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W. Li, H. He, T. Kawakita, S.C. G. Tseng; Basement Membrane Formation of Limbal Corneal Epithelial Cells Expanded on Intact or Denuded Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2078.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previously we reported that limbal epithelial phenotype is preserved by intact amniotic membrane (iAM), while corneal epithelial phenotype is preserved by denuded AM (dAM). We wonder whether the basement membrane (BM) formation may be different during ex vivo expansion on iAM or dAM. Methods: Human limbal explants or limbal corneal epithelial sheets isolated by dispase from eyebank limbal rings, were cultured on iAM or dAM under a submerged manner in SHEM medium for 4 weeks. Cryosections of the limbal explant and the epithelial outgrowth were immunostained for collagen IV, collagen VII, laminin 5, and perlecan, i.e., BM components. Results: In controls, collagen IV, collagen VII, laminin 5, and perlecan were all present in the BM of human corneal and limbal epithelia, but all except for perlecan were present in the BM of human AM. After 4 W culturing, all four BM components were degraded in limbal explants, more so when cultured on dAM than on iAM. Collagen IV, collagen VII, and laminin 5 were strongly stained underneath the epithelial outgrowth when limbal explants or epithelial sheets were cultured on iAM. In contrast, collagen IV, laminin 5, and perlecan were weak and diffuse while collagen VII was negative underneath the epithelial outgrowth when limbal explants and epithelial sheets were cultured on dAM. Perlecan staining was negative when limbal corneal epithelial sheets were cultured on either iAM or dAM, but was much more strongly positive when limbal explants were cultured on iAM than dAM. Conclusions: Intact AM, which retains devitalized amniotic epithelial cells, prevents degradation, thus helping BM formation during ex vivo expansion of corneal limbal epithelial cells. The presence of perlecan in cultured explants but not pure epithelial sheets strongly supports that new BM sythesis is further facilitated by stromal fibroblasts in the explant. This new information helps future refinement of ex vivo expansion.
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