Self-renewing human fetal lung bud tip progenitor organoids differentiate at air-liquid interface to alveolar type I- and II-like cells.Figure 2A) Immunofluorescent staining of fetal lung bud tip progenitor organoids grown in expansion medium co-expressing stem cell markers Sox2 (green) and Sox9 (red). Phalloidin (white) was used to stain actin. Data information: Nuclei are stained with Hoechst B) Immunofluorescent staining of rare HTII-280+ type II pneumocyctes (green) in a subpopulation of fetal lung bud tip progenitor organoids grown in expansion medium. Data information: Nuclei are stained with Hoechst C) Schematic of the 2D air-liquid interface bronchioalveolar-like model. Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express alveolar type I (HOPX, green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express HTI-56, green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express type II cells (HTII-280, green markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express LPCAT1, green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express SFTPC (SPC), green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express basal cell (TP63 (P63), green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm.