Fig. 5. CDH fetal lungs have a multilineage inflammatory signature that is dampened by the administration of AFSC-EVs.

(A) Featureplot of snRNA-seq data split by condition for six inflammatory genes with high expression in CDH+saline lungs. (B) Violin plot of inflammatory signature genes expression split by condition across cell types, as measured by snRNA-seq. (C) Heatmap displaying differential gene expression by major cell type, showing expression all genes ranked by log₂(fold change) and P-adjusted < 0.05 within all conditions. (D) Volcano plots indicating most statistically significant differentially expressed genes by major cell type between CDH+saline-treated and CDH+AFSC-EV–treated groups. FDR, false discovery rate. (E) Representative immunofluorescence images of inflammation marker TNFα in rat fetal lungs from all three conditions, quantified as density per mm². Scale bars, 50 μm. Control+saline (n = 5), CDH+saline (n = 5), and CDH+AFSC-EV (n = 5). AU, arbitrary units. ****P < 0.0001. (F) UMAP of a subset of data that excludes clusters 1 and 2 (overrepresented in CDH+saline group) split by condition. Outlines indicate nuclei or clusters that are represented in CDH+saline group compared to Control+saline and CDH+AFSC-EV groups. Control+saline (n = 30,064), CDH+saline (n = 45,114), and CDH+AFSC-EV (n = 42,193). (G) UMAP of predicted cell types contained in cluster 5 immune cells from (F), generated by machine learning algorithm (scPred) trained on rat adult lungs. Groups were compared using Kruskal-Wallis (post hoc Dunn’s nonparametric comparison) for (E), according to Shapiro-Wilk normality test.