title: Isolation of esophageal stem cells with potential for therapy.
abstract: OBJECTIVE
  Long-gap esophageal atresia represents a significant challenge for pediatric surgeons and current surgical approaches are associated with significant morbidity. A tissue-engineered esophagus, comprising cells seeded onto a scaffold, represents a therapeutic alternative. In this study, we aimed to determine the optimal techniques for isolation and culture of mouse esophageal epithelial cells and to isolate CD34-positive esophageal epithelial stem cells from cadaveric mouse specimens.
  
  
  METHODS
  Primary epithelial cells were isolated from mouse esophagi by enzymatic dissociation from the mucosal layer (Dispase, Trypsin/EDTA) using three different protocols. In protocol A, isolated mucosa was minced and incubated with trypsin once. In protocol B, intact mucosal sheets underwent two trypsin incubations yielding a single-cell suspension. In protocol C, intact mucosa explants were plated epithelial side down. Epithelial cells were cultured on collagen-coated wells.
  
  
  RESULTS
  Initial findings showed that Protocol B gave the best results in terms of yield, viability, and least contamination with different cell types and microbes. Esophageal epithelial cells isolated using Protocol B were stained for CD34 and sorted using fluorescence-activated cell sorting (FACS). Of the total cells sorted, 8.3% (2-11.3) [%median (range)] were CD34 positive.
  
  
  CONCLUSIONS
  Our results demonstrate that mouse esophageal epithelial cells can be successfully isolated from fresh mouse esophagi using two consecutive trypsin incubations of intact mucosal sheets. Furthermore, the cells obtained using this method were successfully stained for CD34, a putative esophageal epithelial stem cell marker. Further research into the factors necessary for the successful proliferation of CD34 positive stem cell lines is needed to progress toward clinical application.
authors.affiliation: Stem Cells and Regenerative Medicine Section, UCL Institute of Child Health and Surgery Unit, Great Ormond Street Hospital, 30 Guilford Street, London, WC1N 1EH, UK.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Maghsoudlou
authors.forename: Daniel
authors.initials: D
authors.lastname: Ditchfield
authors.forename: Dorota H K
authors.initials: DH
authors.lastname: Klepacka
authors.forename: Panicos
authors.initials: P
authors.lastname: Shangaris
authors.forename: Luca
authors.initials: L
authors.lastname: Urbani
authors.forename: Stavros P
authors.initials: SP
authors.lastname: Loukogeorgakis
authors.forename: Simon
authors.initials: S
authors.lastname: Eaton
authors.forename: Paolo
authors.initials: P
authors.lastname: De Coppi
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000166:cytology*; D004933:Esophageal Atresia / Q000628:therapy*; D004947:Esophagus / Q000166:cytology*; D005434:Flow Cytometry; D051379:Mice; D008810:Mice, Inbred C57BL; D013234:Stem Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds