Fig. 2: Endothelial co-culture enhances AEC2 phenotype in the setting of fibroblasts.

a Immunostaining for AEC1 markers RTI-40, AQP5, and AGER in d7 engineered lungs. Arrowheads, cells staining positive for AEC1 markers. b qRT-PCR of AEC1 gene expression in d7 engineered lungs; n = 4 lungs. Gene expression in native P7 and P60 rat lungs is normalized to the average expression in day 0 AEC2 isolates and shown for approximate comparison only; n = 3 lungs. c Immunostaining of AEC2 proteins in AEC2/FB and tri-culture lungs. Arrowheads indicate diffuse cytoplasmic SPB labeling in AEC2/FB co-cultured epithelium. d TEM examining epithelial ultrastructural features in engineered lungs, with a P7 native AEC2 shown for comparison. LB lamellar body, TJ tight junction. Arrowheads, apical microvilli. Arrows, irregular apical projections. Scale bars, 2 μm. Magnified regions, 1 μm. e Quantification of secreted SPB protein in bronchoalveolar lavage (BAL) of adult rat (“Native”) and tri-culture engineered lungs; n = 3 lungs. Representative quasi-static pressure-volume (PV) curves (f) with calculation of compliance (g) for native, decellularized, and tri-culture lungs; n = 3 biological replicates. Scale bars in immunofluorescence images, 25 μm. Magnified regions, 10 μm. Error bars indicate the mean ± SEM. b, e Unpaired two-tailed t-test. g One-way ANOVA with Holm–Sidak’s multiple comparisons tests. ns not significant, *P < 0.05, **P < 0.01.