Expansion of Human Airway Basal Stem Cells and Their Differentiation as 3D Tracheospheres

Robert E Hynds, Colin R Butler, Sam M Janes, Adam Giangreco

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

26 Citations (Scopus)


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.

Original languageEnglish
Title of host publicationOrganoids. Methods in Molecular Biology
PublisherHumana Press
Number of pages11
ISBN (Electronic)978-1-4939-7617-1
ISBN (Print)978-1-4939-7616-4
Publication statusPrint publication - Aug 2016
Externally publishedYes

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
ISSN (Print)1064-3745


  • Cell Culture Techniques/methods
  • Cell Differentiation
  • Cell Proliferation
  • Cells, Cultured
  • Epithelial Cells/cytology
  • Humans
  • Organoids/cytology
  • Stem Cells/cytology
  • Tissue Engineering/methods
  • Trachea/cytology


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