Fig 2: Mesenchyme-rich conditions promote the emergence of secretory-alveolar intermediates, while Sox9+ stressed progenitors emerge across all engineered systems(A and B) Immunofluorescence staining of native adult rat lung reveals rare epithelial progenitor and secretory populations. (A) Sox9+ cells (cyan) are observed near the bronchoalveolar duct junction (BADJ), occasionally co-expressing Scgb1a1 (red). Pdpn (green) marks alveolar type I (ATI) cells. (B) Scgb3a2+ secretory cells are localized to the conducting airways. Arrowheads highlight individual positive cells.(C) UMAP of the integrated day 10 organoid dataset highlights transcriptionally distinct transitional populations, including aRAS/AT0-like, stressed progenitor (Sox9+), and canonical secretory subsets.(D) Boxplot quantifying the percentage of Sox9+ cells (threshold: Sox9 expression >0.1) across five conditions (PD, BAL, PD_3D, Mixed_3D, BAL_3D). Sox9+ cells are rare in the starting populations but expand markedly after 10 days of culture in all conditions. Each point represents an individual biological replicate. Statistical comparisons were performed using two-proportion Z-tests on raw Sox9+ counts, corrected using the Benjamini-Hochberg method (∗∗∗p < 0.001).(E) Scaled heatmap shows differential gene expression across transitional epithelial subtypes. aRAS/AT0-like cells (Scgb3a2+/Scgb1a1+/Sftpc+) are distinct from stressed progenitors, which express stress-associated markers (Cox4i2, Ptges, Stc1) alongside the progenitor marker Sox9.(F) Violin plots show the expression of signature markers distinguishing canonical secretory and activated RAS-like epithelial populations, split by condition.(G) Boxplot shows the percentage of epithelial cells per replicate positive for a RAS-like signature (Scgb1a1+/Scgb3a2+/Sftpc+) across engineered conditions. Signature positivity was determined using scaled expression thresholds: Scgb1a1 > 2, Scgb3a2 > −2.5, and Sftpc > −1.5. Triple-positive cells were observed across all three conditions. Statistical comparison via a Kruskal-Wallis test revealed no significant differences in overall prominence; however, the PD_3D system exhibited greater replicate variability, as demonstrated by the wider interquartile range.(H–J) Immunofluorescence staining of day 10 organoids reveals Sox9+ epithelial cells (cyan) variably distributed across all conditions. Scale bars, 100 μm.(K and L) Immunofluorescence staining of day 10 organoids (PD_3D and Mixed_3D) shows co-expression of Scgb3a2 (green) and Sftpc (magenta) (RAS-like cells). Scale bars, 100 μm (K) and 50 μm (L).

Fig 3: Trajectory analysis reveals dynamic gene programs in alveolar epithelial cellsa, Monocle3 UMAP plot of epithelial cell types; 6 quantiles are used to represent the cell states from AT2 (~Q1, Q2) to Alveolar Transitional (~Q3, Q4) to AT1 (~Q5, Q6). b, Heatmap of relative allelic effect size of significant dynamic eQTLs which fit linear, quadratic, or both models. c-e, Violin plots, where the center line denotes the median, while the 25th and 75th percentile is marked as the lower and upper line of the box, respectively. Whiskers extend 1.5 times from the 25th and 75th percentiles, outliers are represented as dots. The violin width reflects the density. c, Association of normalized and scaled gene expression with their significant interaction eQTL SNPs through the cellular states (6 quantiles). Three representative genes fitting a linear (SCGB3A2, FAM13A, AKR1C1) model of allelic effect change are shown. Chromosome and position of the variants are based on hg38. 0 = reference allele, while 1 = alternative allele. d, Top plot is scaled Regulon activity of CEBPD(+) through cell states. Bottom plot shows normalized expression of FOXP2, which is a part of the CEBPD(+) regulon. e, Association of normalized expression of SFTPA2 with interaction eQTL, rs1617662, across the cell states, rs1617662 is part of a binding motif for FOXP2, where the alternative allele A has the consensus sequence, while the reference allele, T, is predicted to have a weaker binding, based on motifbreakR.