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Stem Cells 2004;22:355-366 www.StemCells.com
© 2004 AlphaMed Press

Characterization of Putative Stem Cell Phenotype in Human Limbal Epithelia

Zhuo Chena, Cintia S. de Paivaa, Lihui Luoa, Francis L. Kretzera,b, Stephen C. Pflugfeldera, De-Quan Lia

a Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, and
b Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA

Key Words. Cornea • Limbus • Epithelium • Stem cells • Stem cell phenotype

De-Quan Li, M.D., Ph.D., Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, 6565 Fannin Street, NC-205, Houston, Texas 77030, USA. Telephone: 713-798-1123; Fax: 713-798-1457; e-mail: dequanl{at}bcm.tmc.edu

This study evaluated proposed molecular markers related to stem cell (SC) properties with the intention of characterizing a putative SC phenotype in human limbal epithelia. Human corneal and limbal tissues were cut in the vertical and horizontal meridians for histology, transmission electron microscopy (TEM), and immunostaining. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization were used to evaluate gene expression. TEM showed that the limbal basal cells were small primitive cells. Immunostaining disclosed that p63, ABCG2 and integrin {alpha}9 were primarily expressed by the basal epithelial cells of limbus. Antibodies against integrin ß1, epidermal growth factor receptor (EGFR), K19, enolase-{alpha}, and CD71 stained the basal cells of the limbus more brightly than the suprabasal epithelia. Integrin {alpha}6, nestin, E-cadherin and connexin 43 did not stain the limbal basal cells, but the suprabasal epithelia of the cornea and limbus showed strong immunoreactivity. K3 and involucrin stained only corneal and limbal superficial cells. RT-PCR showed higher levels of p63, ABCG2 and integrin {alpha}9 mRNA, but lower levels of K3, K12 and connexin 43 expressed in the limbal epithelia than the corneal epithelia. In situ hybridization showed that p63 transcripts were located in basal layer of the limbal epithelium. This work suggests that the basal epithelial cells of the limbus are p63, ABCG2 and integrin {alpha}9 positive, and nestin, E-cadherin, connexin 43, involucrin, K3, and K12 negative, with relatively higher expression of integrin ß1, EGFR, K19, and enolase-{alpha}. This putative SC phenotype may facilitate the identification and isolation of limbal epithelial SCs.




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