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Journal: Cell Reports Medicine
Article Title: Small molecule-directed differentiation of submerged-cultured human nasal airway epithelia for respiratory disease modeling
doi: 10.1016/j.xcrm.2026.102692
Figure Lengend Snippet: Conversion of S-diff HNECs into 3D organoids and CFTR function measurements (A) Graphic illustration showing the workflow of generating airway organoids from epithelial fragments of S-diff HNECs, and application in forskolin-induced swelling assays. (B) Representative bright-field images of cystic airway organoids formed after 48 h from S-diff monolayer cultures. Scale bar, 250 μm. (C) Immunofluorescent (top) and bright-field (bottom) images of S-diff HNEC-derived organoids of β-tubulin IV (yellow, left top), MUC5AC (purple, middle top), p63 (red, right top), and DAPI (cyan). Scale bar, 10 μm. (D) Representative images of forskolin-induced swelling determined with calcein green AM ester-stained HC and CF (F508del/F508del) organoids, with images taken before stimulation with forskolin (t = 0, left) and 60 min after stimulation (t = 60 min, right). Scale bar, 500 μm. (E and F) Comparison of FIS between HC and CF airway organoids derived from S-diff HNECs (both n = 3 independent donors) that were unstimulated or stimulated with forskolin (Fsk). (G) CF F508del homozygous organoids derived from S-diff HNECs ( n = 3 independent donors) were pre-treated with vehicle or CFTR correctors, VX-809, or VX-661/VX-445 for 48 h. Afterward, FIS was determined following acute stimulation with Fsk, VX-770, or vehicle. (H) FIS responses in CF F508del/S1251N organoids ( n = 3 donors) following acute stimulation with VX-770. (I) Schematic overview of the comparison between FIS responses in airway organoids derived from ALI- vs. S-diff cultures. (J) Correlation plot of FIS responses between ALI-derived and S-diff-derived organoids from the same CF donors (Pearson r = 0.90, 95% confidence interval, 0.70–0.97; p < 0.0001). (K) FIS in organoids generated from HC basal cells expanded to passage 3 or 7 before S-diff and organoid formation ( n = 2 donors). FIS results are depicted as (E) the percentage change in surface area relative to t = 0 (normalized area) measured at 15-min time intervals for 60 min or (F–H and K) as area under the curve (AUC) plots (t = 60 min). Data are presented as mean ± SD and individual data point. Statistical significance was tested using a (F and I) two-way ANOVA with Dunnett’s multiple comparison test, (G and H) Tukey’s multiple comparison test, (J) Pearson correlation analysis. ns, non-significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.
Article Snippet:
Techniques: Derivative Assay, Staining, Comparison, Generated