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D.–Q. Li, L.C. Z. Sharpe, C.S. de Paiva, L.–H. Luo, S.C. Pflugfelder; Characterization of Putative Stem Cell Phenotype in Human Limbal Epithelia . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3392.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate proposed molecular markers related to stem cell properties with the intention of characterizing a putative stem cell phenotype in human limbal epithelia. Methods: Human corneal and limbal tissues were cut in different meridians for histology and transmission electron microscopy (TEM). Immunostaining and laser scanning confocal microscopy were performed to localize the expression of stem cell associated markers. Semiquantitative RT––PCR and in situ hybridization were used to evaluate gene expression. Results: The limbal basal cells were observed to be small primitive cells by TEM. Immunostaining disclosed that p63, ABCG2 and integrin α9 were primarily expressed by the basal epithelial cells of the limbus. Antibodies against integrin ß1, EGFR, K19, enolase α and CD71 stained the basal cells of the limbus more brightly than the suprabasal epithelia. Integrin α6, nestin, E–cadherin and connexin 43 antibodies did not stain the limbal basal cells, but the suprabasal epithelia of the cornea and limbus showed strong immunoreactivity. K3 and involucrin antibodies stained only corneal and limbal superficial cells. Confocal microscopy confirmed that limbal basal cells are p63, ABCG2 and integrin α9 positive, but E–cadherin, connexin 43 and K3 negative. RT–PCR showed higher levels of p63, ABCG2 and integrin α9 mRNA, but lower expression of K3, K12 and connexin 43 by the limbal epithelia than the corneal epithelia. In situ hybridization showed that p63 transcripts were located in basal layer of limbal epithelium. Conclusions: Human limbal epithelial basal cells are p63, ABCG2 and integrin α9 positive, and nestin, E–cadherin, connexin 43, involucrin, K3 and K12 negative, with relatively higher expression of integrin ß1, EGFR, K19 and enolase α. This putative stem cell phenotype may facilitate further identification and isolation of limbal epithelial stem cells.
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