Characterization of putative stem cell phenotype in human limbal epithelia.
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 alpha9 were primarily expressed by the basal epithelial cells of limbus. Antibodies against integrin beta1, epidermal growth factor receptor (EGFR), K19, enolase-alpha, and CD71 stained the basal cells of the limbus more brightly than the suprabasal epithelia. Integrin alpha6, 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 alpha9 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 alpha9 positive, and nestin, E-cadherin, connexin 43, involucrin, K3, and K12 negative, with relatively higher expression of integrin beta1, EGFR, K19, and enolase-alpha. This putative SC phenotype may facilitate the identification and isolation of limbal epithelial SCs.
Chen
Zhuo
Z
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas 77030, USA.
de Paiva
Cintia S
CS
Luo
Lihui
L
Kretzer
Francis L
FL
Pflugfelder
Stephen C
SC
Li
De-Quan
DQ
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D016667:Bacterial Capsules; D014408:Biomarkers, Tumor / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D018031:Connexin 43 / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure*; D019573:Epithelium, Corneal / Q000378:metabolism / Q000648:ultrastructure*; D018773:Genes, erbB-1 / Q000235:genetics; D006801:Humans; D016023:Integrins / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000378:metabolism; D016850:Limbus Corneae / Q000378:metabolism / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D009363:Neoplasm Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015703:Antigens, CD; D014408:Biomarkers, Tumor; C068126:CD71 antigen; C081876:CKAP4 protein, human; D015820:Cadherins; D018031:Connexin 43; D004268:DNA-Binding Proteins; D016023:Integrins; D007381:Intermediate Filament Proteins; D008565:Membrane Proteins; C577484:NES protein, human; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D064231:Nestin; D011135:Polysaccharides, Bacterial; D011990:Receptors, Transferrin; C111200:SECTM1 protein, human; D025521:Tumor Suppressor Proteins; C098418:capsular polysaccharide K3; C095157:ENO1 protein, human; D010751:Phosphopyruvate Hydratase