Supplementary Figure 1: Efficient generation of AOs via isolated NKX2-1+ lung progenitor cells. | Nature Methods

Supplementary Figure 1: Efficient generation of AOs via isolated NKX2-1+ lung progenitor cells.

From: Long-term expansion of alveolar stem cells derived from human iPS cells in organoids

Supplementary Figure 1

(a) Optimization of the medium for expansion of NKX2-1+ ventralized anterior foregut endoderm (VAFE) cells on day 21. IBMX, 3-isobutyl-1-methylxanthine. (b) Immunofluorescence imaging of NKX2-1+ cell expansion under each of the medium conditions described in (a) on day 21 (n = 3 repeated experiments, each). Scale bar, 100 μm. (c) Induction efficiency of NKX2-1+ cells on day 21 under each of the medium conditions calculated by the averaged ratio of the NKX2-1+ cells to the total cells in 10 randomly selected fields. CFKD treatment was found to be significantly more effective than FGF10 treatment by Kruskal-Wallis test combined with Dunn’s post hoc multiple comparison test. *P < 0.05. Error bars indicate the mean ± s.e.m (n = 3 cell cultures) B2-3 hiPSC line was used. (d) Optimizing the duration of expansion of NKX2-1+ VAFE cells in CFKD medium. (e) RT-qPCR of SFTPC for each duration of NKX2-1+ VAFE cells expansion after day 14. The expression of SFTPC in AOs after one-week CFKD treatment was significantly greater than that in untreated cells by Kruskal-Wallis test combined with Dunn’s post hoc multiple comparison test. *P < 0.05. The expression of SFTPC was normalized to that of ACTB. The expression of SFTPC in human fetal lung was set at 1. Error bars indicate the mean ± s.e.m (n = 3 cell cultures, each). B2-3 hiPSC line was used. (f) A flow cytometric analysis (n = 5 repeated experiments) of the CPM-based sorting of cells into three cell populations (CPMhi, CPMlo and CPM-). Mouse IgG1 isotype was used as a negative control. H9 ESC line was used. (g) Immunofluorescence images of NKX2-1+ cells of cytospin samples in sorted CPMhi, CPMlo and CPM- cells (n = 3 repeated experiments). H9 ESC line was used. Scale bar, 100 μm. (h) The ratio of NKX2-1+ cells in cells sorted based on CPM in multiple PSC lines. Error bars indicate the mean ± s.e.m (n = 3 cell cultures). The ratio of NKX2.1+ cells in CPMhi cells was found to be significantly greater than that in CPM- cells by Friedman test combined with Dunn’s post hoc multiple comparison test. *P < 0.05. (i) A histogram of the accumulation of LysoTracker Red in SFTPC+ cells in AOs. Numbers indicate the mean ± s.e.m. (n = 3 independent organoids). (j) A gating strategy for flow cytometric analysis of AOs. (k, l) Flow cytometry analysis (k) and graphs (l) of induction efficiency of SFTPC+ cells forming AOs cocultured with various fibroblasts. Numbers and error bars indicate the mean ± s.e.m (n = 5 independent organoids, each). (m) An immunofluorescent image of AOs stained with anti-GFP (SFTPC+ cells), anti-EpCAM (epithelial cells), and anti-CD31 (endothelial cells) (n = 3 repeated experiments). Scale bar, 25 μm.

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