexa Fluor 546 goat anti-mouse was applied as secondary antibody and flat mounts were analyzed by means of laser confocal microscopy. Positive nuclei were counted in eight 200x200 µm² areas of each micrograph. Results: HLEC 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, HLEC cultured on plastic showed a centrifugal reduction of p63 labeling from the central (87.59%) toward peripheral areas (52.37%). After PMA treatment, a decrease of p63 expression in HLEC cultured on AM to 54.39% and 52.37% was noted (center and periphery, respectively), whereas the immunoreactivity to p63 of HLEC cultured on plastic diminished dramatically to 28.07% in central and 22.69% in the peripheral areas associated with marked morphological changes characterized usually by an increased cellular size with signs of desquamation. Conclusion: AM supports p63 protein expression of HLEC and maintains a higher resistance against phorbol ester indicating that characteristic of limbal epithelial progenitor cells might be preserved during ex-vivo expansion on AM. This data provides support to the use of the ex-vivo expansion of HLEC as an alternative therapeutic strategy for corneal surface reconstruction in distinct ocular surface diseases.
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