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Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells


Alveolar epithelial type II cells (AEC2s) are the facultative progenitors of lung alveoli and serve as the surfactant-producing cells of air-breathing organisms. Although primary human AEC2s are difficult to maintain stably in cell cultures, recent advances have facilitated the derivation of AEC2-like cells from human pluripotent stem cells (hPSCs) in vitro. Here, we provide a detailed protocol for the directed differentiation of hPSCs into self-renewing AEC2-like cells that can be maintained for up to 1 year in culture as epithelial-only spheres without the need for supporting mesenchymal feeder cells. The month-long protocol requires recapitulation of the sequence of milestones associated with in vivo development of the distal lung, beginning with differentiation of cells into anterior foregut endoderm, which is followed by their lineage specification into NKX2-1+ lung progenitors and then distal/alveolar differentiation to produce progeny that express transcripts and possess functional properties associated with AEC2s.

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Fig. 1: Overview of PSC-derived alveolosphere differentiation protocol.
Fig. 2: Representative images and flow cytometric profiles throughout PSC-derived alveolosphere differentiation protocol.
Fig. 3: Identification of new cell-surface markers for NKX2-1+ cells.
Fig. 4: Maintaining alveolospheres in long-term culture.
Fig. 5: Plating NKX2-1+ cells at lower densities in Matrigel results in improved specification of SFTPCtdTomato+ cells.
Fig. 6: Single-cell sequencing analysis of alveolospheres.
Fig. 7: Recommended sorting strategies for day 14 lung progenitors and day 30 alveolospheres.

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Data availability

This article includes weblinks to publicly available datasets, such as bioinformatics files deposited at the Gene Expression Ombinus (GEO accession number: GSE103918). There are no restrictions on data sharing, and any raw data, cell lines, or protocol downloads are available through the corresponding author by email request or through his website portal at


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We wish to thank all members of the Kotton and Hawkins laboratories for helpful discussions and optimization of the presented protocol. We are grateful to B.R. Tilton of the BUSM Flow Cytometry Core, supported by NIH grant 1UL1TR001430, for technical assistance; and G. Miller, A. Iyer, and M. James of the CReM, supported by grants R24HL123828 and U01TR001810. This work was supported by grants TL1TR001410 and F31HL134274 (A.J.), HAWKIN15XX0 and R01HL139799 (F.H.), the I.M. Rosenzweig Junior Investigator Award from the Pulmonary Fibrosis Foundation (K.-D.A.), and grants U01HL099997, U01HL134745, R01HL095993, R01HL122442, R01HL128172, and U01HL134766 (D.N.K.). We thank A. Brivanlou, Rockefeller University, for the generous gift of RUES2, an embryonic stem cell line.

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Authors and Affiliations



A.J., M.V., and D.N.K. designed and performed experiments and wrote the manuscript. D.C.T. and F.H. performed serial-dilution experiments. C.V.-M. performed bioinformatics single-cell RNA sequencing analysis. D.A.R. and K.-D.A. performed experiments demonstrating clonality.

Corresponding author

Correspondence to Darrell N. Kotton.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Amy Ryan (Firth) and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Jacob, A. et al. Cell Stem Cell 21, 472–488.e10 (2017):

McCauley, K. B. et al. Stem Cell Rep. 10, 1579–1595 (2018):

Hawkins, F. et al. J. Clin. Invest. 127, 2277–2294 (2017):

Integrated supplementary information

Supplementary Fig. 1 Genomic stability of passaged alveolospheres.

All panels show array-based Comparative Genomic Hybridization (aCGH) analysis of alveolospheres at various points in long term maintenance. a) SPC2-ST-B2 iPSC line at alveolosphere passage 2, day 100 of differentiation. b) SPC2-ST-B2 at alveolosphere passage 12, day 214 of differentiation. c) BU3-NGST iPSC line at alveolosphere passage 3 post SFTPCtdTomato sort without prior CHIR withdrawal. d) BU3-NGST at alveolosphere passage 12 post SFTPCtdTomato sort without prior CHIR withdrawal. Note: Changes in BU3-NGST passage 3 alveolospheres, as well as the CNV in the SPC2-ST passage 3 alveolospheres, were present in the starting iPSCs in both cases.

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Jacob, A., Vedaie, M., Roberts, D.A. et al. Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells. Nat Protoc 14, 3303–3332 (2019).

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