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Kyung-Sun Na, Jeewon Mok, Jungmook Lyu, Choun-Ki Joo; Wnt promotes proliferation of corneal epithelial progenitor cells in xeno-feeder free cultivation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):995.
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This study was to establish a simple, xeno-feeder free method for cultivating human corneal limbal explants , and also to explore the effect of Wnt signaling on epithelial progenitor cell proliferation in this cultivation system in vitro and in vivo.
The limbal tissue explants from cadaveric donor was cultured in Isocove’s Modified Dulbecco’s Medium (IMDM) and low calcium Panserin 801 medium in 1:1 ratio. The outgrowing cells were examined to characterize with flow cytometry, immunohistochemistry, and real-time PCR (RT-PCR). Sprague-Dawley male rats after alkali injuries using 1N NaOH were used for in vivo verification, after which cultivated epithelial sheets were transplanted. Corneal opacity, re-epithelialization, and neovasculazation was observed for 2 week, and the tissue sections analyzed with hematoxylin and eosin stain (HE stain). Conditioned media from L cells secreting Wnt-3a (Wnt3a-L-CM) was used in the cultivation system, and morphological changes and gene expression level were observed.
There was migration of fibroblast like stromal cells from limbal explants initially, and then, epithelial cells migrated and grown on stromal cells as an autofeeder layer, which was revealed by morphological and immunohistochemical methods. RT-PCR showed that the expression of epithelial progenitor cells are more intense compared to fresh limbal tissue. Side population (SP) cells were detected 0.43 ± 0.04 % (n=5) of the primary culture. Flow cytometry resulted 49.12% of E-cadherin, 40.44% of p63, and 44.55% of ABCG2 identified in the cells from explants. Maintaining corneal transparency without neovascularization was observed in rats after cultivated epithelial sheets transplantation for 2 weeks. Predominant increased tightly packed epithelial cells with Wnt3a-L-CM were observed compare to the control CM. ABCG2, p63, Lef1, and CK3 was increased in Wnt3a-L-CM.
This explants culture system with combining media to use stromal cells as autofeeder layer, showed to expand sufficient limbal epithelial progenitor cells in vitro and to be transplanted restoring transparency. Also, these findings demonstrated that Wnt signaling play an important role in the proliferation of limbal epithelial progenitor in the proposed cultivation system. This study may have clinical impact on the expansion of corneal epithelial progenitor cells for ocular surface reconstruction.
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