naa a22 4500 510739121 CHVBK 20180411083021.0 cr unu---uuuuu 180411e20131201xx s 000 0 eng 10.1007/s12015-013-9451-6 doi (NATIONALLICENCE)springer-10.1007/s12015-013-9451-6 Suitability of Human Tenon's Fibroblasts as Feeder Cells for Culturing Human Limbal Epithelial Stem Cells [Elektronische Daten] [Gaia Scafetta, Eleonora Tricoli, Camilla Siciliano, Chiara Napoletano, Rosa Puca, Enzo Vingolo, Giuseppe Cavallaro, Andrea Polistena, Giacomo Frati, Elena De Falco] Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α+LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture. Springer Science+Business Media New York, 2013 Limbal stem cell deficiency nationallicence Cell therapy nationallicence Limbal stem cells nationallicence Feeder layers nationallicence 3T3-J2 nationallicence Human Tenon's Fibroblasts nationallicence ΔNp63α nationallicence Cytokines/growth factors nationallicence Scafetta Gaia Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut Tricoli Eleonora Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut Siciliano Camilla Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut Napoletano Chiara Department of Experimental Medicine, University of Rome "Sapienza”, V. le Regina Elena 324, 00161, Rome, Italy aut Puca Rosa Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut Vingolo Enzo Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut Cavallaro Giuseppe Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut Polistena Andrea UOC General Surgery University of Rome "Sapienza”, AUSL Latina, Latina, Italy aut Frati Giacomo Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut De Falco Elena Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut Stem Cell Reviews and Reports Springer US; http://www.springer-ny.com 9/6(2013-12-01), 847-857 1550-8943 9:6<847 2013 9 12015 https://doi.org/10.1007/s12015-013-9451-6 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s12015-013-9451-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Scafetta Gaia Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut NATIONALLICENCE 700 1- Tricoli Eleonora Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut NATIONALLICENCE 700 1- Siciliano Camilla Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut NATIONALLICENCE 700 1- Napoletano Chiara Department of Experimental Medicine, University of Rome "Sapienza”, V. le Regina Elena 324, 00161, Rome, Italy aut NATIONALLICENCE 700 1- Puca Rosa Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut NATIONALLICENCE 700 1- Vingolo Enzo Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut NATIONALLICENCE 700 1- Cavallaro Giuseppe Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut NATIONALLICENCE 700 1- Polistena Andrea UOC General Surgery University of Rome "Sapienza”, AUSL Latina, Latina, Italy aut NATIONALLICENCE 700 1- Frati Giacomo Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut NATIONALLICENCE 700 1- De Falco Elena Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza”, C.so della Repubblica 79, 04100, Latina, Italy aut NATIONALLICENCE 773 0- Stem Cell Reviews and Reports Springer US; http://www.springer-ny.com 9/6(2013-12-01), 847-857 1550-8943 9:6<847 2013 9 12015 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer