Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
Organoids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1576))

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 139.00
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hogan BL, Barkauskas CE, Chapman HA, Epstein JA, Jain R, Hsia CC, Niklason L, Calle E, Le A, Randell SH, Rock J, Snitow M, Krummel M, Stripp BR, Vu T, White ES, Whitsett JA, Morrisey EE (2014) Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15:123–138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Rock JR, Onaitis MW, Rawlins EL, Lu Y, Clark CP, Xue Y, Randell SH, Hogan BL (2009) Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci U S A 106:12771–12775

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Teixeira VH, Nadarajan P, Graham TA, Pipinikas CP, Brown JM, Falzon M, Nye E, Poulsom R, Lawrence D, Wright NA, McDonald S, Giangreco A, Simons BD, Janes SM (2013) Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors. Elife 2, e00966

    Article  PubMed  PubMed Central  Google Scholar 

  4. Hynds RE, Giangreco A (2013) Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine. Stem Cells 31:417–422

    Article  CAS  PubMed  Google Scholar 

  5. de Jong PM, van Sterkenburg MA, Hesseling SC, Kempenaar JA, Mulder AA, Mommaas AM, Dijkman JH, Ponec M (1994) Ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface. Am J Respir Cell Mol Biol 10:271–277

    Article  PubMed  Google Scholar 

  6. Fulcher ML, Gabriel S, Burns KA, Yankaskas JR, Randell SH (2005) Well-differentiated human airway epithelial cell cultures. Methods Mol Med 107:183–206

    CAS  PubMed  Google Scholar 

  7. Mathis C, Poussin C, Weisensee D, Gebel S, Hengstermann A, Sewer A, Belcastro V, Xiang Y, Ansari S, Wagner S, Hoeng J, Peitsch MC (2013) Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers. Am J Physiol Lung Cell Mol Physiol 304:L489–L503

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343

    Article  CAS  PubMed  Google Scholar 

  9. Chapman S, Liu X, Meyers C, Schlegel R, McBride AA (2010) Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. J Clin Invest 120:2619–2626

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Suprynowicz FA, Upadhyay G, Krawczyk E, Kramer SC, Hebert JD, Liu X, Yuan H, Cheluvaraju C, Clapp PW, Boucher RC Jr, Kamonjoh CM, Randell SH, Schlegel R (2012) Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells. Proc Natl Acad Sci U S A 109:20035–20040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, Timofeeva OA, Nealon C, Dakic A, Simic V, Haddad BR, Rhim JS, Dritschilo A, Riegel A, McBride A, Schlegel R (2012) ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol 180:599–607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Butler CR, Hynds RE, Gowers KH, Lee Ddo H, Brown JM, Crowley C, Teixeira VH, Smith CM, Urbani L, Hamilton NJ, Thakrar RM, Booth HL, Birchall MA, De Coppi P, Giangreco A, O'Callaghan C, Janes SM (2016) Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering. Am J Respir Crit Care Med194(2):156–168. doi:https://doi.org/10.1164/rccm.201507-1414OC, PubMed PMID: 26840431

  13. Wu X, Peters-Hall JR, Bose S, Pena MT, Rose MC (2011) Human bronchial epithelial cells differentiate to 3D glandular acini on basement membrane matrix. Am J Respir Cell Mol Biol 44:914–921

    Article  CAS  PubMed  Google Scholar 

  14. Hegab AE, Ha VL, Darmawan DO, Gilbert JL, Ooi AT, Attiga YS, Bisht B, Nickerson DW, Gomperts BN (2012) Isolation and in vitro characterization of basal and submucosal gland duct stem/progenitor cells from human proximal airways. Stem Cells Transl Med 1:719–724

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Danahay H, Pessotti AD, Coote J, Montgomery BE, Xia D, Wilson A, Yang H, Wang Z, Bevan L, Thomas C, Petit S, London A, LeMotte P, Doelemeyer A, Velez-Reyes GL, Bernasconi P, Fryer CJ, Edwards M, Capodieci P, Chen A, Hild M, Jaffe AB (2015) Notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the lung. Cell Rep 10:239–252

    Article  CAS  PubMed  Google Scholar 

  16. Gao X, Bali AS, Randell SH, Hogan BL (2015) GRHL2 coordinates regeneration of a polarized mucociliary epithelium from basal stem cells. J Cell Biol 211:669–682

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Prof. Richard Schlegel (Georgetown University, USA), Xuefeng Liu (Georgetown University, USA), and Dr. Henry Danahay (University of Sussex, UK) for sharing their laboratories protocols during the development of those described here.

This work was supported by a BBSRC-CASE studentship with industrial support from Unilever (R.E.H.), a Wellcome Trust Clinical Research Fellowship (C.R.B.), an ERC Starting Grant (A.G.), a Wellcome Trust Senior Fellowship (S.M.J.) and was undertaken at UCLH/UCL who receive funding from the Department of Health’s NIHR Biomedical Research Centre’s funding scheme and the UCL Experimental Cancer Medicine Centre (S.M.J.).

Author information

Authors and Affiliations

  1. Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK

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

Authors
  1. Robert E. Hynds
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Colin R. Butler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sam M. Janes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Adam Giangreco
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Adam Giangreco .

Editor information

Editors and Affiliations

  1. Ottawa Hospital Research Institute, Ottawa, ON, Canada

    Kursad Turksen

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Hynds, R.E., Butler, C.R., Janes, S.M., Giangreco, A. (2016). Expansion of Human Airway Basal Stem Cells and Their Differentiation as 3D Tracheospheres. In: Turksen, K. (eds) Organoids. Methods in Molecular Biology, vol 1576. Humana, New York, NY. https://doi.org/10.1007/7651_2016_5

Download citation

  • DOI: https://doi.org/10.1007/7651_2016_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-7616-4

  • Online ISBN: 978-1-4939-7617-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation