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Generation of lung organoids from human pluripotent stem cells in vitro


The lung epithelium is derived from the endodermal germ layer, which undergoes a complex series of endoderm–mesoderm-mediated signaling events to generate the final arborized network of conducting airways (bronchi, bronchioles) and gas-exchanging units (alveoli). These stages include endoderm induction, anterior–posterior and dorsal–ventral patterning, lung specification, lung budding, branching morphogenesis, and, finally, maturation. Here we describe a protocol that recapitulates several of these milestones in order to differentiate human pluripotent stem cells (hPSCs) into ventral–anterior foregut spheroids and further into two distinct types of organoids: human lung organoids and bud tip progenitor organoids. The resulting human lung organoids possess cell types and structures that resemble the bronchi/bronchioles of the developing human airway surrounded by lung mesenchyme and cells expressing alveolar-cell markers. The bud tip progenitor organoids possess a population of highly proliferative multipotent cells with in vitro multilineage differentiation potential and in vivo engraftment potential. Human lung organoids can be generated from hPSCs in 50–85 d, and bud tip progenitor organoids can be generated in 22 d. The two hPSC-derived models presented here have been benchmarked with human fetal tissue and found to be representative of human fetal-like tissue. The bud tip progenitor organoids are thus ideal for exploring epithelial fate decisions, while the human lung organoids can be used to model epithelial–mesenchymal cross-talk during human lung development. In addition to their applications in developmental biology, human lung organoids and bud tip progenitor organoids may be implemented in regenerative medicine, tissue engineering, and pharmaceutical safety and efficacy testing.

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Fig. 1: Schematic of protocol and timeline.
Fig. 2: Applications of the protocol.
Fig. 3: Human pluripotent stem cell splitting and directed differentiation.
Fig. 4: Common errors and troubleshooting.
Fig. 5: Expected outcomes of the protocol.

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Data availability

Some of the data presented in the current study were generated for and published in previous reports. Original data used for figures in this paper are available at the following links: Fig. 2b–d (Dye et al.16),; Figs. 2e and 5g–j (Dye et al.15),; Figs. 2g–i and 5l–r (Miller et al.17),


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Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under award number R01HL119215 to J.R.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations



A.J.M., B.R.D. and J.R.S. conceived the studies. A.J.M., B.R.D., D.F.-T., D.R.H. and A.W.O. performed the experiments. A.J.M., B.R.D., D.F.-T., A.W.O., L.D.S. and J.R.S. analyzed the results. A.J.M. and J.R.S. wrote the manuscript. A.J.M., B.R.D., D.F.-T., A.W.O., D.R.H., L.D.S. and J.R.S. read and edited the manuscript.

Corresponding author

Correspondence to Jason R. Spence.

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Competing interests

J.R.S., B.R.D. and A.J.M. hold patents pertaining to the lung organoid technologies described. The other authors declare no competing interests.

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Key references using this protocol

Miller, A. J. et al. Stem Cell Rep. 10, 101–119 (2018):

Dye, B. R. et al. eLife 5, e19732 (2016):

Dye, B. R. et al. eLife 4, e05098 (2015):

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Miller, A.J., Dye, B.R., Ferrer-Torres, D. et al. Generation of lung organoids from human pluripotent stem cells in vitro. Nat Protoc 14, 518–540 (2019).

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