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The in vitro multilineage differentiation and maturation of lung and airway cells from human pluripotent stem cell–derived lung progenitors in 3D

Nature Protocols volume 16, pages 1802–1829 (2021)Cite this article

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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 mature lung and airway epithelial cells obtained through maturation of NKX2.1+ hPSC-derived lung progenitors in a 3D matrix of collagen I in the absence of glycogen synthase kinase 3 inhibition. This protocol is an extension of our previously published protocol on the directed differentiation of lung and airway epithelium from hPSCs that modifies the technique and offers additional applications. This protocol is conducted in defined media conditions, has a duration of 50–80 d, does not require reporter lines and results in cultures containing mature alveolar type II and I cells as well as airway basal, ciliated, club and neuroendocrine cells. We also present a flow cytometry strategy to assess maturation in the cultures. Several of these populations, including mature NGFR+ basal cells, can be prospectively isolated by cell sorting and expanded for further investigation.

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Fig. 1: Differentiation of hPSCs to AFE.
Fig. 2: Differentiation of hPSCs to LPs.
Fig. 3: Practical aspects of the culture protocol.
Fig. 4: Differentiation of hESC-derived LPs to airway and distal cells at day 50 of the differentiation protocol.
Fig. 5: Differentiation of hiPSC-derived LPs to airway and distal cells at day 50 of the differentiation protocol.
Fig. 6: Extension of the maturation period yields more mature basal cells.
Fig. 7: Expression of mature lung and airway markers in cultures carried to day 80.
Fig. 8: Assessment of the culture by flow cytometry.
Fig. 9: Applications of the differentiation protocol.

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Data availability

The RNA sequencing and single-cell RNA sequencing data are deposited in GEO under accession number GSE101558. Example flow cytometry data sets have been uploaded to the flow cytometry repository (http://flowrepository.org) under the following repository IDs: FR-FCM-Z2VH, FR-FCM-Z2VM, FR-FCM-Z2VN, FR-FCM-Z3Z6 and FR-FCM-Z3Z7. Raw data supporting the findings presented in the figures are available in the accompanying source data files.

References

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

    Article  CAS  PubMed  Google Scholar 

  2. 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  PubMed  PubMed Central  Google Scholar 

  3. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. Firth, A. L. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Konishi, S. et al. Directed induction of functional multi-ciliated cells in proximal airway epithelial spheroids from human pluripotent stem cells. Stem Cell Rep. 6, 18–25 (2016).

    Article  CAS  Google Scholar 

  9. Chen, Y.-W. et al. A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat. Cell Biol. 19, 542–549 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. McCauley, K. B. et al. Efficient derivation of functional human airway epithelium from pluripotent stem cells via temporal regulation of Wnt signaling. Cell Stem Cell 20, 844–857.e6 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Jacob, A. et al. Differentiation of human pluripotent stem cells into functional lung alveolar epithelial cells. Cell Stem Cell 21, 472–488.e10 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yamamoto, Y. et al. Long-term expansion of alveolar stem cells derived from human iPS cells in organoids. Nat. Methods 14, 1097–1106 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. de Carvalho, A. L. R. T. et al. Glycogen synthase kinase 3 induces multilineage maturation of human pluripotent stem cell-derived lung progenitors in 3D culture. Development 146, dev171652 (2019).

    PubMed  PubMed Central  Google Scholar 

  14. Dye, B. R. et al. In vitro generation of human pluripotent stem cell derived lung organoids. eLife 4, e05098 (2015).

    Article  PubMed Central  Google Scholar 

  15. Miller, A. J. et al. Generation of lung organoids from human pluripotent stem cells in vitro. Nat. Protoc. 14, 518–540 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dye, B. R., Miller, A. J. & Spence, J. R. How to grow a lung: applying principles of developmental biology to generate lung lineages from human pluripotent stem cells. Curr. Pathobiol. Rep. 4, 47–57 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Hawkins, F. & Kotton, D. N. Embryonic and induced pluripotent stem cells for lung regeneration. Ann. Am. Thorac. Soc. 12, S50–S53 (2015).

    Article  PubMed  Google Scholar 

  18. Swarr, D. T. & Morrisey, E. E. Lung endoderm morphogenesis: gasping for form and function. Annu. Rev. Cell Dev. Biol. 31, 553–573 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Huang, S. X. L. et al. The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells. Nat. Protoc. 10, 413–425 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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  PubMed  Google Scholar 

  21. Hawkins, F. et al. Prospective isolation of NKX2-1–expressing human lung progenitors derived from pluripotent stem cells. J. Clin. Invest 127, 2277–2294 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Strikoudis, A. et al. Modeling of fibrotic lung disease using 3D organoids derived from human pluripotent stem cells. Cell Rep. 27, 3709–3723.e5 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dye, B. R. et al. A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids. eLife 5, e19732 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Butler, C. R. et al. Rapid expansion of human epithelial stem cells suitable for airway tissue engineering. Am. J. Respir. Crit. Care Med 194, 156–168 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ramani, S., Crawford, S. E., Blutt, S. E. & Estes, M. K. Human organoid cultures: transformative new tools for human virus studies. Curr. Opin. Virol. 29, 79–86 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ciancanelli, M. J. et al. Infectious disease. Life-threatening influenza and impaired interferon amplification in human IRF7 deficiency. Science 348, 448–453 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rafeeq, M. M. & Murad, H. A. S. Cystic fibrosis: current therapeutic targets and future approaches. J. Transl. Med. 15, 84 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Kaur, A., Mathai, S. K. & Schwartz, D. A. Genetics in idiopathic pulmonary fibrosis pathogenesis, prognosis, and treatment. Front. Med 4, 154 (2017).

    Article  Google Scholar 

  29. 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  PubMed  PubMed Central  Google Scholar 

  30. Kim, C. F. B. et al. Identification of bronchioalveolar stem cells in normal lung and lung. cancer Cell 121, 823–835 (2005).

    CAS  Google Scholar 

  31. 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  PubMed  Google Scholar 

  32. Chen, H. J. et al. Generation of pulmonary neuroendocrine cells and SCLC-like tumors from human embryonic stem cells. J. Exp. Med 216, 674–687 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chen, Z. et al. Genetic engineering of human embryonic stem cells for precise cell fate tracing during human lineage development. Stem Cell Rep. 11, 1257–1271 (2018).

    Article  CAS  Google Scholar 

  34. An, W. F. et al. Discovery of Potent and Highly Selective Inhibitors of GSK3b (National Center for Biotechnology Information, 2010).

  35. 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  PubMed  Google Scholar 

  36. Hynds, R. E., Butler, C. R., Janes, S. M. & Giangreco, A. Expansion of human airway basal stem cells and their differentiation as 3D tracheospheres. Methods Mol. Biol. 1576, 43–53 (2019).

    Article  CAS  PubMed  Google Scholar 

  37. Gonzalez, R. F., Allen, L., Gonzales, L., Ballard, P. L. & Dobbs, L. G. HTII-280, a biomarker specific to the apical plasma membrane of human lung alveolar type II cells. J. Histochem. Cytochem. 58, 891–901 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Dobbs, L. G., Gonzalez, R. F., Allen, L. & Froh, D. K. HTI 56, an integral membrane protein specific to human alveolar type I cells. J. Histochem. Cytochem 47, 129–137 (1999).

    Article  CAS  PubMed  Google Scholar 

  40. Jarrard, J. A. et al. MUC1 is a novel marker for the type II pneumocyte lineage during lung carcinogenesis. Cancer Res 58, 5582–5589 (1998).

    CAS  PubMed  Google Scholar 

  41. Ramirez, M. I. et al. T1α, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolus formation at birth. Dev. Biol. 256, 62–73 (2003).

    Article  Google Scholar 

  42. Rock, J. R. et al. Notch-dependent differentiation of adult airway basal stem cells. Cell Stem Cell 8, 639–648 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kim, K. C. & Lillehoj, E. P. MUC1 mucin: a peacemaker in the lung. Am. J. Respir. Cell Mol. Biol. 39, 644–647 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. McCauley, K. B. et al. Single-cell transcriptomic profiling of pluripotent stem cell-derived SCGB3A2+ airway epithelium. Stem Cell Rep. 10, 1579–1595 (2018).

    Article  CAS  Google Scholar 

  45. Roost, M. S. et al. KeyGenes, a tool to probe tissue differentiation using a human fetal transcriptional atlas. Stem Cell Rep. 4, 1112–1124 (2015).

    Article  CAS  Google Scholar 

  46. Lindahl, M., Ståhlbom, B. & Tagesson, C. Newly identified proteins in human nasal and bronchoalveolar lavage fluids: potential biomedical and clinical applications. Electrophoresis 20, 3670–3676 (1999).

    Article  CAS  PubMed  Google Scholar 

  47. Porotto, M. et al. Authentic modeling of human respiratory virus infection in human pluripotent stem cell-derived lung organoids. mBio 10, e00723-19 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Branche, A. & Falsey, A. Parainfluenza virus infection. Semin. Respir. Crit. Care Med 37, 538–554 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Henrickson, K. J. Parainfluenza viruses. Clin. Microbiol. Rev. 16, 242–264 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Schomacker, H., Schaap-Nutt, A., Collins, P. L. & Schmidt, A. C. Pathogenesis of acute respiratory illness caused by human parainfluenza viruses. Curr. Opin. Virol. 2, 294–299 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Zhang, L. et al. Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium. J. Virol. 79, 1113–1124 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the US National Institutes of Health grants HL120046 and 1U01HL134760 (to H.-W.S.) and AI31971 (to A.M.), the Thomas R. Kully IPF Research Fund (to H.-W.S.) and the Fundação para a Ciência e a Tecnologia (fellowship PD/BD/52320/2013 to A.L.R.T.d.C.). Flow cytometry was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056.

Author information

Authors and Affiliations

  1. Columbia Center for Human Development, Columbia University Medical Center, New York, NY, USA

    Ana Luisa Rodrigues Toste de Carvalho, Hsiao-Yun Liu, Ya-Wen Chen & Hans-Willem Snoeck

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

    Ana Luisa Rodrigues Toste de Carvalho, Hsiao-Yun Liu, Ya-Wen Chen & Hans-Willem Snoeck

  3. Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

    Ana Luisa Rodrigues Toste de Carvalho

  4. ICVS/3B’s, PT Government Associate Laboratory, Guimarães, Portugal

    Ana Luisa Rodrigues Toste de Carvalho

  5. Department of Pediatrics, Columbia University Medical Center, New York, NY, USA

    Matteo Porotto & Anne Moscona

  6. Center for Host–Pathogen Interaction, Columbia University Medical Center, New York, NY, USA

    Matteo Porotto & Anne Moscona

  7. Department of Experimental Medicine, University of Campania ‘Luigi Vanvitelli’, Caserta, Italy

    Matteo Porotto

  8. Department of Microbiology & Immunology, Columbia University Medical Center, New York, NY, USA

    Anne Moscona

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

    Anne Moscona

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

    Hans-Willem Snoeck

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Contributions

A.L.R.T.d.C. developed the lung maturation protocol, contributed to the concept and co-wrote the manuscript. Y.-W.C. and H.-Y.L. contributed to the development of the protocol. A.M. and M.P. generated and provided virology reagents, contributed to the design and provided instructions for HPIV infection experiments. H.-W.S. developed the concept, contributed to protocol development and co-wrote the manuscript with A.L.R.T.d.C.

Corresponding author

Correspondence to Hans-Willem Snoeck.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

de Carvalho, A. L. R. T. et al. Development 146, dev171652 (2019): https://doi.org/10.1242/DEV.171652

Huang, S. X. L. et al. Nat. Biotechnol. 32, 84–91 (2014): https://doi.org/10.1038/nbt.2754

This protocol is an extension to: Nat. Protoc. 10, 413–425 (2015): https://doi.org/10.1038/nprot.2015.023

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Supplementary Tables 1–3 and Supplementary Methods.

Reporting Summary

Supplementary Data 1

Source data for Supplementary Table 2 (flow cytometry) and Supplementary Table 3 (flow cytometry).

Source data

Source Data Fig. 8

RT-aPCR data for Fig. 8d.

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Rodrigues Toste de Carvalho, A.L., Liu, HY., Chen, YW. et al. The in vitro multilineage differentiation and maturation of lung and airway cells from human pluripotent stem cell–derived lung progenitors in 3D. Nat Protoc 16, 1802–1829 (2021). https://doi.org/10.1038/s41596-020-00476-z

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