O2140: Human Amniotic Fluid Stem Cell Extracellular Vesicles Rescue Features of Lung Development in Human Hypoplastic Fetal Lungs

Background: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is a devastating condition characterized by impaired fetal lung growth and maturation. We have previously shown that administration of extracellular vesicles derived from rat amniotic fluid stem cells (AFSC-EVs) rescues lung development in rat, mouse, and rabbit models of CDH. To evaluate if these findings in rodent models translate to the human condition, we tested human AFSC-EVs on a human model of pulmonary hypoplasia. Herein, we aimed to investigate the effects of human AFSC-EVs on fetal human hypoplastic lungs at a cellular and molecular level.

Methods: AFSC-EVs: Human AFSC-EVs were separated from human AFSC conditioned medium by ultracentrifugation, and characterized for size (Nanoparticle tracking analysis), morphology (transmission electron microscopy), and Western blot for canonical surface markers of EVs (CD63, FLOT1, TSG101). Lung samples (IRB: #10-0128-E) were harvested from medical terminations of healthy fetuses at 18-19 weeks of gestation.
Human pulmonary hypoplasia model: Specimens were cultured separately as explants and treated with medium alone (control group) or NSC23766 (an inhibitor of RAC1, a Rho GTPase that modulates lung branching morphogenesis) to induce pulmonary hypoplasia. We treated NSC23766-injured lung explants with medium alone (NSC group) or human AFSC-EVs at 48h and 72h (NSC+AFSC-EV group) and harvested at 96h.
Fetal lung growth and maturation: Branching morphogenesis was assessed with radial airspace count (RAC) and mean linear intercept (MLI, parenchyma:airspace ratio) by histology (H&E). Lung maturation was assessed using immunofluorescence for surfactant protein C (SPC) expression levels.
Single nucleus RNA sequencing (snRNA-seq): We included three biological replicates with three experimental groups (n=9 samples total) for snRNA-seq studies. Nuclei were isolated and subjected to 10X genomics library preparation and sequencing pipeline. Cell identity was assigned based on specific gene expression profiles using Seurat.

Results: Administration of NSC23766 to human fetal lungs resulted in fewer airspaces with increased parenchyma (lower RAC and higher MLI) and less mature epithelium (reduced SPC expression in the distal airways compared to control; Fig.A). Human AFSC-EV administration to hypoplastic lung explants restored RAC and MLI, and improved expression of SPC (Fig.A). Using snRNA-seq, we identified the four major cell populations of the fetal lung, i.e., epithelial, endothelial, mesenchymal, and immune lineages (Fig.B). Hypoplastic human fetal lungs had marked changes in the gene expression levels of several clusters, which were altered by the administration of human AFSC-EVs.

Conclusion: This is the first study demonstrating single-cell level changes in an experimental model of human hypoplastic fetal lungs. Treatment with human AFSC-EVs rescues features of pulmonary hypoplasia, likely by altering the expression of various cells in hypoplastic lungs. Antenatal administration of human AFSC-EVs represents a promising approach to treating pulmonary hypoplasia secondary to CDH.