title: Expansion of Human Airway Basal Stem Cells and Their Differentiation as 3D Tracheospheres.
abstract: Although basal cells function as human airway epithelial stem cells, analysis of these cells is limited by in vitro culture techniques that permit only minimal cell growth and differentiation. Here, we report a protocol that dramatically increases the long-term expansion of primary human airway basal cells while maintaining their genomic stability using 3T3-J2 fibroblast coculture and ROCK inhibition. We also describe techniques for the differentiation and imaging of these expanded airway stem cells as three-dimensional tracheospheres containing basal, ciliated, and mucosecretory cells. These procedures allow investigation of the airway epithelium under more physiologically relevant conditions than those found in undifferentiated monolayer cultures. Together these methods represent a novel platform for improved airway stem cell growth and differentiation that is compatible with high-throughput, high-content translational lung research as well as human airway tissue engineering and clinical cellular therapy.
authors.affiliation: Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Hynds
authors.affiliation: Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK.
authors.forename: Colin R
authors.initials: CR
authors.lastname: Butler
authors.affiliation: Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK.
authors.forename: Sam M
authors.initials: SM
authors.lastname: Janes
authors.affiliation: Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK. a.giangreco@ucl.ac.uk.
authors.forename: Adam
authors.initials: A
authors.lastname: Giangreco
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology*; D006801:Humans; D009940:Organoids / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods*; D014132:Trachea / Q000166:cytology*
keywords: Adult stem cells; Cell culture techniques; Cilia; Epithelial cells; Goblet cells; Lung; Primary cell culture; Stem cells