title: Simulation of an in vitro niche environment that preserves conjunctival progenitor cells.
abstract: OBJECTIVE
  To evaluate a serum-free system where mitotically active subconjunctival fibroblasts were co-cultured with conjunctival epithelial cells to mimic a niche environment for conjunctival progenitor cells.
  
  
  METHODS
  Human conjunctival epithelial cells were expanded in vitro and evaluated for their colony-forming efficiency and clonal ability. The cells were then transferred to a serum-free co-culture system and cultured in the presence of mitotically active subconjunctival fibroblasts (human conjunctival epithelial cells and human bulbar subconjunctival fibroblasts [HCEC-HCF]). Cells were evaluated by Ki67 staining, total colony-forming efficiency and the number of colonies with a surface area of more than 10 mm(2). The expression of putative progenitor cell markers p63α, ABCG2 and CK15, and the presence of MUC5AC- and periodic acid-Schiff-positive cells was compared with standard culture conditions (HCEC-3T3).
  
  
  RESULTS
  Conjunctival epithelial cells cultured under HCEC-HCF and HCEC-3T3 conditions demonstrated strong immunoreactivity to p63α and ABCG2. Co-localization of CK15 and p63α revealed a subpopulation of CK15-positive cells under HCEC-3T3 conditions compared with only a few CK15-positive cells found under HCEC-HCF conditions. MUC5AC- and periodic acid-Schiff-positive cells were much more common under HCEC-3T3 conditions than under HCEC-HCF conditions. These results were confirmed by reverse transcription-PCR. Cells in HCEC-HCF conditions demonstrated a significantly higher total colony-forming efficiency and a significantly higher percentage of colonies with holoclone-like morphology.
  
  
  CONCLUSIONS
  The simulation of a niche environment in vitro by co-culturing mitotically active subconjunctival fibroblasts with conjunctival epithelial cells supports the maintenance of conjunctival cells with progenitor cell characteristics and therefore might be a useful tool to expand conjunctival epithelial progenitor cells in vitro for clinical use.
authors.affiliation: Cells for Sight Transplantation & Research Programme, Department of Ocular Biology & Therapeutics, UCL Institute of Ophthalmology, London, UK. mail@stefanschrader.de
authors.forename: Stefan
authors.initials: S
authors.lastname: Schrader
authors.forename: Maria
authors.initials: M
authors.lastname: Notara
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Tuft
authors.forename: Michele
authors.initials: M
authors.lastname: Beaconsfield
authors.forename: Gerd
authors.initials: G
authors.lastname: Geerling
authors.forename: Julie T
authors.initials: JT
authors.lastname: Daniels
mesh_terms: D016475:3T3 Cells; D000368:Aged; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D003228:Conjunctiva / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology; D020397:Goblet Cells / Q000166:cytology; D006801:Humans; D019394:Ki-67 Antigen; D051379:Mice; D008954:Models, Biological; D009077:Mucins / Q000378:metabolism; D055153:Stem Cell Niche / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*
chemical_list: D015415:Biomarkers; D019394:Ki-67 Antigen; D009077:Mucins