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Bo Han, Yingting Zhu, Suzhen Zhang, Scheffer Tseng, Cornea; Immobilized HC-HA Preserves Limbal Niche Cell Phenotype to Prevent Limbal Epithelial Progenitor Cells from Differentiation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1003.
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© ARVO (1962-2015); The Authors (2016-present)
We have recently reported the success of isolating limbal niche cells (LNC) that can prevent limbal epithelial progenitor cells (LEPC) from differentiation in a reunion assay in 3-dimentional (3D) Matrigel. Amniotic membrane (AM) alone can help expand residual limbal stem cells in vitro and in vivo. Because we have successfully isolated heavy chain-hyaluronan complex (HC-HA) from AM, we wonder whether HC-HA can preserve LNC to prevent LEPC from differentiation.
LNC were cultured in MESCM on plastics, 3D Matrigel, or immobilized HC-HA. qRT-PCR and immunostaining were used to compare expression of markers that have been reported for LNC. AMD 3100 was used to block SDF1/CXCR4 signaling. LEPC were added to LNC aggregates formed on immobilized HC-HA in MESCM and compared to the reunion assay in 3D Matrigel for 7 days. qRT-PCR, Western blotting, and immunostaining were used to characterize expression of markers for LEPC and corneal epithelial differentiation.
LNC aggregated on 2D immobilized HC-HA at day 1 with small and round cells and expressed 2- to 4-fold higher ESC markers such as Nanog, Oct4, Rex1, and Sox2 at day 7 than plastic and 3D Matrigel. Such aggregation was mediated by 3 to 4-fold higher expression of SDF1 and CXCR4 and could be abolished by AMD3100 added on day 0 but not day 4. Presumably because of high expression of SDF1 and CXCR4, LNC attracted reunion with LEPC on immobilized HC-HA, similar to our recent report that LNC attracted reunion with LEPC in 3D Matrigel. We speculate that such a reunion with LEPC will prevent LEPC from differentiation.
Limbal niche cells form aggregates on HC-HA via SDF1/CXCR4 signaling, express higher levels of ESC markers, and attract reunion with LEPC. Studies are underway to characterize whether such reunion on immobilized HC-HA may prevent LEPC from differentiation.
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