(a) The self-renewal rate of FD-SFTPC+ cells in FD-AOs. A representative flow cytometric analysis is shown. P1, Passage number 1. “P1 FD-AO” indicates AOs derived from P0 FD-SFTPC+ cells. The values were calculated as the ratio of SFTPC+ cells to EpCAM+ cells. Error bars indicate the mean ± s.e.m (n = 3 independent organoids, each). (b) Linear increase in the population doubling level (PDL) of FD-SFTPC+ cells was observed. PDL was calculated as log10 (N/N0) x 3.33. N is the number of EpCAM+ cells. N0 is the number of SFTPC+ cells and was fixed at 1.0 x 104 in the present study. Linear regression analysis with Pearson’s correlation coefficient was used to examine relationship between the increase in the culture period and PDL. r, Pearson’s correlation coefficient. Error bars indicate the mean ± s.e.m (n = 3 independent organoids, each). (c) Normal karyotypes of P0 and P5 FD-SFTPC+ cells. (d) Confocal immunofluorescence imaging of P3 FD-AOs (n = 5 collected images). Scale bar, 25 μm. (e) Confocal immunofluorescence imaging of SFTPC-PDPN+AQP5+ AT1-like cells in P3 FD-AOs (n = 5 collected images). Scale bar, 25 μm. (f) Flow cytometric analysis of FD-AOs for the quantification of maturation of lamellar bodies with calculation of the mean signal intensity of LysoTracker. Error bars indicate the mean ± s.e.m (n = 3 independent organoids, each). *P < 0.05, Kruskal-Wallis test combined with Dunn’s post hoc multiple comparison test. B2-3 hiPSC line was used for all of the experiments.