Spatial mapping of phenotypic CD34⁺ HSPCs in the lung. (A) Immunofluorescence imaging of putative HSCs (Lin⁻/CD34⁺/CD90⁺) in the human lung and BM. Left panel: representative section of lung showing a Lin⁻/CD34⁺/CD90⁺ in the interstitial space. Right panel: representative section of BM showing 2 Lin⁻/CD34⁺/CD90⁺ cells. (B) Spatial transcriptomics analysis workflow. smFISH was performed to visualize gene expression in human lung tissue. Transcripts were assigned to individual cells after cell segmentation and cells were annotated based on marker gene expression (supplemental Figure 10A-D). HSPC candidate cells were computationally identified based on their gene signature and visually validated (supplemental Figure 10E-F; supplemental Figure 11A-B). (C) Representative image of a putative HSPC in its pulmonary niche. Upper panel (left to right): DAPI staining, QuPath segmentation, zoom on putative HSC (arrow). Selected transcripts are shown. Lower panel (left to right): all transcripts, pseudocoloring of cell types in the lung tissue based on marker clustering (supplemental Figure 10). Zoom on putative HSPC in niche. Scale bar, 250 μm. (D) Anatomic location of candidate cells in the lung. Representative images of phenotypic HSPCs in 4 major locations (alveolar interstitium, peribronchial, perivascular, or intravascular) and proportion of cells in each location. Scale bar, 150 μm. (E) Squidpy co-occurrence score computed every 2 μm between putative HSPCs and the rest of the clusters across lung tissue sections from 4 organ donors. High score values indicate greater co-occurrence probability; endothelial cells (red) co-occur with the HSPCs at short distances. (F) Pie graphs showing the proportion of neighboring cells within a radius of 20 μm from the putative HSPCs in the major anatomic locations. Alv, alveolar space; br, bronchus; DAPI, 4′,6-diamidino-2-phenylindole; smFISH, single-molecule fluorescence in situ hybridization; vasc, vasculature.