title: Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures.
abstract: Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin beta1) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin beta1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells were identified in 2.3+/-0.7% of explant cultures and 3.73+/-1.5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine,6565 Fannin Street, NC-205, Houston, TX 77030, USA.
authors.forename: Hyun-Seung
authors.initials: HS
authors.lastname: Kim
authors.forename: Xiu
authors.initials: X
authors.lastname: Jun Song
authors.forename: Cintia S
authors.initials: CS
authors.lastname: de Paiva
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Chen
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Pflugfelder
authors.forename: De-Quan
authors.initials: DQ
authors.lastname: Li
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016667:Bacterial Capsules; D015415:Biomarkers / Q000032:analysis; D001973:Bromodeoxyuridine; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D018031:Connexin 43 / Q000032:analysis; D004268:DNA-Binding Proteins; D019573:Epithelium, Corneal / Q000166:cytology*; D066246:ErbB Receptors / Q000032:analysis; D016147:Genes, Tumor Suppressor; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D019012:Integrin beta1 / Q000032:analysis; D016850:Limbus Corneae / Q000166:cytology; D008875:Middle Aged; D010641:Phenotype; D010750:Phosphoproteins / Q000032:analysis; D011135:Polysaccharides, Bacterial / Q000032:analysis; D011498:Protein Precursors / Q000032:analysis; D013234:Stem Cells / Q000166:cytology*; D015534:Trans-Activators / Q000032:analysis; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins
chemical_list: D015415:Biomarkers; D018031:Connexin 43; D004268:DNA-Binding Proteins; D019012:Integrin beta1; D010750:Phosphoproteins; D011135:Polysaccharides, Bacterial; D011498:Protein Precursors; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C098418:capsular polysaccharide K3; C032253:involucrin; D066246:ErbB Receptors; D001973:Bromodeoxyuridine