O1120: Gmp-grade Human Amniotic Fluid Stem Cell Extracellular Vesicles Promote Fetal Lung Branching Morphogenesis and Maturation in a Novel Experimental Human Model of Pulmonary Hypoplasia

Background: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is a devastating condition with high mortality and morbidity rates. We have previously shown that administration of extracellular vesicles derived from rat amniotic fluid stem cells (rAFSC-EVs) rescues lung development in fetal rodent models of congenital diaphragmatic hernia (CDH). Herein, we assessed whether EVs derived from human AFSCs (hAFSC-EVs) obtained under good manufacturing practices (GMP) could restore markers of branching morphogenesis and maturation of the distal airways in a novel model of human fetal pulmonary hypoplasia.

Methods: 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 (REB:#10-0128-E) were harvested from seven medical terminations of healthy fetuses at 15-18 weeks of gestation. Specimens were cultured 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 hAFSC-EVs at 48h and 72h (NSC+hAFSC-EV group) and harvested at 96h. Uninjured untreated explants served as control. Explants were analyzed for expression of markers of branching morphogenesis (FGF10) using RT-qPCR, as well as fetal lung maturation (PDPN; alveolar type 1, and SPC; alveolar type 2) via RT-qPCR and Western blotting. Statistics: One-way (qPCR) ANOVA.

Results: Hypoplastic human fetal lungs had significantly lower gene expression levels of FGF10 and gene and protein levels of PDPN and SPC levels (Fig.A-C). Administration of hAFSC-EVs to hypoplastic fetal lung explants restored expression levels of all three of these markers back to normal levels (Fig.A-C).

Conclusion: This is the first study demonstrating impaired expression of key markers of branching morphogenesis and fetal lung maturation of the distal airways in a novel human model of pulmonary hypoplasia. Treatment with GMP-grade hAFSC-EVs rescues these key markers of lung development to control levels. Antenatal administration of hAFSC-EVs could be a promising approach to treat pulmonary hypoplasia secondary to CDH.