Fig. 1: AEP-derived alveolar organoids clonally expand and pattern complex, polarized alveolar-like cavities.

A Schematic of experimental design and overview. Live/CD31⁻/CD45⁻/CD326⁺(EpCAM⁺)/TdTomato⁺(Axin2⁺) cells (AEPs) were mixed with mouse lung fibroblasts from P28 mice and cultured for up to 35 days, followed by analysis via high content imaging. B H&E of 5 µm sections of FFPE day 35 Axin2⁺ organoids, showing cellular morphologies typical of both AT1 and AT2 cells. C–G Whole-mount immunofluorescence time course of Axin2⁺ organoids showing expansion of SFTPC⁺ AT2 cells (red), increased differentiation into RAGE⁺ AT1 cells (green) and increased structural complexity. H Imaris 3D reconstruction of day 35 Axin2⁺ organoid (z-depth = 174.13 µm) showing cellular arrangement/organization within mature organoids. I Click-iT EdU (green) whole-mount day 25 Axin2⁺ organoids, with proliferating cells primarily on outer edges or ‘buds’ growing outward from the organoid. J, K Electron microscopy of day 28 organoids. J Image of properly polarized AT2 cell with apical microvilli (black arrowhead) secreting surfactant (blue arrowhead) into a lumen. K Image of AT2 cell with lamellar bodies (black arrowhead) adjacent to an AT1 cell (green arrowhead, right). L, M Comparison of in vivo mouse lung (9-month C57BL/6J mouse) and in vitro day 25 Axin2⁺ organoids. Data throughout the figure represents at least 5 biological replicates with 3 technical replicates per experiment. [Scale bars = 50 µm, except for electron microscopy (J, K) scale bars = 2.5 µm]. (RAGE Receptor for Advanced Glycation End-products [AT1 cell marker], SFTPC Surfactant Protein C [AT2 cell marker], EdU 5-ethynyl-2’-deoxyuridine, FFPE formalin-fixed, paraffin-embedded). Schematics created with Biorender.com.