K. Khalaj1, M. Duci1, A. Zani1,2 1The Hospital for Sick Children, Developmental & Stem Cell Biology Program, Toronto, ONTARIO, Canada 2The Hospital for Sick Children, Division Of General And Thoracic Surgery, Toronto, ONTARIO, Canada
Introduction: Congenital diaphragmatic hernia (CDH) is a devastating life-threatening condition characterized by pulmonary hypoplasia with impaired fetal lung growth and maturation, whose management remains challenging. We have previously shown that administration of extracellular vesicles derived from rat amniotic fluid stem cells (AFSC-EVs) rescues lung development in fetal rodent models of CDH. Towards clinical translation, we established a human ex-vivo epithelial organoid model of pulmonary hypoplasia that replicates the epithelial phenotype typical of CDH babies. Herein, we investigated: 1) fetal lung maturation in a human epithelial organoid model of pulmonary hypoplasia; and 2) the effects of human amniotic fluid stem cell-derived extracellular vesicles on epithelial cells.
Methods: EVs were separated from human AFSC (hAFSC-EVs) conditioned medium by ultracentrifugation, and characterized for size (Nanoparticle tracking analysis), morphology (transmission electron microscopy), and canonical EV protein markers by Western blot. Lung samples (IRB#10-0128-E) were harvested from three medical terminations of healthy fetuses at 18-19 weeks of gestation. Specimens were cultured as explants and treated with medium alone (control) or NSC23766 (an inhibitor of RAC1 that modulates lung branching morphogenesis) to induce pulmonary hypoplasia. NSC23766-injured lung explants were treated with medium alone (NSC) or medium + hAFSC-EVs at 48h and 72h (NSC+hAFSC-EV). Lungs were harvested at 96h, and tissue was digested using collagenase and grown in Matrigel for 14 days as fetal lung organoids. Organoids were stained for alveolar epithelial cell type-2 marker surfactant protein C (SPC) and pan-epithelial marker E-Cadherin (ECAD) and imaged in 3D using confocal microscopy. 3D single-organoid analysis was performed using Imaris software.
Results: Single-organoid analysis for SPC revealed impairment in SPC expression in human hypoplastic epithelial fetal lung organoids compared to control (p=0.003). Human AFSC-EV administration to hypoplastic epithelial fetal lung organoids resulted in upregulation of SPC back to control (p=0.0229; Figure). These findings also matched ECAD expression, as ECAD expression was downregulated in human hypoplastic epithelial fetal lung organoids compared to control (p=0.0281) and rescued by hAFSC-EV administration (p=0.0373; Figure).
Conclusion: This study demonstrates altered fetal lung maturation in a human organoid model of pulmonary hypoplasia. Treatment with human AFSC-EVs promotes fetal lung epithelial maturation and development. Antenatal administration of human AFSC-EVs represents a promising approach to treating fetal lung maturation in pulmonary hypoplasia secondary to CDH.