Fig. 1: Characterization of differentiated alveolar organoids.

a A schematic graph outlines the generation of alveolar organoids. Photomicrographs display live lung organoids (LO) and differentiated alveolar organoids (AlvO); scale bar, 100 µm. Confocal images present LO and AlvO labeled with DAPI (blue) and Phalloidin (white); scale bar, 20 µm. See also Supplementary Figs. S1 and S2. b Parental LOs and the differentiated AlvOs were assessed for the expression level of AT1- and AT2-cell marker genes. Data represent means ± SD of a representative experiment in one organoid line, n = 2. Two-tailed unpaired Student’s t-test. See also Supplementary Figs. S3–S6. c AlvOs were subjected to immunofluorescence staining to label AQP5⁺ AT1 cells (red), SFTPB⁺ (green, top), or HTII-280⁺ (green, bottom) AT2 cells. Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar, 20 µm. d Parental LOs and differentiated AlvOs were applied to flow cytometry to examine the abundance of AQP5⁺ AT1 and SFTPC⁺/LysoTracker red⁺/HTII-280⁺ AT2 cells. Representative histograms are shown on the top. Red, cells stained with specific antibodies or dyes; blue, cells stained with isotype controls or mock-stained. Data underneath represent means ± SD of a representative experiment in one organoid line, n = 3 for AQP5 and SFTPC, n = 2 for LysoTracker red and HTII-280. Two-tailed unpaired Student’s t-test. See also Supplementary Fig. S7. e–i AlvOs were examined with transmission electron microscopy. e Cuboidal AT2 cells and thin AT1 cells in an alveolar organoid. Scale bar, 10 µm. f Microvilli on an AT2 cell. Scale bar, 2 µm. g Lamellar bodies in an AT2 cell. Scale bar, 1 µm. h A lamellar body within an AT2 cell. Scale bar, 200 nm. i An AT1 cell with thin cytoplasm. Scale bar, 10 µm. j AlvOs preincubated with BODIPY phosphatidylcholine (green) were further labeled with LysoTracker red (red) and then subjected to live-cell confocal imaging. Organoids were counterstained with Hoechst (blue) and CellMask™ plasma membrane stain (white). Scale bar, 20 µm.