Suitability of human Tenon’s fibroblasts as feeder cells for culturing human limbal epithelial stem cells

Gaia Scafetta, Eleonora Tricoli, Camilla Siciliano, Chiara Napoletano, Rosa Puca, Enzo Maria Vingolo, Giuseppe Cavallaro, Andrea Polistena, Giacomo Frati, Elena De Falco

Research output: Contribution to journalArticlepeer-review

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

Original languageEnglish
Article numberA010
Pages (from-to)847-857
Number of pages11
JournalStem Cell Reviews and Reports
Volume9
Issue number6
DOIs
Publication statusPublished - 2013

Keywords

  • 3T3-J2
  • Cell therapy
  • Cytokines/growth factors
  • Feeder layers
  • Human Tenon’s Fibroblasts
  • Limbal stem cell deficiency
  • Limbal stem cells
  • ΔNp63α

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Developmental Biology

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