Amniotic fluid stem cell extracellular vesicles promote fetal lung branching and cell differentiation in experimental congenital diaphragmatic hernia

Abstract

Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by impaired branching morphogenesis and differentiation. We have previously demonstrated that administration of extracellular vesicles derived from rat amniotic fluid stem cells (AFSC-EVs) rescues development of hypoplastic lungs at the pseudoglandular and alveolar stages in rodent models of CDH. Herein, we tested whether AFSC-EVs exert their regenerative effects at the canalicular and saccular stages, as these are translationally relevant for clinical intervention. To induce fetal pulmonary hypoplasia, we gavaged rat dams with nitrofen at embryonic day 9.5 and demonstrated that nitrofen-exposed lungs had impaired branching morphogenesis, dysregulated signaling pathways relevant to lung development (FGF10/FGFR2, ROBO/SLIT, Ephrin, Neuropilin 1, beta-catenin) and impaired epithelial and mesenchymal cell marker expression at both stages. AFSC-EVs administered to nitrofen-exposed lung explants rescued airspace density and increased the expression levels of key factors responsible for branching morphogenesis. Moreover, AFSC-EVs rescued the expression of alveolar type 1 and 2 cell markers at both canalicular and saccular stages, and restored markers of club, ciliated epithelial, and pulmonary neuroendocrine cells at the saccular stage. AFSC-EV treated lungs also had restored markers of lipofibroblasts and PDGFRA+ cells to control levels at both stages. EV tracking showed uptake of AFSC-EV RNA cargo throughout the fetal lung and an mRNA-miRNA network analysis identified that several miRNAs responsible for regulating lung development processes were contained in the AFSC-EV cargo. These findings suggest that AFSC-EV based therapies hold potential for restoring fetal lung growth and maturation in babies with pulmonary hypoplasia secondary to CDH.

Fetal lung regeneration using stem cell‐derived extracellular vesicles: A new frontier for pulmonary hypoplasia secondary to congenital diaphragmatic hernia

“…As they typically appear during the canalicular stage, we evaluated the expression of lipofibroblast markers at the canalicular, saccular, and alveolar stages. We confirmed that fetal hypoplastic lungs had reduced density of lipofibroblasts at all these stages of lung development, and that AFSC‐EV administration rescued their expression to control levels 29,64 …”

“…64 Specifically, we observed an increase in number of branches and airspaces, as well as a rescue of expression levels of genes involved in branching morphogenesis. 64 When we assessed the last stage of lung development, we found that the lungs of fetal rabbit pups treated with AFSC-EVs had a restored number of alveoli and decreased alveolar thickness, as well as normalized levels of BMP signaling (BMP, BMP2, BMP4, and Id1), a critical pathway for alveolarization. 29 Next, we evaluated the effects of AFSC-EVs on lung epithelial maturation.…”

“…At the canalicular and saccular stages, we observed that AFSC‐EV administration to fetal hypoplastic lung explants rescued the expression of ATI and ATII cell markers 64 . Moreover, at the saccular stage, AFSC‐EVs rescued the expression markers of club, ciliated, basal, and neuroendocrine cells 64 …”

“…We also observed AFSC‐EV beneficial effects on rescuing epithelial cell proliferation and differentiation to ATII and club cells in fetal lung organoids obtained from nitrofen‐injured lungs harvested at the pseudoglandular stage. At the canalicular and saccular stages, we observed that AFSC‐EV administration to fetal hypoplastic lung explants rescued the expression of ATI and ATII cell markers 64 . Moreover, at the saccular stage, AFSC‐EVs rescued the expression markers of club, ciliated, basal, and neuroendocrine cells 64 …”

“…To evaluate the EV ability to rescue fetal lung growth and maturation, we tested the effects of rat AFSC‐EVs during all stages except the embryonic stage, as this occurs at such an early gestational age (4–8 weeks of gestation in human) that it is translationally irrelevant to CDH (Figure 2). 29,63,64 We specifically addressed branching morphogenesis in the pseudoglandular, canalicular, and saccular stages, using the nitrofen fetal rat model of CDH 65 . To study the alveolar stage, we turned to the surgical model of CDH in fetal rabbits, where formation of alveoli begins prenatally like in human babies and unlike rats 62 .…”