Treatment with Amniotic Fluid Stem Cell Extracellular Vesicles Promotes Fetal Lung Branching and Cell Differentiation at Canalicular and Saccular Stages in Experimental Pulmonary Hypoplasia Secondary to 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, β-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 vascular development is disrupted by mechanical compression and rescued by administration of amniotic fluid stem cell extracellular vesicles via regulation of the Hippo signaling pathway

“…We have previously shown that AFSC-EVs restore fetal lung branching morphogenesis and epithelial cell differentiation in rodents and human models of CDH [20][21][22] . The current study provides evidence that AFSC-EV administration to rat and human compressed fetal lungs rescues also fetal lung vascular development.…”

“…The multifactorial pathogenesis of arrested lung development in CDH necessitates an antenatal therapy that addresses all aspects of impaired fetal lung growth, maturation, and vascularization 8 . We have previously shown that AFSC-EVs restore fetal lung branching morphogenesis and epithelial cell differentiation in rodents and human models of CDH 20-22 . The current study provides evidence that AFSC-EV administration to rat and human compressed fetal lungs rescues also fetal lung vascular development.…”

“…Ideally, an optimal therapy would be one that addresses all aspects of fetal lung underdevelopment, that is lung growth, maturation, and vascularization. Experimentally, administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs) has been reported to rescue branching morphogenesis and epithelial and mesenchymal cell differentiation in rodent and human models of CDH [20][21][22] . The beneficial effect of AFSC-EVs on hypoplastic lungs is attributed at least in part to the delivery of their microRNA cargo 20,21 .…”

Single cell-guided prenatal derivation of primary epithelial organoids from the human amniotic and tracheal fluids

“…This timeline is further shortened by the fact that AF / chorionic villi sampling is not normally performed until the end of the first trimester; and furthermore, the majority of prenatal therapies are currently ideally delivered prior to 30 weeks. Several examples of iPSCs-organoid derivation highlight the need of at least 21 weeks to produce the organoids 73 . Our approach benefits from the already committed progenitors present in the fetal fluids, which require minimal manipulation to lead to the production of a large amount of primary autologous fetal organoids in approximately 4 to 6 weeks.…”

Association of Fetal Lung Development Disorders with Adult Diseases: A Comprehensive Review

“…Regenerative therapies, such as stem cell-based approaches, are another prospective area for therapeutic development. These novel treatments could lung tissue damage incurred by fetal lung development disorders and promote lung regeneration, a concept backed by emerging preclinical and clinical studies demonstrating the potential benefits of stem cell therapies for lung diseases [238][239][240].…”