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. 2023 Jan 16;25(2):351–365. doi: 10.1038/s41556-022-01064-x

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 4 — (a) Balloon-plot of known mesenchymal markers (COL1A2-COL14A1), together with the top-5 cluster markers of the mesenchymal dataset (17 donors). General: COL1A2², TBX4¹⁵, immature: RSPO2¹¹⁷, Smooth Muscle (SM): TAGLN, ACTA2², Chondroblast: COL2A1, SOX9, SOX6^118,119, Pericyte: PDGFRB¹⁰⁵, Mesothelial: WT1¹²⁰, MSLN¹²¹, Proliferating: MKI67¹¹⁴, PCNA¹²², Lipofibroblast: APOE, FST, PLIN2², Adventitial-fibroblast: SERPINF1, SFRP2², Alveolar-fibroblast: GPC3, SPINT2², Myofibroblast: ASPN, WIF1², Fibromyocyte: SCX, LGR6², COL13A1^pos-fibroblast: COL13A1³¹ and COL14A1^pos-fibroblast: COL14A1³¹. From the differentially expressed genes (adjusted p-value < 0.001, MAST, Bonferroni corrected), the top-10 (log2 fold-change) were sorted according to proportion of positive cells in the cluster and the top-5 of these were plotted. (b) Stereoscope assigned distribution of (i) mesechymal1 (cl-0), (ii) mesenchymal2 (cl-2) and (iii) mesenchymal5 (cl-6) cells in three timepoints. Red numbers: the highest percent of the indicated cell-state. Dark red: high, gray: zero. H&E staining: tissue structure. Scale-bar: 400 µm. (c) scVelo-analysis, using a dataset subset (441 cells/cluster) from all donors. Arrow direction: future state, arrow size: transition possibility. (d) HybISS analysis of a 5 pcw lung section showing the mesothelial marker WT1 mRNA expression in tissue periphery^120,121 (top) and the prediction of mesothelial-cell spatial distribution, according to PciSeq (bottom). Representative data in: https://hdca-sweden.scilifelab.se/tissues-overview/lung/ (e) Immunofluorescence for α-SMA (cyan, SM), Ecad (magenta, epithelium) and MKI67 (yellow, proliferating cells) on 8.5 (left), 12 (middle) and 14 (right) pcw lungs, in proximal-large (top), stalk (middle) and distal (bottom) airways. Nuclei (blue, DAPI). Scale-bars: 50 µm. (f) scVelo-analysis of the proliferation (cl-20) and maturation (cl-12 and −13) airway SM-trajectories. Colors as in ‘B’. (g) Balloon-plot of ACTA2 and TAGLN (SM), COL9A1, MATN2, FBLN7, FBN2 and FBN3 (extracellular matrix) and MKI67 and PCNA (proliferation). In Balloon-plots, size: percent of positives. Color intensity: scaled expression. Blue: high, Gray: low. ‘arw’: airway, ‘prox.’: proximal, ‘tr’: trachea, br-v bundle: bronchovascular bundle.