Abstract
Durable and functional regeneration of the airway epithelium in vivo with transplanted stem cells has the potential to reconstitute healthy tissue in diseased airways, such as in cystic fibrosis or primary ciliary dyskinesia. Here, we present detailed protocols for the preparation and culture expansion of murine primary and induced pluripotent stem cell-derived airway basal stem cells (iBCs) and methods for their intra-airway transplantation into polidocanol-conditioned murine recipients to achieve durable in vivo airway regeneration. Reconstitution of the airway tissue resident epithelial stem cell compartment of immunocompetent mice with syngeneic donor cells leverages the extensive self-renewal and multipotent differentiation properties of basal stem cells (BCs) to durably generate a broad diversity of mature airway epithelial lineages in vivo. Engrafted donor-derived cells re-establish planar cell polarity as well as functional ciliary transport. By using this same approach, human primary BCs or iBCs transplanted into NOD-SCID gamma recipient mice similarly display engraftment and multilineage airway epithelial differentiation in vivo. The time to generate mouse or human iBCs takes ~60 d, which can be reduced to ~20 d if previously differentiated cells are thawed from cryopreserved iBC archives. The tracheal conditioning regimen and cell transplantation procedure is completed in 1 d. A competent graduate student or postdoctoral trainee should be able to perform the procedures listed in this protocol.
Key points
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This protocol outlines methods for the culture of primary or pluripotent stem cell-derived basal cells in vitro, the engraftment of these cells in vivo into immunocompetent mice and similarly the xenotransplantion of human primary or pluripotent stem cell-derived basal cells into immunocompromised mouse recipients.
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The approach enables reconstitution of the in vivo airway stem cell compartment and, importantly, uses syngeneic cell transplantation into mice with normal immune function rather than into immunodeficient recipients, simulating future autologous cell-based therapy.
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Data availability
This article includes publicly available transcriptomic datasets, and the bioinformatics files have been deposited at the Gene Expression Ombinus (accession number: GSE232545). 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 our website portal at www.kottonlab.com.
Change history
31 March 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41596-025-01180-6
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Acknowledgements
We thank B. R. Tilton and the entire BUSM Flow Cytometry Core and Y. Alekseyev and the entire BUSM Microarray and Sequencing Resource Core for their technical assistance. We thank the staff of the Marsico Lung Institute Tissue Procurement and Cell Culture Core for primary human cells and media. We thank the entire Kotton and Hawkins Laboratories and the Center for Regenerative Medicine for their support and suggestions throughout the course of this research. We are indebted to G. Miller and M. James for overall laboratory support as well as for reprogramming and characterization of iPSC lines. We are also grateful to L. Ikonomou for his insight and input throughout the project. This work was supported by NHLBI Progenitor Cell Translational Consortium (PCTC) Jumpstart Award to L.M.; Boston University Kilachand Multicellular Design Program Accelerator Grants to L.M., M.J.H. and D.N.K.; NIH grants U01HL134745, U01HL134766, U01HL148692, R01HL095993 and P01HL170952 to D.N.K.; NIH grant R01HL139799 and Cystic Fibrosis Foundation Grant HAWKIN20XX2 to F.J.H.; and NIH grants R01HL124392 and R21HD094012 to X.V. Human cell biobanking and sharing were supported by NHLBI grant NO1: 75N92020C00005 to D.N.K. Schematics in figures were created with Biorender.com.
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Contributions
L.M., F.J.H. and D.N.K. conceptualized the project. L.M. and M.J.H. developed methods for mouse donor cell preparation. J.A.L.S. developed methods for human donor cell preparation. L.M. and B.R.T. developed methods for donor cell transplantation. L.M. and A.T.-L. performed recipient animal analysis in the manuscript. P.S.B., F.W. and L.M. performed bioinformatic analysis. L.M. and D.N.K. wrote the manuscript. X.V., F.J.H. and D.N.K. supervised the project.
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Key references using this protocol
Ma, L. et al. Cell Stem Cell 30, 1199–1216.e7 (2023): https://doi.org/10.1016/j.stem.2023.07.014
Herriges, M. J. et al. Cell Stem Cell 30, 1217–1234.e7 (2023): https://doi.org/10.1016/j.stem.2023.07.016
Hawkins, F. J. et al. Cell Stem Cell 28, 79–95.e8 (2021): https://doi.org/10.1016/j.stem.2020.09.017
McCauley, K. B. et al. Cell Stem Cell 20, 844–857.e6 (2017): https://doi.org/10.1016/j.stem.2017.03.001
Serra, M. et al. Development 144, 3879–3893 (2017): https://doi.org/10.1242/dev.150193
Supplementary information
Supplementary Information
Figure S1. Donor-derived cells do not alter the overall recipient airway structure or cause excessive immune influx. H&E and immunofluorescence confocal microscopy (GFP, CD45 and α-SMA) staining of an old (≥1 y after transplantation) recipient and a control animal that never had airway injury or donor cell transplantation. Both coronal and longitudinal samples are shown. Scale bar = 200 μm.
Supplementary Video 1
Video S1. Mucociliary clearance assay in iBC recipient.
Supplementary Video 2
Video S2. Mucociliary clearance bead assay control.
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Ma, L., Thapa, B.R., Le Suer, J.A. et al. Life-long functional regeneration of in vivo airway epithelium by the engraftment of airway basal stem cells. Nat Protoc 20, 810–842 (2025). https://doi.org/10.1038/s41596-024-01067-y
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DOI: https://doi.org/10.1038/s41596-024-01067-y
