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Human distal lung maps and lineage hierarchies reveal a bipotent progenitor

Nature volume 604, pages 111–119 (2022)Cite this article

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Abstract

Mapping the spatial distribution and molecular identity of constituent cells is essential for understanding tissue dynamics in health and disease. We lack a comprehensive map of human distal airways, including the terminal and respiratory bronchioles (TRBs), which are implicated in respiratory diseases1,2,3,4. Here, using spatial transcriptomics and single-cell profiling of microdissected distal airways, we identify molecularly distinct TRB cell types that have not—to our knowledge—been previously characterized. These include airway-associated LGR5+ fibroblasts and TRB-specific alveolar type-0 (AT0) cells and TRB secretory cells (TRB-SCs). Connectome maps and organoid-based co-cultures reveal that LGR5+ fibroblasts form a signalling hub in the airway niche. AT0 cells and TRB-SCs are conserved in primates and emerge dynamically during human lung development. Using a non-human primate model of lung injury, together with human organoids and tissue specimens, we show that alveolar type-2 cells in regenerating lungs transiently acquire an AT0 state from which they can differentiate into either alveolar type-1 cells or TRB-SCs. This differentiation programme is distinct from that identified in the mouse lung5,6,7. Our study also reveals mechanisms that drive the differentiation of the bipotent AT0 cell state into normal or pathological states. In sum, our findings revise human lung cell maps and lineage trajectories, and implicate an epithelial transitional state in primate lung regeneration and disease.

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Fig. 1: Spatial and single-cell transcriptomics reveal distinct epithelial cell types in distal airways.
Fig. 2: LGR5 marks a unique fibroblast population in airways.
Fig. 3: Dynamics of AT0 and unique cell trajectories in human alveolar epithelium.
Fig. 4: Revised lineage hierarchies in primate alveolar regeneration and disease.

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Data availability

scRNA-seq data generated in this study are available from the Gene Expression Omnibus (GEO) under accession code GSE178360 (human airways) and GSE178519 (organoids). Spatial transcriptomics data generated in this study are available under accession code GSE178361. snRNA-seq data from monkey lungs45 are available at the GSA (accession code CRA002577). Human lung scRNA-seq datasets from Travaglini et al.11 are available through Synapse (https://www.synapse.org/#!Synapse:syn21041850); Habermann et al.12 from the GEO (accession code GSE135893); Deprez et al.23 through the UCSC Cell Browser (and also in the European Genome-Phenome Archive; https://www.ebi.ac.uk/ega/datasets/EGAD00001005714); and Miller et al.28 on the EMBL-EBI Array Express database (accession code E-MTAB-8221). Source data are provided with this paper.

Code availability

All analyses performed in this study used pre-existing packages mentioned in the Methods. No custom code was generated.

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Acknowledgements

We thank B. Hogan for advice and critical reading of the manuscript; members of the P.R.T. laboratory for discussions; the Human Cell Atlas community for discussions and feedback; and members of the S.H.R. laboratory at UNC Chapel Hill for providing human tissues (supported by grant BOUCHER19R0 from the CF Foundation and NIH grant DK065988). We acknowledge support from the Duke University Molecular Genomics Core Facility (for help with Visium experiments); and from the Duke BRPC shared resource (for providing lung tissue specimens). V.S. is supported by a fellowship from Regeneration Next initiative at Duke University. Y.K. was supported by a fellowship from Japan Society for the Promotion of Science Overseas Research. Human tissue procurement was supported by funding from the CF Foundation (BOUCHER19R0) and the NIH (DK065988). J.R.S. is supported by grant CZF2019-002440 from the Chan Zuckerberg Initiative DAF, an advised fund of the Silicon Valley Community Foundation, and by the National Heart, Lung and Blood Institute (NHLBI; R01HL119215). We also thank I. Glass and the University of Washington Laboratory of Developmental Biology, which is supported by NIH award 5R24HD000836 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). This work was supported by funds from the NIH/NHLBI (R01 HL127002, U01HL134766) to J.R.R.; the NIH (P51OD011107) to L.A.M.; the NIH (R01 DK047967-30) to J.F.E.; a Pathways to Independence award from the NHLBI/NIH (R00HL127181, R01HL146557 and R01HL153375); a NCI/NIH P30 Cancer Center Support Grant (P30CA014236); funds from the Chan Zuckerberg Initiative–Human Lung Cell Atlas pilot award to P.R.T.; and funds from Regeneration NeXT and Kaganov–MEDx Pulmonary Initiative to P.R.T. at Duke University. This work was partially supported by funds from the Whitehead foundation and P.R.T. is a Whitehead Scholar at Duke University.

Author information

Author notes

  1. These authors contributed equally: Preetish Kadur Lakshminarasimha Murthy, Vishwaraj Sontake

Authors and Affiliations

  1. Department of Cell Biology, Duke University School of Medicine, Durham, NC, USA

    Preetish Kadur Lakshminarasimha Murthy, Vishwaraj Sontake, Aleksandra Tata, Yoshihiko Kobayashi, Lauren Macadlo & Purushothama Rao Tata

  2. Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Kenichi Okuda, Satoko Nakano & Richard C. Boucher

  3. Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, USA

    Ansley S. Conchola

  4. Department of Neurology, Duke University Medical Center, Durham, NC, USA

    Simon Gregory

  5. California National Primate Research Center, Davis, CA, USA

    Lisa A. Miller

  6. Department of Anatomy, Physiology and Cell biology, School of Veterinary Medicine, University of California, Davis, CA, USA

    Lisa A. Miller

  7. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA

    Jason R. Spence

  8. Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    John F. Engelhardt

  9. Department of Anatomy & Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    John F. Engelhardt

  10. Department of Immunology Discovery, Genentech, South San Francisco, CA, USA

    Jason R. Rock

  11. Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Scott H. Randell

  12. Duke Cancer Institute, Duke University School of Medicine, Durham, NC, USA

    Purushothama Rao Tata

  13. Duke Regeneration Center, Duke University School of Medicine, Durham, NC, USA

    Purushothama Rao Tata

  14. Division of Pulmonary Critical Care, Department of Medicine, Duke University School of Medicine, Durham, NC, USA

    Purushothama Rao Tata

  15. Center for Advanced Genomic Technologies, Duke University, Durham, NC, USA

    Purushothama Rao Tata

  16. Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan

    Yoshihiko Kobayashi

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  1. Preetish Kadur Lakshminarasimha Murthy
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Contributions

P.K.L.M. co-designed and performed scRNA-seq, Visium, RNAScope, qRT–PCR, plasmid cloning, FACS and non-human primate, human fetal, pediatric and adult lung characterization experiments, analysed data, performed computational analysis and co-wrote the manuscript; V.S. co-designed and performed airway dissociation, plasmid cloning, FACS, qRT–PCR, organoid culture and basal and fibroblast co-culture experiments, performed immunostaining on lung tissues from patients with acute lung injury, COPD and IPF and co-wrote the manuscript. A.T. performed microdissection and dissociation of airways, performed immunostaining on human (healthy and IPF) and non-human primate lung tissues and co-wrote the manuscript. Y.K. performed scRNA-seq and RNA-FISH (PLISH) experiments and assisted with computational analysis. L.M. assisted with human tissue processing, sectioning of human and monkey lungs and immunostaining. K.O. performed RNAScope experiments. K.O. and S.N. performed cilia length measurements. A.S.C. and J.R.S. provided fetal lung samples. S.G. supervised tissue optimization for Visium experiments. L.A.M. and J.R.R. supervised bleomycin-induced injury in non-human primates and provided samples. J.F.E. provided insights into distal airway anatomy across species. R.C.B. supervised RNAScope experiments. S.H.R. provided human lung tissues. P.R.T. co-designed, conceived and supervised the work and co-wrote the manuscript. All authors reviewed and edited the manuscript.

Corresponding author

Correspondence to Purushothama Rao Tata.

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Competing interests

P.R.T. serves as a consultant for Surrozen, Cellarity and Celldom on work not related to the contents of this manuscript. J.R.R. is an employee of Genentech. The other authors declare no competing interests.

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Nature thanks Qing Nie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Spatial transcriptomics of cartilaginous airway and distal lung.

a. Heat map shows differentially expressed genes in each cluster of ST assay from proximal airway. b. ST spots show expression of indicated genes from proximal airway. c. Heat map indicates differentially expressed genes in each cluster of ST assay from the distal lung. d. ST spots indicate the expression of indicated genes from the distal lung.

Extended Data Fig. 2 scRNA-seq of human distal airways and alveoli.

a. UMAPs show the expression of cell class specific marker genes. b. Dot plot indicates expression of cell type specific markers. c. UMAP shows the annotation of cell types from blood (c), endothelial (d), and mesenchymal (e) lineages. f. H&E reference sections and ST spots show the expression of RNASE1 in proximal (top) and distal airway (bottom). g. ST spots show the expression of MUC5AC, SFTPB, MUC5B and SCGB3A2 in proximal and distal airways as indicated. h. PLISH for RNASE1 (grey), MUC5B (green) and SFTPB (red) in proximal (top) and distal airways (bottom). blue (DAPI) indicates nuclei. Scale bar: 50 µm. i. Visualization of indicated epithelial cell transcriptomic signatures on ST tissue section from distal lung.

Extended Data Fig. 3 Molecularly distinct cell types and zones in human airways.

a. Immunostaining for MUC5B (blue), MUC5AC (red), SCGB1A1 (green) and SCGB3A2 (grey) (bottom panel). Lines illustrate described zones. Scale bar: 100 µm. b. Quantification of distal airway epithelial cell types across the airway zones. Data are presented as mean ± SEM. (n = 4 for zone-1 and n = 3 for zones-2, 3 and 4). c. Co-staining for SCGB3A2 (green), SCGB1A1 (red), SFTPB (grey) in different regions of the human airway. Scale bar: 100 µm. d. Staining for SCGB1A1 (blue), MUC5B (green), SFTPB (red) and SCGB3A2 (grey) in different regions of the airway. Scale bar: 100 µm (main panel) and 20 µm (inset). e. Box plot showing the difference in cell populations identified in this study (red bar) and Habermann et al., (turquoise bar). Line in the box: median; box edges: 25/75 percentile; whiskers: 1.5 times interquartile range; points: outliers. f. Dot plot shows expression of indicated genes in TRB-epithelial cells (this study) but not in Deprez et al., scRNA-seq data from airway epithelial cells (annotated as distal airway). In panels c and d, images were stitched to show large area. Insets show zoomed images of regions highlighted in boxes. g. UMAP plots show the expression of indicated genes in epithelial cells. h. UMAP plots show the expression of indicated genes in epithelial cells. i. (top) Immunostaining for SCGB3A2 (green), SCGB1A1 (grey) and NAPSA (red) showing the expression of NAPSA in TRB-SC and TB-SC. Scale bar: 10 and 20 µm. (bottom) RNA-FISH for SCGB3A2 (green), TMEM45A (red) and SCGB1A1 (grey) showing the expression of TMEM45A in TRB-SC and TB-SC. blue (DAPI) indicates nuclei. Scale bar: 20 µm. j. PLISH for SCGB3A2 (green), SFTPB (red) and KDR (grey). Scale bar 50 µm. k. UMAPs show the expression of indicated genes in epithelial cell types. l. Bar plot showing the pathways enriched in TRB-SCs.

Source Data

Extended Data Fig. 4 Gene expression signatures and pathway analysis of AT0s, distal-BCs and SCGB3A2-CCs.

a. Volcano plot shows differential gene expression between TRB-SC and AT0s. b. Volcano plot shows differential gene expression between AT2 and AT0s. c. Immunostaining for ACTA2 (green), SFTPB (red) and SCGB3A2 (grey) (left and top right) showing rare AT0s found in alveolar septae in healthy lungs. Immunostaining for SFTPB (green), SFTPC (red) and SCGB3A2 (grey) (bottom right) showing alveolar sacs. blue (DAPI) indicates nuclei. Scale bar: 50 and 20 µm. d. UMAP plot and immunostaining for SOX2 (green), SFTPC (red) show the expression of SOX2 in AT0s near respiratory bronchioles. Scale bar: 50 µm. Inset: individual channels of regions indicated by a black box. e. Violin plots show expression patterns of genes encoding indicated transcription factors in TRB-SC, AT0, and AT2s. f. Heat map shows expression of indicated genes in deuterosomal, ciliated, SCGB3A2-CC and TRB-SC cell populations. (Scale shows z-score). g. Volcano plot shows differential gene expression between SCGB3A2-CC and deuterosomal cell populations. h. Quantification of SCGB3A2-CC cell type across the airway zones. Data are presented as mean ± SEM (n = 4 for zones-1, 2 and 4; n = 3 for zone-3). i. Heat map shows expression of indicated genes in basal, distal-BC-1 and distal-BC-2 cell populations. (rows were hierarchically clustered and scale shows z-score). j. Violin plot shows the expression of indicated genes in basal cell populations. k. Violin plot demonstrates the expression of indicated genes in different basal cell populations. l. Quantification of distal basal cell types across the airway zones. Data are presented as mean ± SEM (n = 4 for zone-1, n = 5 for zone-2, n = 3 for zone-3 and n = 4 for zone-4). m. Volcano plot shows differential gene expression between IPF basaloid (from Habermann et al.,) and distal-BC-2 cell populations. n and o. UMAPs show the expression of TP63, SFTPB and FOXJ1 and SCGB3A2 in distal, intermediate and proximal airway cells profiled in Deprez et al.

Source Data

Extended Data Fig. 5 Transcriptional regulation of TRB-specific epithelial cells.

a. Heat map showing the enrichment of transcription factor regulons in epithelial cell types. b. Dot plots indicate the expression of transcription factors in epithelial cell types.

Extended Data Fig. 6 Molecular and phenotypic differences of basal cells isolated from proximal and distal airways.

a. Schematic depicting the location of airway for sampling the basal cells (top, proximal airways and bottom, distal airways) for organoid cultures and ALI cultures. b. Representative basal cell organoid images of proximal and distal airways. c. Quantification of the colony formation efficiency (CFE). Data are presented as mean ± SEM. NS (p = 0.5462) (unpaired two-tailed t-test) (n = 3). d. SFTPB (green), TP63 (red) and CK5 expression (grey) on 2D cultures. blue (DAPI) indicates nuclei. Scale bar 50 µm. e. Representative whole mount immunostaining image for SCGB3A2 (green), SCGB1A1 (red) and SFTPB (red) expression in basal cell ALI culture of proximal and distal airways at day 28.blue (DAPI) indicates nuclei. Scale bar: 20 µm. f. Relative mRNA expression of TP63, SFTPB, KRT5 and RNASE1 in basal cell cultures. Data are presented as mean ± SEM. (** p = 0.0073 for TP63), (* p = 0.0490 for KRT5), (* p = 0.0130 for SFTPB) and (*** p = 0.0006 for RNASE1) (unpaired two-tailed t-test) (n = 3, distal airway; n = 4, proximal airway, SFTPB mRNA was not detected in two biological replicates of proximal airway). g. Relative mRNA expression of TP63, SFTPB, RNASE1, SCGB1A1, SCGB3A2, and FOXJ1 in basal cell ALI culture of proximal and distal airways at day 28. Data are presented as mean ± SEM. (* p = 0.0312 for TP63), (*** p = 0.0003 for SFTPB), (** p = 0.0028 for RNASE1), (* p = 0.0234 for SCGB3A2), (** p = 0.0071 for SCGB1A1), and (NS p = 0.75 for FOXJ1) (unpaired two-tailed t-test with Welch’s correction) (n = 5, distal airway; n = 4, proximal airway). h. Representative histology images showing the difference in ciliary length in proximal and distal airways. Scale bar: 10 µm. i. Representative histology images showing the difference in ciliary length in basal cell ALI culture proximal and distal airways at day 28. Scale bar: 10 µm. j. Quantification of cilia length and cilia beating frequency in basal cell ALI culture of proximal and distal airways. Data are presented as mean ± SEM. * (p = 0.0359) (unpaired two-tailed t-test) (n = 6).

Source Data

Extended Data Fig. 7 Transcriptional and signalling activity of newly identified mesenchymal cells.

a. ST spots show the expression of FGF14 and WNT2. b. Representative RNA-FISH images showing the expression of SFTPB (green), LGR5 (red) and PDGFRA (grey) (left) and SCGB3A2 (green), LGR5 (red) and PDGFRA (grey) (right) and Scale bar: 50 µm and 20 µm. DAPI stains nuclei (blue). Insets show magnified regions. c. Quantification of LGR5+ fibroblasts in large and distal airway. Data are presented as mean ± SEM. NS (p = 0.3152) (unpaired two-tailed t-test) (n = 3) d. Heat map showing net outgoing and incoming signals in respective cell types as predicted by the CellChat algorithm. e. FGF and PDGF signalling activity from (top panel) and towards (bottom panel) LGR5+ cells and their interaction with epithelial cells as predicted using the CellChat algorithm. f. Heat map shows the enrichment of transcription factor regulons in mesenchymal cell types. g. Heat map shows LGR5+ fibroblast specific transcription factor expression pattern in mesenchymal cell types (scale shows z-score).

Source Data

Extended Data Fig. 8 LGR5-mRFP reporter validation and co-culture.

a. Schematic depicting pLKO-LGR5-mRFP reporter construct. b. Representative gating for FACS sorting of LGR5-mRFP reporter fibroblasts used for organoid co-cultures. c. RNA-FISH for LGR5 (left), and PDGFRα (right) on LGR5-mRFP fibroblasts. d. Relative mRNA expression of LGR5, WNT5A, WNT2, CA3, and FGF7 in cultured LGR5-mRFP fibroblasts vs alveolar fibroblasts. Data are presented as mean ± SEM. (** p = 0.0055 for LGR5), (* p = 0.0187 for WNT5A), (NS p = 0.0972 for CA3), (NS p = 0.1216 for WNT2), and (NS p = 0.1525 for FGF7) (unpaired two-tailed t-test with Welch’s correction) (n = 3). e. Immunostaining for SFTPB (green), TP63 (red) and KRT5 (grey) in basal cell organoid co-cultured with LGR5-mRFP fibroblasts or alveolar fibroblasts. blue (DAPI) indicates nuclei. Scale bar: 50 µm. f. Relative mRNA expression of SCGB3A2 and SOX2 in AT2s co-cultured with LGR5-mRFP fibroblasts. Data are presented as mean ± SEM. NS p = 0.2383, and *p = 0.0383 and (unpaired two-tailed t-test with Welch’s correction) (n = 3).

Extended Data Fig. 9 Expression pattern of markers of distal airway cells in fetal and postnatal human airways.

a. UMAP shows expression pattern of indicated genes in fetal lung epithelial scRNA-seq data from Miller et al. b. Schematic representation of human fetal airway and distal tip bud. c. Immunostaining for SCGB3A2 (green) and SFTPB (grey) on 19-20 weeks old human fetal lung. Scale bar 100 µm. Insets show magnified regions. d. Immunostaining for SCGB3A2 (green), TP63 (red) and SFTPB (grey) in human fetal extrapulmonary airway. Scale bar: 100 µm. e. Representative RNA-FISH images showing the expression of SCGB3A2 (green), LGR5 (red) and PDGFRA (grey). Scale bar: 50 µm. DAPI stains nuclei (blue). f. Schematic representation of human postnatal lung showing TRBs and distal alveoli. g. Co-staining for SCGB3A2 (green) and SFTPB (grey) in 7-month-old postnatal human lung. Scale bar 100 µm. Insets show magnified regions. h. Immunostaining for SCGB1A1 (blue), SCGB3A2 (grey) and SFTPB (green) expression in postnatal human lung. Scale bar: 100 µm. i. Co-staining for SCGB3A2 (green), TP63 (red) and SFTPB (grey) expression in 7–12 months old postnatal lung. Scale bar: 100 µm. j–m. Representative immunostaining images from 7 months old postnatal human lung show the expression of (j) KRT5 (green), TP63 (red) and SFTPB (grey). Scale bars: 20 µm (top), 10 µm (bottom left) and 5 µm (bottom right). (k) SFTPB (green), FOXJ1 (red) and SCGB3A2 (grey). Scale bar: 10 µm. (l) SCGB3A2 (green) and SFTPB (red). Scale bar 10 µm. (m) SCGB3A2 (green) and SFTPC (red). Scale bar 20 µm. DAPI stains nuclei (blue). c, e–m: Insets indicate individual colour channels of regions marked in white boxes.

Extended Data Fig. 10 Computational prediction of AT2 cellular transition dynamics.

a. PAGA graph of AT0, AT1, Immature AT1, AT2 and TRB-SC cells from healthy adult human lung. b. Cellular trajectory on AT0, AT1, Immature AT1, AT2 and TRB-SC cells as inferred by scVelo and PAGA algorithms. c. Cellular trajectory (left) and cells ordered along pseudotime (right) as inferred by Monocle 3 algorithm on of AT0, AT1, Immature AT1, AT2 and TRB-SC cells. d. Overall energy landscape view of AT0, AT1, Immature AT1, AT2 and TRB-SC cells as predicted by scEpath algorithm. e. MuTrans analysis of AT0, AT1, Immature AT1, AT2 and TRB-SC cells. Plots showing (from left to right) entropy, SFTPC level and SCGB3A2 level. f. Heat map showing expression of genes along trajectory from AT2 to AT1 predicted by Slingshot algorithm. Genes enriched in AT0s are highlighted by the black outline. g. Line plots showing smoothened expression (Loess smoothened) of select genes along Slingshot predicted AT2 to AT1 trajectory. Grey indicates 95% confidence interval derived from the smoothened fit. h. Line plots showing smoothened expression (Loess smoothened) of select genes along Slingshot predicted AT2 to TRB-SC trajectory. Grey indicates 95% confidence interval derived from the smoothened fit.

Extended Data Fig. 11 Organoid models reveal cell trajectories in human lungs.

a. Schematic depicting experimental workflow for signalling modulation in organoid cultures. b. Representative immunofluorescence images show expression of SCGB3A2 (green), SFTPB (red), and SFTPC (grey) in organoids cultured in CHIR, FGF10, SB43152 and EGF depletion conditions for 21 days. Scale bar: 20 µm. c. Staining for SOX2 (green), SFTPB (red), and SFTPC (grey) in control and EGF depleted organoids. Scale bar: 20 µm. d. Staining for SFTPC (green), SFTPB (red), and SCGB3A2 (grey) expression in organoid cultures treated with Erlotinib (2 µm) at day 7. Scale bar: 20 µm. e. Relative mRNA expression of SFTPC, and SCGB3A2 in AT2 cultures treated with indicated small molecules for 7 days. Data are presented as mean ± SEM. *** (p = 0.0005 for GSK690693), NS (p = 0.6722 for BAY73-4506), NS (p = 0.2167 for PD0325901), NS (p = 0.1279 for SP600125) for SFTPC. * (p = 0.0133 for GSK690693), ** (p = 0.0081 for BAY73-4506), *** (p = 0.0003 for PD0325901), NS (p = 0.0940 for SP600125) for SCGB3A2. (Dunnett’s multiple comparisons test) (n = 3). f. (left) Schematic depicting the experimental workflow to study AT2 to AT1 differentiation. (right) Immunostaining for SCGB3A2 (green) and HTI-56 (red), and SFTPB (grey) on organoids cultured in ADM for 6 days. Scale bar: 20 µm. DAPI stains nuclei (blue). g. Representative gating for FACS sorting of AT0s based on HTI-56 levels. h. Immunostaining for SCGB3A2 (green) and HTI-56 (red), and SFTPC (grey) on AT0s cultured in ADM for 7 days.

Extended Data Fig. 12 scRNA-seq on human lung organoids from different treatments and AT0 sorting.

a. Schematic showing experimental workflow for organoid scRNA-seq. b. UMAPs show expression pattern of indicated genes in specific cell populations as revealed by scRNA-seq from different culture conditions. c. Scatterplot showing the relative expression of genes in AT0s when compared to AT2s in organoid cultures (EGF depletion or serum addition) or in vivo. Linear regression is shown by the black line and Pearson correlation is shown with 95% confidence interval indicated by grey colour.

Extended Data Fig. 13 TRB regions of non-human primates.

a. Immunostaining for SFTPB (green) and SCGB3A2 (red) in terminal and respiratory bronchioles of Rhesus macaque. Scale bar 100 µm. Images from adjacent regions were aligned to show large area. b. Representative immunofluorescence images from terminal and respiratory bronchioles of Rhesus macaque show the expression of KRT5 (green), TP63 (red) and SFTPB (grey). Scale bar 12.5 µm (left and right) and 15 µm (middle). c. Quantification of AT0s in TRB regions in control and bleomycin treated lungs. Data are presented as mean ± SEM (p = 0.496) (unpaired two-tailed t-test) (n = 3). d. Immunostaining for SCGB3A2 (green), SFTPB (red), and SFTPC (grey) in COPD lung. Scale bar: 50 µm. (left). e. Staining for SCGB3A2 (green), ACTA2 (red), and SFTPC (grey) in mild fibrotic (left) and bronchiolized region (right) of the IPF lung. Scale bar: 50 µm. f. Staining for SOX2 (green) and SFTPC (red) in mild fibrotic (left) and bronchiolized region (right) of the IPF lung. Scale bar: 50 µm. Maximum intensity projections of z-stacks are shown. g. Staining for SCGB3A2 (green), KI67 (red) and SFTPC (grey) in healthy human lung and in acute lung injury. Scale bar: 50 µm. h. Quantification of proliferating (Ki67+) cells in control and acute lung injury lungs. Data are presented as mean ± SEM (n = 3). In panels – d-g, insets indicate individual colour channels of regions marked in white boxes.

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Supplementary information

Supplementary Figure 1

FACS gating strategy. a, Gating strategy to sort LGR5-mRFP cells. b, Gating strategy to sort alveolar fibroblasts. Right panel shows unstained control. c, Gating strategy to sort AT0 cells from serum treated AT2 cultures.

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Supplementary Table 1

Enriched markers in all cell clusters. Each tab contains a cell class. Non-parametric Wilcoxon rank-sum test was used with Bonferroni correction for multiple comparisons.

Supplementary Table 2

Human tissue metadata. Age, gender, smoking history, existing lung disease, cause of death, tissue use and enzymes used for dissociation are provided.

Supplementary Table 3

Probe sequences used for PLISH. Probe names and gene-specific and detection-probe-specific sequences are provided.

Supplementary Table 4

qPCR primers and LGR5 promoter PCR primers. Primer names and forward and reverse sequences are provided.

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Kadur Lakshminarasimha Murthy, P., Sontake, V., Tata, A. et al. Human distal lung maps and lineage hierarchies reveal a bipotent progenitor. Nature 604, 111–119 (2022). https://doi.org/10.1038/s41586-022-04541-3

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