Fig. 2: Single-cell composition and epithelial-mesenchymal interactions in alveolar organoids over time course of differentiation.
A UMAP of all cell populations combining d14, d21 and d28 AEP-derived organoid scRNAseq datasets. B Cell population proportions at each time point with increasing proportion of epithelial cells. C Heatmap showing expression of top 10 most differentially expressed genes in each population D, E Ligand-receptor analysis of organoid culture demonstrating extensive mesenchymal-epithelial communication in organoids. F Schematic of experimental set-up of live imaging and 3D reconstruction of live day 20 organoids generated using PDGFRαEGFP fibroblasts stained with Hoechst, with data shown in (F, G). F 3D reconstruction of confocal z-stacks of whole wells including Transwell filter, showing the majority of GFP+ fibroblasts are growing on the filter; F’–F”’ Whole-mount immunofluorescence showing lack of PDGFRα+ cells within day 20 organoids, with scattered cells found throughout the surrounding Matrigel. G Whole-mount IHC showing few GFP+ fibroblasts inside of organoids. H CD45 staining of organoids; see also Supplementary Fig. S3I. Data in (F–H) represents 2 biological replicates with 3 technical replicates per experiment. [Scale bars = 50 µm]; (PDGFRα = platelet-derived growth factor receptor alpha, GFP = Green Fluorescent Protein; RAGE = Receptor for Advanced Glycation End-products [AT1 cell marker]; SFTPC = Surfactant Protein C [AT2 cell marker]); AEP = alveolar epithelial progenitor, pAEP = proliferative AEP, AT2tr = AT2 transitional cell, AT2 = alveolar type 2 cell, AT1tr = AT1 transitional cell, AT1 = alveolar type 1 cell, Krt8 = Krt8/DATP/PATS-like transitional cell, Ifn = Interferon responsive alveolar cell, pMes = proliferative mesenchymal cell, AlvFB1 = alveolar fibroblast type1, AlvFB2 = alveolar fibroblast type 2, SM = smooth-muscle like mesenchyme. Schematics created with Biorender.com.
