Fig. 5: Characterization and differentiation of lung AFOs.
a, Phase-contrast images depicting LAFO expansion (scale bar, 200 μm). IF staining highlights proliferative marker Ki67 (scale bar, 50 μm). b, Dot plot showing representative bulk RNA-seq gene expression in LAFOs (n = 12 independent biological samples, n = 21 undifferentiated LAFOs, n = 14 differentiated LAFOs, n = 6 control FLOs). c, IF staining highlighting the presence in LAFOs of lung stem/progenitor cell markers NKX2-1 and SOX2 together with P63 basal cells; counterstaining with phalloidin (F-ACT) (scale bars, 50 μm). d, IF quantification (n = 6 AF samples, ≥4 organoids per sample; mean ± s.e.m.). e, Annotated scRNA-seq UMAPs of LAFOs in expansion (gray; 1,966 cells, n = 4 organoid lines), proximal (top; 3,371 cells, n = 3 organoid lines) and distal differentiation (bottom; 1,351 cells, n = 3 organoid lines). f, IF staining on proximally differentiated LAFOs reveals polarized expression of ciliary protein Ac-αTUB, and ciliated cell marker FOXJ1. The panel also shows expression of basal cell markers P63, KRT5, presence of mucin 5AC goblet cells and maintenance of SOX2 progenitor cells (scale bars, 50 μm). g, Quantification of FOXJ1-positive cells within LAFOs in expansion (CT) versus proximal differentiation (DIFF) (n = 5 independent biological samples, ≥4 organoids per sample; mean ± s.e.m.; **P = 0.0016, two-tailed paired t-test). h, Violin plot showing gene expression of proximal airway markers FOXJ1, TUBA1A, SCGB1A1, MUC5AC and KRT5 in proximal LAFOs (DIFF) compared to undifferentiated controls (CT) based on the RNA-seq plot presented in b (n ≥ 7 independent biological samples; median and quartiles; SCGB1A1 ***P = 0.0003, ****P < 0.0001, Holm–Šídák multiple unpaired t-test). i, TEM images showing proximal LAFOs with cilia inside the lumen (asterisk) (scale bar, 2 μm); in cross-section, axonemes showing outer (red arrow) and inner (white arrow) dynein arms (scale bar, 100 nm). j, Distalized LAFOs showing surfactant-secreting cells (SFTPB) with granular and luminal secretion; Hoechst-counterstained nuclei (scale bars, 50 μm). k, Violin plot showing surfactant-related gene expression in distalized LAFOs (DIST DIFF) compared to control in expansion (CT) based on the RNA-seq plot presented in b (n ≥ 7 independent biological samples; median and quartiles; NS, non-significant; SFTPA1 *P = 0.0107, SFTPA2 **P = 0.0002, SFTPB *P = 0.0168, SFTPD *P = 0.0028, Holm–Šídák multiple unpaired t-test). l, Left: TEM of distalized LAFOs showing lumen (*) and cells containing lamellar bodies (red arrows). Right: magnification of lamellar body containing multi-lamellar membranes. Scale bars, 1 μm and 500 nm, respectively.
