Fig. 2
From: In vitro platform to model the function of ionocytes in the human airway epithelium
ALI culture induces differentiation towards mature AECs with similar properties to HBECs. A: Brightfield image of lung progenitors in 3D organoid culture. The scale bar is 250 μm. B: Schematic of ALI culture. Organoids were dissociated and cells seeded in Transwell® inserts and cultured with medium on both sides. Once cells were confluent, medium from the top compartment was removed to form an ALI with the apical membrane of cells in contact with air. DAPT was added to the maturation medium in the bottom compartment and the cells cultured for a further 14 days, followed by another 14 days of culture with PALI medium. C: Brightfield image of hiPSC-AECs in a Transwell® insert after establishing an ALI. The scale bar is 500 μm. D: mRNA expression of AEC markers in cells in expansion conditions (D0) and in ALI culture (D28). ALI cultured HBECs were used as a control. Filled circles represent individual values and columns are means ± SD (n = 3 independent experiments); *P < 0.05 vs. D0; one-way ANOVA with Tukey’s post-test. E: Immunocytochemical analysis of mature AEC markers in ALI cultures. The scale bars are 50–100 μm as indicated. F: Immunofluorescence staining of a histological section through an ALI culture showing polarization of the airway epithelium. Cilia at the apical side are labelled with Acetylated tubulin (AcTub) and mature basal cells on the basal side are labelled with CK5. G: Rt measurements of polarized epithelia formed by hiPSC-AECs. Filled circles represent the average of three readings of the same sample and columns are means ± SD (n = 3 independent experiments). H: Ciliary beat frequency (CBF) measurements in hiPSC-AECs and HBECs. Filled circles represent the average of 20 FOVs from one sample and columns are means ± SD (n = 3 independent experiments); Student’s t-test. I: Area covered by cilia in hiPSC-AECs ALI cultures is significantly smaller compared to HBECs. Filled circles represent the average of 20 FOVs from one sample and columns are means ± SD (n = 3 independent experiments); **P < 0.01; Student’s t-test