title: Suitability of human Tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells.
abstract: 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.
authors.affiliation: Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza", C.so della Repubblica 79, 04100, Latina, Italy.
authors.forename: Gaia
authors.initials: G
authors.lastname: Scafetta
authors.forename: Eleonora
authors.initials: E
authors.lastname: Tricoli
authors.forename: Camilla
authors.initials: C
authors.lastname: Siciliano
authors.forename: Chiara
authors.initials: C
authors.lastname: Napoletano
authors.forename: Rosa
authors.initials: R
authors.lastname: Puca
authors.forename: Enzo Maria
authors.initials: EM
authors.lastname: Vingolo
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Cavallaro
authors.forename: Andrea
authors.initials: A
authors.lastname: Polistena
authors.forename: Giacomo
authors.initials: G
authors.lastname: Frati
authors.forename: Elena
authors.initials: E
authors.lastname: De Falco
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D003470:Culture Media / Q000494:pharmacology; D020405:Dermis / Q000166:cytology; D061252:Feeder Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology; D012995:Solubility; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D058475:Tenon Capsule / Q000166:cytology*; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D015415:Biomarkers; D003470:Culture Media; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D016685:Mitomycin