Contents lists available at ScienceDirect Contact Lens and Anterior Eye journal homepage: www.elsevier.com/locate/clae Cultivation and characterization of pterygium as an ex vivo study model for disease and therapy Natasha Josifovska a , Dóra Júlia Szabó a , Richárd Nagymihály a , Zoltán Veréb a , Andrea Facskó a , Ketil Eriksen b , Morten C. Moe b , Goran Petrovski a,b, ⁎ a Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Koranyi Fasor 10-11, 6720 Szeged, Hungary b Center for Eye Research, Department of Ophthalmology, Oslo University Hospital and University of Oslo, Kirkeveien 166, N-0407 Oslo, Norway ARTICLE INFO Keywords: Pterygium Long-term cultures Tissue engineering Mitomycin C IL-6 IL-8 ABSTRACT Purpose: Development of ex vivo model to study pathogenesis, inflammation and treatment modalities for pterygium. Methods: Pterygium obtained from surgery was cultivated (3 months). Gravitational attachment method using viscoelastic facilitated adherence of graft and outgrowing cells. Medium contained serum as the only growth supplement with no use of scaffolds. Surface profiling of the multi-layered cells for hematopoietic- and mesenchymal stem cell markers was performed. Examination of cells by immunohistochemistry using pluripotency, oxidative stress, stemness, migration and proliferation, epithelial and secretory markers was performed. The effect of anti-proliferative agent Mitomycin C upon secretion of pro-inflammatory cytokines IL-6 and IL-8 was assessed. Results: Cells showed high expression of migration- (CXCR4), secretory- (MUC1, MUC4) and oxidative damage- (8-OHdG) markers, and low expression of hypoxia- (HIF-1α) and proliferation- (Ki-67) markers. Moderate and low expression of the pluripotency markers (Vimentin and ΔNp63) was present, respectively, while the putative markers of stemness (Sox2, Oct4, ABCG-2) and epithelial cell markers- (CK19, CK8-18) were weak. The surface marker profile of the outgrowing cells revealed high expression of the hematopoietic marker CD47, mesenchymal markers CD90 and CD73, minor or less positivity for the hematopoietic marker CD34, mesenchymal marker CD105, progenitor marker CD117 and attachment protein markers while low levels of IL-6 and IL-8 secretion ex vivo, were inhibited upon Mitomycin C treatment. Conclusion: Ex vivo tissue engineered pterygium consists of a mixture of cells of different lineage origin, suitable for use as a disease model for studying pathogenesis ex vivo, while opening possibilities for new treatment and prevention modalities. 1. Introduction Pterygium is a common ocular surface disorder characterized by an active wing-shaped overgrowth of epithelial and fibrovascular tissue growing from the conjunctiva towards the limbus and onto the cornea, thus giving cosmetic alterations to the ocular surface and in advanced cases impairing vision [1–4]. It consists of a head which falls onto the anterior cornea, a neck which covers the superficial limbus, and a body which overlies the sclera. The first sign of pterygium is formation of a cap which appears as halo in front of the head and goes deep into the epithelium without respecting the limbal epithelium stem cell border [5]. It is not fully known whether the stem cell deficiency alone or other factors drive the centripetal migration of the pterygium cells onto the cornea, or how sclera support may play a role in the process. The pathogenesis of pterygium remains uncertain, although it is thought to be mainly caused by ultraviolet (UV) radiation. A wide range of alternative pathogenic factors have been proposed, including viral infections, epigenetic aberrations, epithelial-mesenchymal transition, immunologic and anti-apoptotic mechanisms, angiogenic and lymphan- giogenic stimulation, deregulation of extracellular matrix (ECM) mod- ulators and growth factors, inflammation cascades, recruitment of bone-marrow-derived stem- and progenitor cells, and modifications in the cholesterol metabolism. Most of these factors are rather related to the development and maintenance of the disease than to its origin [6]. In the present study, cells were adherently cultivated and grown out of the pterygium using gravitational force from viscoelastic material for more than three months or until they formed multi-layered structures. Surface marker profiling using hematopoietic- and mesenchymal stem http://dx.doi.org/10.1016/j.clae.2017.04.002 Received 6 October 2016; Received in revised form 30 March 2017; Accepted 19 April 2017 ⁎ Corresponding author at: Department of Ophthalmology, University of Szeged, Korányi fasor 10-11, 6720, Szeged, Hungary. E-mail address: petrovski.goran@med.u-szeged.hu (G. Petrovski). Contact Lens and Anterior Eye 40 (2017) 283–292 1367-0484/ © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved. MARK