(A) Adult human lung, tHLOs and 80d HLOs possess SOX2+ (green) epithelium marked by ECAD (white). Only adult lung airways and tHLO airway-like structures possessed a pseudostratified epithelium (ECAD, white). Scale bars represent 50 µm in low mag images and 10 µm in high mag. (B) Measurements of cell height were taken from adult human lung (n = 3), tHLO (n = 5), and HLO (n = 6) airways of cells facing toward the lumen. Averages were adult: 69.59 µm ± 1.65, tHLO: 75.69 µm ± 4.74, HLO = 36.39 µm ± 3.39. *** represents p<.0005 and error bars represent SEM. All HLOs were derived from hESC line UM63-1. (C–E) Adult human lung, tHLOs, and HLOs (65d, 80d) expressed the basal cell markers P63 (C, green), cytokeratin5 (CK5, D, green), and NGFR (E, green) Scale bars represent 10 µm. (F) 3D rendering of z-stack images revealed tube-like structures with cells lining the tube expressing the basal cell marker P63 (green) and cells within the tube cells expressing the ciliated cell marker FOXJ1 (white). A cross section of the z-stack images through the tube revealed that P63 (green) lines the tube while FOXJ1+ cells (white) are within the tube. Scale bars represent 100 µm. (G) Adult human lung, tHLO, and 80d HLO possessed ciliated cells labeled by ACTTUB (white) with the cilia facing in toward the lumen. Scale bars represent 10 µm. (H) NKX2.1+ airway-like structures within each tHLO (n = 9) that contained NKX2.1+ACTTUB+(ciliated cells), NKX2.1+CC10+ (club cells), or NKX2.1+MUC5AC+ (goblet cells) in each tHLO. Data was quantified from UM63-1 hESC-derived tHLOs transplanted for 8 weeks. Each independent tHLO counted is represented by a different color. (n = 9, error bars represent SEM).