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C.-C. Sun, Y.-F. Lin, H.-T. Chiu, J.-H. S. Pang; Differential Expression of β Integrin Subunits in Intact Amniotic Membrane-Expanded Human Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3764.
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The corneal epithelial stem cells are located at the limbus. Ex vivo expansion of limbal explants on amniotic membrane (AM) are capable of reconstructing the ocular surface with limbal stem cell deficiency (LSCD). Cell-cell matrix interaction plays an important role in this model. The purpose of this study was to investigate the expression of integrins, one of the major adhesion molecule families, in limbal explant outgrowth on AM.
Corneoscleral buttons from human donor eyes were cut into 1.5 x 2 x 3 mm3 pieces and cultured on intact or denuded AM for 3 weeks. The extent of each outgrowth was monitored with a phase contrast microscope. Expression of various β subunits of integrin was determined by Western blotting, immunofluorescent staining and reverse transcription-polymerase chain reaction (RT-PCR). In addition, the expression of adult stem cell marker Musashi-1 and corneal epithelial differentiation marker cytokeratin K3 was assessed by immunofluorescent staining.
All β subunits of integrin except for β2 were expressed at transcriptional level as determined by RT-PCR, but only β4 subunit was demonstrable by Western blot. Immunofluorescent staining showed that β1, β4 and β6 subunits were expressed in intact AM-expanded human limbal epithelial cells. Intense β4 staining was preferentially located at the migration edge of outgrowths. In contrast, β1 was selectively expressed in cells not in contact with AM epithelial cells, which was in consistent with the expression pattern of K3 cytokeratin. Expanded limbal epithelial cells immediate adjacent to intact AM (negative β1 staining cells) were positively stained by Musashi-1. Moreover, enhanced β1 expression was noted in all limbal epithelial cells expanded on denuded AM.
We demonstrate the expression profile of β integrin in ex vivo expanded limbal epithelial cells on intact AM. Our study also imply a possible role for β4 integrin in mediating limbal epithelilal outgrowths, while β1 integrin might be involved in limbal epithelial progenitor cell differentiation.
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