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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


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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  1. 1.

    et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat. Biotechnol. 29, 267–272 (2011).

  2. 2.

    et al. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat. Biotechnol. 32, 84–91 (2014).

  3. 3.

    & Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008).

  4. 4.

    & Preparing for the first breath: genetic and cellular mechanisms in lung development. Dev. Cell 18, 8–23 (2010).

  5. 5.

    , & Stem cells of the respiratory system: from identification to differentiation into functional epithelium. BioEssays 35, 261–270 (2013).

  6. 6.

    , & Alveolar surfactant homeostasis and the pathogenesis of pulmonary disease. Annu. Rev. Med. 61, 105–119 (2010).

  7. 7.

    , & Genetic disorders influencing lung formation and function at birth. Hum. Mol. Genet. 13 (Spec No 2): R207–R215 (2004).

  8. 8.

    et al. Evidence for type II cells as cells of origin of K-Ras-induced distal lung adenocarcinoma. Proc. Natl. Acad. Sci. USA 109, 4910–4915 (2012).

  9. 9.

    & The acute respiratory distress syndrome. N. Engl. J. Med. 342, 1334–1349 (2000).

  10. 10.

    , & An overview of pulmonary surfactant in the neonate: genetics, metabolism, and the role of surfactant in health and disease. Mol. Genet. Metab. 97, 95–101 (2009).

  11. 11.

    et al. Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells. Cloning Stem Cells 10, 49–64 (2008).

  12. 12.

    , , , & A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 104, 4449–4454 (2007).

  13. 13.

    et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 10, 398–411 (2012).

  14. 14.

    et al. Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix. J. Clin. Investig. 123, 4950–4962 (2013).

  15. 15.

    et al. Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs. Cell Stem Cell 10, 385–397 (2012).

  16. 16.

    et al. Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells. Proc. Natl. Acad. Sci. USA 111, E1723–E1730 (2014).

  17. 17.

    et al. Generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells. Stem Cell Rep. 3, 394–403 (2014).

  18. 18.

    et al. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTRTR protein. Nat. Biotechnol. 30, 876–882 (2012).

  19. 19.

    et al. Signaling through BMP receptors promotes respiratory identity in the foregut via repression of Sox2. Development 138, 971–981 (2011).

  20. 20.

    , , , & Bmp4 is required for tracheal formation: a novel mouse model for tracheal agenesis. Dev. Biol. 322, 145–155 (2008).

  21. 21.

    et al. Development of definitive endoderm from embryonic stem cells in culture. Development 131, 1651–1662 (2004).

  22. 22.

    et al. Stage-specific signaling through TGF-β family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells. Development 138, 861–871 (2011).

  23. 23.

    et al. Wnt2/2b and β-catenin signaling are necessary and sufficient to specify lung progenitors in the foregut. Dev. Cell 17, 290–298 (2009).

  24. 24.

    , , , & Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development 122, 1693–1702 (1996).

  25. 25.

    et al. A retinoic acid–dependent network in the foregut controls formation of the mouse lung primordium. J. Clin. Investig. 120, 2040–2048 (2010).

  26. 26.

    , , , & Differentiation of human pulmonary type II cells in vitro by glucocorticoid plus cAMP. Am. J. Physiol. 283, L940–L951 (2002).

  27. 27.

    et al. BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat. Biotechnol. 24, 1402–1411 (2006).

  28. 28.

    et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc. Natl. Acad. Sci. USA 106, 12771–12775 (2009).

  29. 29.

    , & Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature 507, 190–194 (2014).

  30. 30.

    et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotechnol. 26, 313–315 (2008).

  31. 31.

    et al. A functionally characterized test set of human induced pluripotent stem cells. Nat. Biotechnol. 29, 279–286 (2011).

  32. 32.

    et al. Reference maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell 144, 439–452 (2011).

  33. 33.

    et al. Derivation of novel human ground state naive pluripotent stem cells. Nature 504, 282–286 (2013).

  34. 34.

    et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158, 1254–1269 (2014).

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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


  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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S.X.L.H. developed the lung and airway differentiation protocol and co-wrote the manuscript; M.D.G. developed the AFE generation protocol; 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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