Fig. 4

FOXI1 knock-out (KO) in hiPSCs leads to hiPSC-AEC cultures lacking ionocytes. A: FOXI1 gene targeting and differentiation strategy. Following FOXI1 KO in hiPSCs, WT and KO clones were selected from the targeted pool and differentiated in parallel towards AECs. FOXI1 KO cells were not expected to generate ionocytes. Targeted but unedited WT cells served as an isogenic control. B: Each hiPSC line was targeted with a different sgRNA (Strategy #1 for FS13B and Strategy #2 for CF17/NKX2.1-GFP), testing two different genetic backgrounds and two different targeting strategies in the same study. The diagram shows the sgRNA used on each line to target the DNA binding domain in Exon 1, the PAM region highlighted in blue and the indels in the selected KO clones in red. C: Relative mRNA expression of key markers at different time points during the first stages of differentiation. Filled circles represent individual data points and bars are means ± SD (n = 3 independent experiments); two-way ANOVA with Sidak multiple comparison test. The dotted line indicates the level of the normalized reference-gene expression average value. D: Relative mRNA expression of key mature AEC markers of cells in expansion (D0) and after maturation in ALI cultures (D28). Filled circles represent individual data points and bars are means ± SD (n = 3 independent experiments), two-way ANOVA with Sidak multiple comparison test. E: Representative immunocytochemical staining of FOXI1, CFTR and DAPI in mature hiPSC-AECs after 28 days in ALI culture in FOXI1 WT and KO cells. The scale bar is 20 μm. F: Z-stack panel with orthogonal views of FOXI1 WT cells from E. G: Cropped representative Western blot images of FOXI1 expression in WT and KO hiPSC-AECs (right panel), undifferentiated hiPSCs were used as a negative control and MCF7 cells were used as a positive control (left panel). Vinculin was used as a loading control