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The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells

Nature Protocols volume 10, pages 413425 (2015) | Download Citation

Abstract

Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here we describe a strategy for directed differentiation of hPSCs into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development, and it takes 50 d. First, definitive endoderm (DE) is induced in the presence of high concentrations of activin A. Subsequently, lung-biased anterior foregut endoderm (AFE) is specified by sequential inhibition of bone morphogenetic protein (BMP), transforming growth factor-β (TGF-β) and Wnt signaling. AFE is then ventralized by applying Wnt, BMP, fibroblast growth factor (FGF) and retinoic acid (RA) signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, cAMP and glucocorticoid agonism. This protocol is conducted in defined conditions, it does not involve genetic manipulation of the cells and it results in cultures in which the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells.

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Acknowledgements

This work was supported by Price Center for Comprehensive Chest Care at Columbia University Medical Center, and by a US National Institutes of Health grant 1R01HL120046 to H.-W.S.

Author information

Affiliations

  1. Columbia Center for Translational Immunology, Columbia University Medical Center, New York, New York, USA.

    • Sarah X L Huang
    • , Michael D Green
    • , Ana Toste de Carvalho
    • , Melanie Mumau
    • , Ya-Wen Chen
    •  & Hans-Willem Snoeck
  2. Department of Medicine, Columbia University Medical Center, New York, New York, USA.

    • Sarah X L Huang
    • , Michael D Green
    • , Ana Toste de Carvalho
    • , Melanie Mumau
    • , Ya-Wen Chen
    •  & Hans-Willem Snoeck
  3. Department of Developmental and Regenerative Biology, Black Family Stem Cell Institute, Experimental Therapeutic Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

    • Sunita L D'Souza
  4. Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA.

    • Hans-Willem Snoeck

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Contributions

S.X.L.H. developed the lung and airway differentiation protocol and co-wrote the manuscript; M.D.G. developed the AFE generation protocol; A.T.de C., M.M. and Y.-W.C. contributed to the development of the protocol; S.L.D. provided cells used in differentiation assays; H.-W.S. developed the concept, contributed to protocol development and co-wrote the manuscript with S.X.L.H.

Competing interests

The authors have filed patent applications PCT/US11/33751 and IRCU13340.

Corresponding author

Correspondence to Hans-Willem Snoeck.

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https://doi.org/10.1038/nprot.2015.023

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