Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Efficient generation of lung and airway epithelial cells from human pluripotent stem cells

Nature Biotechnology volume 32, pages 84–91 (2014)Cite this article

Subjects

Abstract

The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP—agonists to signaling pathways critical for early lung development in the mouse—recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Establishment of a protocol of lung field induction.
Figure 2: Requirement of morphogens for lung field induction from hPSCs.
Figure 3: In vivo potential of hPSC-derived lung and airway progenitors.
Figure 4: Further differentiation of hPSC-derived lung and airway progenitors.
Figure 5: Terminal differentiation of hPSCS-derived lung and airway progenitors.
Figure 6: Morphology and function of hPSC-derived lung and airway epithelium.

Similar content being viewed by others

References

  1. Green, M.D., Huang, S.X. & Snoeck, H.W. Stem cells of the respiratory system: from identification to differentiation into functional epithelium. Bioessays 35, 261–270 (2013).

    Article  CAS  Google Scholar 

  2. Rock, J.R. & Hogan, B.L. Epithelial progenitor cells in lung development, maintenance, repair, and disease. Annu. Rev. Cell Dev. Biol. 27, 493–512 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Rawlins, E.L., Clark, C.P., Xue, Y. & Hogan, B.L. The Id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells. Development 136, 3741–3745 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Mou, H. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Nostro, M.C. & Keller, G. Generation of beta cells from human pluripotent stem cells: potential for regenerative medicine. Semin. Cell Dev. Biol. 23, 701–710 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. D'Amour, K.A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Bellusci, S., Grindley, J., Emoto, H., Itoh, N. & Hogan, B.L. Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 124, 4867–4878 (1997).

    CAS  PubMed  Google Scholar 

  15. Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. & Hogan, B.L. 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).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Li, Y., Gordon, J., Manley, N.R., Litingtung, Y. & Chiang, C. Bmp4 is required for tracheal formation: a novel mouse model for tracheal agenesis. Dev. Biol. 322, 145–155 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Yamamoto, M. et al. Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo. Nature 428, 387–392 (2004).

    Article  CAS  Google Scholar 

  20. Perea-Gomez, A. et al. Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks. Dev. Cell 3, 745–756 (2002).

    Article  CAS  Google Scholar 

  21. del Barco Barrantes, I., Davidson, G., Grone, H.J., Westphal, H. & Niehrs, C. Dkk1 and noggin cooperate in mammalian head induction. Genes Dev. 17, 2239–2244 (2003).

    Article  Google Scholar 

  22. Yu, P.B. et al. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat. Chem. Biol. 4, 33–41 (2008).

    Article  CAS  Google Scholar 

  23. Inman, G.J. et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62, 65–74 (2002).

    Article  CAS  Google Scholar 

  24. Chen, B. et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol. 5, 100–107 (2009).

    Article  CAS  Google Scholar 

  25. Bennett, C.N. et al. Regulation of Wnt signaling during adipogenesis. J. Biol. Chem. 277, 30998–31004 (2002).

    Article  CAS  Google Scholar 

  26. Fusaki, N., Ban, H., Nishiyama, A., Saeki, K. & Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 85, 348–362 (2009).

    Article  CAS  Google Scholar 

  27. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630 (2010).

    Article  CAS  Google Scholar 

  28. Kimura, S. et al. The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev. 10, 60–69 (1996).

    Article  CAS  Google Scholar 

  29. Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547–551 (2011).

    Article  CAS  Google Scholar 

  30. Harris-Johnson, K.S., Domyan, E.T., Vezina, C.M. & Sun, X. beta-Catenin promotes respiratory progenitor identity in mouse foregut. Proc. Natl. Acad. Sci. USA 106, 16287–16292 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Weaver, M., Yingling, J.M., Dunn, N.R., Bellusci, S. & Hogan, B.L. Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development. Development 126, 4005–4015 (1999).

    CAS  PubMed  Google Scholar 

  33. Shu, W. et al. Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung. Dev. Biol. 283, 226–239 (2005).

    Article  CAS  Google Scholar 

  34. Post, M. et al. Keratinocyte growth factor and its receptor are involved in regulating early lung branching. Development 122, 3107–3115 (1996).

    CAS  PubMed  Google Scholar 

  35. Malpel, S., Mendelsohn, C. & Cardoso, W.V. Regulation of retinoic acid signaling during lung morphogenesis. Development 127, 3057–3067 (2000).

    CAS  PubMed  Google Scholar 

  36. Wongtrakool, C. et al. Down-regulation of retinoic acid receptor alpha signaling is required for sacculation and type I cell formation in the developing lung. J. Biol. Chem. 278, 46911–46918 (2003).

    Article  CAS  Google Scholar 

  37. Gonzales, L.W., Guttentag, S.H., Wade, K.C., Postle, A.D. & Ballard, P.L. Differentiation of human pulmonary type II cells in vitro by glucocorticoid plus cAMP. Am. J. Physiol. Lung Cell. Mol. Physiol. 283, L940–L951 (2002).

    Article  CAS  Google Scholar 

  38. Sanchez-Esteban, J. et al. Mechanical stretch promotes alveolar epithelial type II cell differentiation. J. Appl. Physiol. 91, 589–595 (2001).

    Article  CAS  Google Scholar 

  39. Whitsett, J.A., Wert, S.E. & Weaver, T.E. Alveolar surfactant homeostasis and the pathogenesis of pulmonary disease. Annu. Rev. Med. 61, 105–119 (2010).

    Article  CAS  Google Scholar 

  40. Rooney, S.A. Regulation of surfactant secretion. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 129, 233–243 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  45. Blauwkamp, T.A., Nigam, S., Ardehali, R., Weissman, I.L. & Nusse, R. Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors. Nat. Commun. 3, 1070 (2012).

    Article  Google Scholar 

  46. Barkauskas, C.E. et al. Type 2 alveolar cells are stem cells in adult lung. J. Clin. Invest. 123, 3025–3036 (2013).

    Article  CAS  Google Scholar 

  47. Xu, X. 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).

    Article  CAS  Google Scholar 

  48. O'Neill, J.D. et al. Decellularization of human and porcine lung tissues for pulmonary tissue engineering. Ann. Thorac. Surg. 96, 1046–1056 (2013).

    Article  Google Scholar 

Download references

Acknowledgements

S.X.L.H. is a Druckenmiller Fellow of the New York Stem Cell Foundation. The authors wish to thank J. Sonnet and the Columbia Lung Regeneration Team, D. Farber and J. Thome for providing samples of human lung tissue; K. Brown for kind help with electron microscopy; S.-H. Ho for assistance with microscopy.

Author information

Authors and Affiliations

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

    Sarah X L Huang, Zheng Hu, Yong-Guang Yang, Ya-Wen Chen, Melanie Mumau, Michael D Green & Hans-Willem Snoeck

  2. Department of Medicine, Columbia University Medical Center, New York, New York, USA

    Sarah X L Huang, Mohammad Naimul Islam, Yong-Guang Yang, Ya-Wen Chen, Melanie Mumau, Gordana Vunjak-Novakovic, Jahar Bhattacharya & Hans-Willem Snoeck

  3. Department of Biomedical Engineering, Columbia University, New York, New York, USA

    John O'Neill & Gordana Vunjak-Novakovic

  4. Department of Physiology & Cellular Biophysics, Columbia University Medical Center, New York, New York, USA

    Jahar Bhattacharya

  5. Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA

    Hans-Willem Snoeck

Authors
  1. Sarah X L Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mohammad Naimul Islam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John O'Neill
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zheng Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yong-Guang Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ya-Wen Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Melanie Mumau
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael D Green
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gordana Vunjak-Novakovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jahar Bhattacharya
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Hans-Willem Snoeck
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.X.L.H. performed most experiments, developed this protocol and co-wrote the manuscript; M.N.I. performed functional analysis of ATII cells supervised by J.B.; M.M., M.D.G. and Y.-W.C. gave technical advice and assisted S.X.L.H. experimentally; Z.H. performed kidney capsule transplantations supervised by Y.-G.Y.; J.O.N. provided human lung human decellularized extracellular lung matrix and these experiments were supervised by G.V.-N.; H.-W.S. developed the concept, co-analyzed primary data and co-wrote the manuscript with S.X.L.H.

Corresponding author

Correspondence to Hans-Willem Snoeck.

Ethics declarations

Competing interests

The authors have filed patent application IRCU13340.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1 and 2 (PDF 2840 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huang, S., Islam, M., O'Neill, J. et al. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat Biotechnol 32, 84–91 (2014). https://doi.org/10.1038/nbt.2754

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2754

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing