Extended Culture of Endodermal Cells on Lung Scaffolds Promotes Differentiation to Ciliated, Club, and Basal Airway Epithelial Cells
(A) Schematic representation of ESC differentiation to airway epithelial populations.
(B and C) RT-PCR analysis reveals the expression of airway epithelial genes Foxj1, Scgb1a1, TRP63, and Cftr. Alveolar epithelial markers Aqp5, Sftpb, and Sftpc were detected at much lower levels, although Aqp5 and Sftpb increased with extended culture. Gene expression is presented relative to adult lungs, mean ± SEM, n = 4 experiments, ∗p < 0.001.
(D) IF staining and confocal microscopic analysis of day 21 cell-matrix cultures showed mature airway epithelial populations: ciliated cells (TUBB4A+), club cells (SCGB1A1+), and basal cells (KRT5+). Scale bar represents 50 μm.
(E) Cell proportions were quantified as percent of total cell population at days 7, 14, and 21, mean ± SEM, n = 3 experiments.
(F and G) Scanning EM image of the surface epithelia of day 21 cultures (F, scale bar represents 2.5 μm) displays mature tight junction-coupled (white arrowhead) ciliated and nonciliated cells with similar morphology to native mouse airways (G, scale bar represents 5 μm).
(H and I) Transmission EM analysis of day 21 tissue sections confirms the presence of tight junction-coupled (white arrowhead) ciliated cells (ci), club cells (cc), and basal cells (bc). (H) Scale bar represents 4 μm. (I) Scale bar represents 2 μm.
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