Extended Data Fig. 3: Expression differences and localization of lung cell states and canonical epithelial and endothelial subtypes.

a, Proliferative signature score (based on expression of indicated genes in cells from 10x dataset; cell numbers are in Supplementary Table 2) of each cluster of basal cells, T and natural killer cells, and macrophages. Three clusters had high scores: proliferating basal cells (Bas-p), proliferating natural killer/T cells (NK/T-p), and proliferating macrophages. b, Dot plot of mean level of expression (dot intensity, grey scale) of indicated basal cell markers and percent of cells in population with detected expression (dot size) for 10x dataset. Note partial overlap of markers among different basal populations. c, Immunostaining of adult human pseudostratified airway for differentiation marker HES1 (green) in basal cells (marked by KRT5, red) with DAPI (nuclear) counter stain (blue). Scale bars, 10 μm. Note apical processes extending from HES1⁺ basal cells (arrowheads) indicating migration away from basal lamina as they differentiate. Other HES1⁺ cells have turned off basal marker KRT5. Dashed outlines, basal cell nuclei. Quantification shows fraction of basal cells (cuboidal KRT5⁺ cells on basement membrane) and differentiating basal (Bas-d) cells (KRT5⁺ cells with apical processes) that were HES1⁺. n denotes KRT5⁺ cells scored in sections of two human lungs with staining repeated on four participants. d, Immunostaining of adult human pseudostratified airway for proliferation marker MKI67 (green) in basal cells (marked by KRT5, red) with DAPI counter stain (blue). Scale bars, 5 μm. Quantification shows abundance of proliferating (MKI67-expressing) basal cells in pseudostratified (pseudo) and simple epithelial airways; n denotes KRT5⁺ cells scored in sections of two human lungs with staining repeated on four participants. e, Relative abundance of epithelial and stromal cell types in scRNA-seq analysis of human lung samples obtained from proximal (blue; 10x cells from P3) and distal (red; 10x cells from D1a, D1b, D2, D3) lung sites. In addition to the expected proximal enrichment of some airway cell types (goblet cells, ionocytes, neuroendocrine cells) and distal enrichment of alveolar cell types (AT1, AT2, AT2-signalling, myofibroblasts), note three bracketed pairs of related cell types (ciliated and proximal ciliated; basal and proximal basal (Bas-px) cells; myofibroblasts and fibromyocytes) with one of them proximally enriched. Relative enrichment values are provisional because they can be influenced by efficiency of collection during cell dissociation and isolation. Cell number for proximal cells are (from left to right): 357, 275, 73, 175, 153, 191, 39, 145, 57, 24, 20, 10, 328, 1,505, 235, 25 and 70; and for distal cells are: 537, 806, 15, 197, 4, 58, 6, 14, 336, 0, 2, 1, 467, 2,095, 434, 198 and 28. f, RNAscope smFISH and quantification for general basal marker KRT5 (red) and proximal basal cell marker SERPINB3 (white) with DAPI counter stain (blue) and ECM autofluorescence (green) on proximal, pseudostratified bronchi and distal, simple bronchioles. Scale bars, 20 μm (inset, 10 μm). Note enrichment of proximal basal cells (KRT5 SERPINB3 double positive, yellow arrowhead and box) enrichment at base of pseudostratified airways. SERPINB3 was not detected in simple airways, indicating that basal cells (but not proximal basal cells) are present there. Staining repeated on two participants. g, Dot plot of expression in ciliated and proximal ciliated cells of canonical (general) ciliated cell markers and specific proximal ciliated markers (in 10x dataset). h, smFISH and quantification of human pseudostratified epithelial (left) and simple epithelial (right) airways for general ciliated marker C20orf85 (white) and proximal ciliated marker DHRS9 (red) with DAPI counterstain (blue) and ECM autofluorescence (green). Note restriction of proximal ciliated cells to pseudostratified airways. Scale bars, 10 μm. Staining repeated on two particpants. i, Heat map of expression of representative general AT2, AT2 selective, and AT2-signalling selective marker genes in AT2 and AT2-signalling human lung cells (SS2 data). AT2 selective markers include negative regulators of Hedgehog and Wnt signalling pathways (for example, HHIP and WIF1, highlighted red) and AT2-signalling selective markers include Wnt ligands, receptors and transcription factors (for example, WNT5A, LRP5 and TFC7L2 highlighted green). Values shown are ln(CPM + 1) for 50 randomly selected cells in each cluster (SS2 data). j, Dot plot of expression of endothelial markers (10x dataset). k, Micrograph (low magnification, left) of bronchial vessel (boxed region) showing vessel location near airway (dotted outline). smFISH for general endothelial marker CLDN5 (red, centre), bronchial vessel-specific markers MYC (green) and Bro1-specific marker ACKR1 (red, right) on serial sections of bronchial vessel cells (arrowheads), co-stained for DAPI (blue). Scale bar, 10 μm. Quantification shows relative abundance of Bro1 and Bro2 cells. Staining repeated on two participants. l–n, smFISH and quantification of vessel types indicated (dotted outlines) showing vein marker ACKR1 (red; l), artery marker GJA5 (red; m), lymphatic marker CCL21 (red; n), and general endothelial marker CLDN5 with DAPI counter stain (blue) and ECM autofluorescence (green). Scale bars, 50 μm (l), 30 μm (m) and 40 μm (n). Staining repeated on two participants. For more details on statistics and reproducibility, see Methods.

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