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A distal lung organoid model to study interstitial lung disease, viral infection and human lung development

Nature Protocols volume 18, pages 2283–2312 (2023)Cite this article

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Abstract

Organoids have been an exciting advancement in stem cell research. Here we describe a strategy for directed differentiation of human pluripotent stem cells into distal lung organoids. This protocol recapitulates lung development by sequentially specifying human pluripotent stem cells to definitive endoderm, anterior foregut endoderm, ventral anterior foregut endoderm, lung bud organoids and finally lung organoids. The organoids take ~40 d to generate and can be maintained more than 180 d, while progressively maturing up to a stage consistent with the second trimester of human gestation. They are unique because of their branching morphology, the near absence of non-lung endodermal lineages, presence of mesenchyme and capacity to recapitulate interstitial lung diseases. This protocol can be performed by anyone familiar with cell culture techniques, is conducted in serum-free conditions and does not require lineage-specific reporters or enrichment steps. We also provide a protocol for the generation of single-cell suspensions for single-cell RNA sequencing.

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Fig. 1: Overview of the protocol.
Fig. 2: FBS-containing media does not maintain organoid cultures.
Fig. 3: Maturation of organoids and selection of suspension organoids for embedding.
Fig. 4: Morphology of lung organoids embedded in Col I gel.
Fig. 5: IF analysis of organoids at different stages.
Fig. 6: RT–qPCR analysis of the core and tip areas of a lung organoid.
Fig. 7: Integrated scRNAseq analysis.
Fig. 8: Merged scRNAseq analysis.
Fig. 9: Troubleshooting of LBO formation.

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Data availability

scRNAseq data are available in the Gene Expression Omnibus database (Gene Expression Omnibus Submission (GSE215825), access code Urihimewhncrbql). The RNAseq datasets that support the findings of this study are from ref. 20, and available from the Sequence Read Archive. The Sequence Read Archive accession number for d25 LBOs sequencing is SRP073749 and SRR4295269 for d170 LBOs. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants NIH HL120046 (H.-W.S.), NIH 1U01HL134760 (H.-W.S.), NIH S10 OD032447 (H.-W.S.), NIH 5T32HL105323 (R.T.S., principal investigator: J. Bhattacharya) and the Parker B. Francis Fellowship (Y.-W.C.), and by the Thomas R. Kully IPF Research Fund.

Author information

Author notes

  1. These authors contributed equally: Ivana Matkovic Leko, Remy T. Schneider, Tania A. Thimraj.

  2. These authors jointly supervised this work: Ya-Wen Chen, Hans-Willem Snoeck.

Authors and Affiliations

  1. Columbia Center for Human Development/Center for Stem Cell Therapies, Columbia University Irving Medical Center, New York, NY, USA

    Ivana Matkovic Leko, Remy T. Schneider, Tania A. Thimraj, Daniel Beitler, Hsiao-Yun Liu & Hans-Willem Snoeck

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

    Ivana Matkovic Leko, Remy T. Schneider, Tania A. Thimraj, Daniel Beitler, Hsiao-Yun Liu & Hans-Willem Snoeck

  3. Department of Genetic and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Nadine Schrode & Kristin Beaumont

  4. The Center for Advanced Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Nadine Schrode & Kristin Beaumont

  5. Department of Otolaryngology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Ya-Wen Chen

  6. Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Ya-Wen Chen

  7. Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Ya-Wen Chen

  8. Institute for Airway Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Ya-Wen Chen

  9. Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, USA

    Hans-Willem Snoeck

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Contributions

Y.-W.C. originally developed the protocol and co-wrote the manuscript with H.-W.S., I.M.L. and R.T.S. played a key role in troubleshooting and perfecting the protocol. R.T.S. generated the images in Fig. 5. T.A.T. analyzed scRNAseq in collaboration with N.S. and K.B. who performed the scRNAseq, D.B. provided technical assistance, H.-Y.L. helped with optimizing and troubleshooting the protocol.

Corresponding authors

Correspondence to Ya-Wen Chen or Hans-Willem Snoeck.

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Competing interests

H.-W.S. and Y.-W.C. hold patents pertaining to the lung organoid technologies described. The other authors declare no competing interests.

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Nature Protocols thanks Shuibing Chen, Robert Hynds and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Chen, Y. W. et al. Nat Cell Biol. 19, 542–549 (2017): https://doi.org/10.1038/ncb3510

Porotto, M. et al. mBio 10, e00723-19 (2019): https://doi.org/10.1128/mBio.00723-19

Strikoudis, A. et al. Cell Rep. 27, 3709–3723.e5 (2019): https://doi.org/10.1016/j.celrep.2019.05.077

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Source data

Source Data Figs. 3a and 6

Raw data and statistical analysis.

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Matkovic Leko, I., Schneider, R.T., Thimraj, T.A. et al. A distal lung organoid model to study interstitial lung disease, viral infection and human lung development. Nat Protoc 18, 2283–2312 (2023). https://doi.org/10.1038/s41596-023-00827-6

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