Extended Data Fig. 1. Characterization of AT2 organoids.

(A) Derivation and establishment of fdAT2 organoids from lung tip progenitor cells (upper panel), or proximal airway progenitors (lower panel) of human fetal lungs at 22 pcw. Upper panel: The isolated tip progenitor cells were immediately transduced and selected based on SFTPC-GFP and EF1a-TagRFP after 48 h of transduction; SFTPC-GFP and EF1a-TagRFP reporter positive cells were efficiently expanded into fdAT2 organoids when grown in AT2 medium for 3 weeks. Lower panel: Proximal airway cells were immediately transduced with SCGB3A2-GFP, EF1a-TagRFP reporter lentivirus and the airway progenitor cells were selectively isolated by SCGB3A2-GFP after 48h of transduction; SCGB3A2-GFP, EF1a-TagRFP reporter positive cells and expanded into small AT2-like organoids when grown in AT2 medium for 3 weeks, but efficiently formed airway organoids when grown in airway medium for the same period. Scale bar, 50 μm (B) Size of organoids expanded from tip progenitors (SFTPC-GFP⁺) or proximal airway progenitors (SCGB3A2-GFP⁺) in AT2 medium was measured; mean ± SD, n=50 (****p<0.0001; unpaired t-test (two-tailed)). (C) Expression of mature SFTPC protein in organoids expanded from tip progenitors or airway progenitors in AT2 medium. DAPI, nuclei. Scale bar, 50 μm. (D) Cultured fdAT2 organoids at early and late passages, stably expressing SFTPC promoter-driven GFP (SFTPC-GFP). Two independent lines of AT2 organoids at P3 and P17, and P4 and P20. Scale bars, 50 μm. (E) qRT-PCR analysis of alveolar type 2 cell lineage markers, NKX2.1, SFTPC, ABCA3, and LAMP3, in 7-9 pcw and 16-22 pcw tip progenitor organoids, and fdAT2 organoids at P12, P20, and P21. Data were normalised to 7-9 pcw tip organoids; mean ± SD, n=3 independent repeats (*p<0.05, **p<0.01, ***p<0.001; One-way ANOVA with Tukey multiple comparison post-test). (F) Immunoblot of mature forms of SFTPC and SFTPB in human fetal lung tip progenitor-derived organoids at 7-9 pcw and 16-22 pcw, respectively, and the fdAT2 organoids. Two independent replicates were used. (G - I) FdAT2 organoids were cultured in the AT2 medium for 2 weeks, in the presence (control) or absence of FGF7 (-FGF7) and AT2 lineage markers were measured by qRT-PCR (H) after 7 and 14 days of culture. Data were normalised to AT2 organoids cultured in the AT2 medium containing FGF7 (control); mean ± SD, n=3 independent repeats (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey multiple comparison post-test). (I) Mature SFTPC protein expression was visualised with a proliferation marker, KI67, and E-cadherin by immunofluorescence staining, at 14 days of culture. DAPI, nuclei. Scale bar, 50 μm.