title: Partial enrichment of a population of human limbal epithelial cells with putative stem cell properties based on collagen type IV adhesiveness.
abstract: The concept that corneal epithelium stem cells reside in limbus has been recognized for more than a decade, but isolation of these stem cells has not been accomplished. This study was an initial attempt to isolate a population of human limbal epithelial cells enriched for certain putative stem cell properties based on their phenotype. Epithelial cells harvested from fresh human limbal rings and their primary cultures were allowed to adhere to collagen IV-coated dishes for 20 min and 2 hr, sequentially. The rapidly adherent cells (RAC), slowly adherent cells and non-adherent cells were evaluated for certain stem cell properties: (a) BrdU-label retention, (b) expression of basal cell (integrin beta1, p63, ABCG2) and differentiation (involucrin, keratin 12) markers, and (c) colony forming efficiency (CFE) and growth capacity on a 3T3 fibroblast feeder layer. Among unfractionated cells and the three selected populations, the RAC, accounting for about 10% of whole population, were enriched 5-fold in BrdU label-retaining cells, displayed the highest number of integrin beta1 and p63 positive and involucrin negative cells, expressed high levels of DeltaNp63 and ABCG2 mRNA, and lacked involucrin and K12 expression, and possessed the greatest CFE and growth capacity. These findings demonstrated for the first time that human limbal epithelial cells with stem cell properties can be partially enriched by their adhesiveness to collagen IV. The RAC population enriched for certain putative stem cell properties may prove useful in the future for transplantation to diseased and damaged corneas with limbal stem cell deficiency.
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, 6565 Fannin Street, NC-205, Houston, TX 77030, USA. dequan1@bcm.tmc.edu
authors.forename: De-Quan
authors.initials: DQ
authors.lastname: Li
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Chen
authors.forename: Xiu Jun
authors.initials: XJ
authors.lastname: Song
authors.forename: Cintia S
authors.initials: CS
authors.lastname: de Paiva
authors.forename: Hyun-Seung
authors.initials: HS
authors.lastname: Kim
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Pflugfelder
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis; D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000032:analysis; D001973:Bromodeoxyuridine / Q000032:analysis; D002448:Cell Adhesion / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000502:physiology*; D004268:DNA-Binding Proteins; D004847:Epithelial Cells / Q000502:physiology; D016147:Genes, Tumor Suppressor; D006801:Humans; D019012:Integrin beta1 / Q000032:analysis; D053551:Keratin-12; D007633:Keratins / Q000032:analysis; D016850:Limbus Corneae / Q000166:cytology*; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000032:analysis; D010641:Phenotype; D010750:Phosphoproteins / Q000032:analysis; D011498:Protein Precursors / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D013234:Stem Cells / Q000502:physiology*; D015534:Trans-Activators / Q000032:analysis; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015415:Biomarkers; D024141:Collagen Type IV; D004268:DNA-Binding Proteins; D019012:Integrin beta1; C507494:KRT12 protein, human; D053551:Keratin-12; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011498:Protein Precursors; D012333:RNA, Messenger; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C032253:involucrin; D007633:Keratins; D001973:Bromodeoxyuridine