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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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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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.
The authors declare no competing interests.
Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.
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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
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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