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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.

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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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