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D. Meller, E.E. Hernandez Galindo, C. Theiss, K.P. Steuhl; P63 Expression in "Label-Retaining" Human Limbal Epithelial Cells Expanded on Intact Human Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1355.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate the co-expression of ΔNp63 protein, a gene product restricted to progenitor epithelial cells, and BrdU, incorporated in human limbal epithelial cells (HLEC). Thereby, we attempt to identify subpopulations in our cell cultures that share characteristic features of limbal epithelial progenitor cells. 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. Afterwards, incorporation was done with 5µM BrdU for 3 days and then chased for additional 14 days in BrdU-free medium. Monoclonal antibody against ΔNp63 was used to study expression of ΔNp63 in HLEC. Furthermore, a polyclonal antibody to Connexin 43 (Cx43) was used as a differentiation-associated marker. Flat mounts were analyzed by means of laser confocal microscopy. HLEC grown on plastic were used as controls. Results: We have observed that 72.8% (± 17.5%) of the ΔNp63 positive HLEC cultured on plastic co-expressed Cx43, in contrast to only 22 % (± 3.7%) of the ΔNp63 positive HLEC cultured on AM (p<0.001) indicating that ΔNp63 protein is typically found in human corneal epithelial cells with high proliferative capacity, including not exclusively limbal epithelial stem cells (SC) but probably also transient amplifying cells (TAC). After PMA pretreatment and subsequent BrdU incorporation with 14 days of chase, HLEC cultured on AM showed a higher labeling index for BrdU (6.7%, ± 3.8%) compared to HLEC growing on plastic (2%, ± 2.8%, p<0.05). Furthermore, 9.5% (± 5%) of ΔNp63 positive HLEC grown on AM were "label-retaining" for BrdU, however, on plastic only 3.1% (± 4%, p<0.05) of ΔNp63 positive HLEC were co-expressing incorporated BrdU. Conclusions: ΔNp63 protein does not exclusively label corneal epithelial stem cells. AM supports ΔNp63 protein expression in Cx43 negative and label-retaining cells of limbal explants indicating that characteristic features 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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