title: Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.
abstract: Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.
authors.affiliation: Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Dóra Júlia
authors.initials: DJ
authors.lastname: Szabó
authors.affiliation: Center of Eye Research, Department of Ophthalmology, Oslo University Hospital and University of Oslo, Oslo, Norway.
authors.forename: Agate
authors.initials: A
authors.lastname: Noer
authors.affiliation: Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Richárd
authors.initials: R
authors.lastname: Nagymihály
authors.affiliation: Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Natasha
authors.initials: N
authors.lastname: Josifovska
authors.affiliation: Eye Hospital, University Medical Centre, Ljubljana, Slovenia.
authors.forename: Sofija
authors.initials: S
authors.lastname: Andjelic
authors.affiliation: Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Veréb
authors.affiliation: Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Andrea
authors.initials: A
authors.lastname: Facskó
authors.affiliation: Center of Eye Research, Department of Ophthalmology, Oslo University Hospital and University of Oslo, Oslo, Norway.
authors.forename: Morten C
authors.initials: MC
authors.lastname: Moe
authors.affiliation: Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Goran
authors.initials: G
authors.lastname: Petrovski
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001344:Autopsy; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007633:Keratins / Q000235:genetics / Q000378:metabolism; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture / Q000379:methods*; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D023822:Tissue Engineering; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015703:Antigens, CD; D015415:Biomarkers; D003470:Culture Media; D019394:Ki-67 Antigen; D009363:Neoplasm Proteins; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014746:Vimentin; D007633:Keratins; D003094:Collagen