O1109: Dysregulated Cell-cell Signaling in Rat Hypoplastic Lungs Secondary to Congenital Diaphragmatic Hernia Can Be Reversed with Administration of Amniotic Fluid Stem Cell Extracellular Vesicles

Background: Hypoplastic lungs of babies with congenital diaphragmatic hernia (CDH) are immature, have thickened alveolar walls, and increased interstitial tissue, leading to poor outcomes. Impaired ligand-receptor crosstalk between different cell-types could be the source of these major changes. We reported that administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues normal lung development in fetal models of pulmonary hypoplasia. Herein, we identified the aberrant cellular crosstalk of CDH lungs and determined the effects of AFSC-EV administration on cell-to-cell signaling.

Methods: AFSC-EV isolation: by ultracentrifugation from rat AFSC conditioned medium; characterization by size (nanoparticle tracking analysis), morphology (transmission electron microscopy), and expression of canonical markers (Western blot, CD63/TSG101/Flo1).
In vivo model: Rat dams received nitrofen to induce CDH or vehicle (control) at embryonic day (E)9.5. At E19.5, fetuses received intra-amniotic injection of saline (control+saline, n=38; CDH+saline, n=64), or AFSC-EVs (CDH+AFSC-EV, n=49). Lungs were harvested at E21.5 and assessed for 1) branching morphogenesis impairment (mean linear intercept, H&E), and 2) gene expression of lung developmental markers FGF-10, Sox9, and SPC.
Single nucleus RNA-sequencing: fetal lungs from control+saline (n=2), CDH+saline (n=3), and CDH+AFSC-EVs (n=3) groups were immediately frozen, nuclei were extracted and subsequently subjected to 10X genomics 3’ v3.1 library preparation and mRNA sequencing pipeline. To identify cell-type specific gene expression profiles and perform differential gene expression analysis, datasets were pooled and analyzed using Seurat. CellChat was used to establish cell-specific expression of ligands and receptors to generate a network identifying key overrepresented pathways throughout all cell clusters.

Results: CDH lungs have unique cell clusters with inflammatory signatures that signal to other cell types of endothelial and epithelial origin (Fig.A). For example, inflamed fibroblasts have high expression of pleiotrophin (ligand) that signals through syndecan-4 (receptor) in multiple cell types. This finding can indicate an upregulation of beta-catenin signaling, leading to epithelial-to-mesenchymal transition in CDH lungs. AFSC-EV administration dramatically altered ligand-receptor signaling networks, including upregulation of Fgf10 (ligand) from inflamed fibroblasts that signals to Fgfr2 (receptor) on alveolar type 2 cells (Fig.B).

Conclusion: Major signaling pathways known to be involved in branching morphogenesis and epithelial-to-mesenchymal transition are dysregulated in CDH lungs. Cell-to-cell communication can be restored with an AFSC-EV based therapy, which can promote proper lung development in CDH lungs.