Fig. 1

hiPSCs differentiate into lung progenitors in 16 days. A: Diagram of the differentiation protocol, fluorescence activated cell sorting, expansion in 3D organoids and maturation at an ALI to form hiPSC-AECs. Abbreviations: AFE, anterior foregut endoderm; BMP4, bone morphogenetic protein 4; CHIR, CHIR-99021; DAPT, (2 S)-N-[2(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl-glycine 1,1-Dimethylethyl ester; DE, definitive endoderm; FGF7/10, fibroblast growth factor 7/10; IBMX, 3-isobutyl-1-methylxanthine; LP, lung progenitor; PALI, PneumaCult™-ALI Medium; RA, retinoic acid; SB, SB431542; Y, Y-27632. B: Relative mRNA expression of key markers at different time points during differentiation. The control (CTL) is human trachea total mRNA. Filled circles represent individual values and columns are means ± SD (n = 4 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. D0; one-way ANOVA with Tukey’s post-test. C: Brightfield image of cells after 16 days of differentiation. The scale bar is 1000 μm. D: Immunofluorescence staining showing lung progenitor marker NKX2.1 expression (red) and nuclear marker DAPI (blue) on day 16 of differentiation. The scale bar is 100 μm. E: Flow cytometry panel showing levels of expression of CPM at day 16 of differentiation (red). The population labelled CPM + was sorted for enrichment of NKX2.1-expressing lung progenitors. HiPSCs stained with anti-CPM antibody served as a negative control (blue). F: Enrichment of NKX2.1 mRNA expression after sorting of CPM + cells. Filled circles represent individual values and columns are means ± SD (n = 5 independent experiments). **P < 0.01 vs. pre-sorted; Student’s t-test