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David Ma, Hung-Chi Chen, Jui-Yang Lai, Kevin Ma, Lung-Kun Yeh, Unique Yang, Jessica Ma; EDC/NHS cross-linked amniotic membrane preferentially preserves corneal epithelial progenitor cells by activating Wnt/β catenin signaling. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1002.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we have shown that EDC/NHS cross-linked denuded amniotic membrane (CLDAM) is compatible for the growth of human limbo-corneal epithelial (HLE) cells in vitro and in vivo (Biomaterials, 2010, 31: 6647-6658), in this study we further investigate whether CLDAM preferentially preserves HLE progenitor cells and the underlying mechanism.
HLE cells expanded from explants were cultured on dish (HLE/dish), on denuded AM (HLE/DAM) and on CLDAM (HLE/CLDAM). When near confluency, cell density, BrdU label retention, and colony formation assay (CFA) were analyzed. Immunoconfocal microscopy, Western blot, and Q-PCR for keratin 12, connexin 43, ABCG2, deltaNp63α, β-catenin and TCF-4 were performed. Finally, selective GSK3β inhibitors SB216763 or SB415286 were added to HLE/dish cultures to evaluate CFA and deltaNp63α expression.
Compared with HLE/dish or HLE/DAM, HLE cells on CLDAM were more compact in morphology, expressed higher level of p63, ABCG2, and lower level of connexin 43 and keratin 12. CFA was highest in HLE/CLDAM, so were the nuclear expression of β-catenin and TCF-4. Addition of GSK3-β inhibitors to HLE/dish cultures not only increased CFE but also the expression of stem cell marker p63.
CLDAM showed tendency to better preserve HLE progenitor cells in vitro, and Wnt/βcatenin signaling may be involved, possibly through the activation of p63.
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