Extended Data Fig. 1. Establishment of feeder-free organoids derived from AT2 cells.

a, Schematics of experimental design for isolation of Sftpc lineage-labelled AT2 cells at indicated time points after tamoxifen treatment. b, Representative bright-field images of organoids derived from lineage-labelled Tomato⁺Sftpc⁺ cells in indicated conditions; complete medium with WNT3A, RSPO1 (R-spondin 1), EGF, FGF7, FGF10, and NOG (Noggin), withdrawal of indicated factors (–FGF10, –FGF7, or –WNT3A/RSPO1). Scale bar, 2,000μm. c, d, Statistical quantification of colony forming efficiency (c, n=5) and passaging numbers (d, n=5) of organoids. Each individual dot represents individual biological replicate and data are presented as mean and s.e.m. Statistical analysis was performed using two-tailed unpaired Student’s t test; n.s; not significant. e, Representative serial bright-field images of a lung organoid growing originated from single Tomato⁺Sftpc⁺ cells at the indicated time points. Magnifications: X20 (day 4 and 7), X10 (day10 and 13), and X4 (day 16, 20, and 30). Scale bars, 400μm. f, A representative immunofluorescence (IF) image of organoids derived from Tomato⁺Sftpc⁺ cells at the first passage under feeder-free condition with complete culture medium. SPC (for AT2 cells, red), Pdpn (for AT1 cells, green), and DAPI (blue). Scale bars, 50μm. g, Representative bright-field images of organoids under feeder-free condition with complete culture medium at passage 5. Insets (left) show high-power view (right). Scale bars, 2,000μm. h, Representative IF images of mixed organoids cultured in complete medium at passage >5. SPC (red), Hopx (white), and DAPI (blue). Scale bars, 50μm.