title: Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering.
abstract: Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts.
  
  
  
  To define a scalable cell culture system to deliver airway epithelium to clinical grafts.
  
  
  
  Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures.
  
  
  
  3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model.
  
  
  
  Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.
authors: 1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.
authors: Colin R
authors: 0000-0003-4192-0519
authors: CR
authors: Butler
authors: 1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.
authors: Robert E
authors: 0000-0002-2170-8791
authors: RE
authors: Hynds
authors: 1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.
authors: Kate H C
authors: KH
authors: Gowers
authors: 2 Respiratory, Critical Care, and Anesthesia, Institute of Child Health, University College London, London, United Kingdom.
authors: Dani Do Hyang
authors: Ddo H
authors: Lee
authors: 1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.
authors: James M
authors: JM
authors: Brown
authors: 3 Stem Cell and Regenerative Medicine Section, Great Ormond Street Hospital and UCL Institute of Child Health, London, United Kingdom.
authors: Claire
authors: C
authors: Crowley
authors: 1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.
authors: Vitor H
authors: VH
authors: Teixeira
authors: 2 Respiratory, Critical Care, and Anesthesia, Institute of Child Health, University College London, London, United Kingdom.
authors: Claire M
authors: CM
authors: Smith
authors: 3 Stem Cell and Regenerative Medicine Section, Great Ormond Street Hospital and UCL Institute of Child Health, London, United Kingdom.
authors: Luca
authors: L
authors: Urbani
authors: 1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.
authors: Nicholas J
authors: NJ
authors: Hamilton
authors: 1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.
authors: Ricky M
authors: RM
authors: Thakrar
authors: 4 Department of Thoracic Medicine, University College London Hospitals, London, United Kingdom; and.
authors: Helen L
authors: HL
authors: Booth
authors: 5 UCL Ear Institute, Royal National Throat, Nose and Ear Hospital, London, United Kingdom.
authors: Martin A
authors: MA
authors: Birchall
authors: 3 Stem Cell and Regenerative Medicine Section, Great Ormond Street Hospital and UCL Institute of Child Health, London, United Kingdom.
authors: Paolo
authors: P
authors: De Coppi
authors: 1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.
authors: Adam
authors: A
authors: Giangreco
authors: 2 Respiratory, Critical Care, and Anesthesia, Institute of Child Health, University College London, London, United Kingdom.
authors: Christopher
authors: C
authors: O'Callaghan
authors: 1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.|4 Department of Thoracic Medicine, University College London Hospitals, London, United Kingdom; and.
authors: Sam M
authors: 0000-0002-6634-5939
authors: SM
authors: Janes
mesh_terms: D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D009079:Mucociliary Clearance / Q000502:physiology; D016133:Polymerase Chain Reaction; D020545:Respiratory Mucosa / Q000502:physiology; D012140:Respiratory Tract Diseases / Q000628:therapy*; D013234:Stem Cells / Q000378:metabolism*; D023822:Tissue Engineering / Q000379:methods*
keywords: adult stem cells; epithelium; respiratory mucosa; tissue engineering; trachea