Amniotic Fluid Stem-Cell Extracellular Vesicles Improve Pulmonary Vasculature in Experimental Congenital Diaphragmatic Hernia via the Release of RNA Cargo

Rationale: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development. Objectives: To evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms. Methods: EVs were isolated from c-kit+ AFSC conditioned medium by ultracentrifugation and characterized for size, morphology, and EV markers. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. Expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1/p62) was analyzed in vitro (rat and human fetal lung explants) and in vivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy. Measurements and Main Results: Rat and human models of fetal pulmonary hypoplasia showed reduced autophagy mainly at pseudoglandular and canalicular stages. AFSC-EV administration restored autophagy in both pulmonary hypoplasia models by transferring miR-17~92 cluster members contained in the EV cargo. Conclusions: AFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through miRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in CDH babies.

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

10.1101/2022.01.10.475632 ◽

2022 ◽

Author(s):

Kasra Khalaj ◽

Rebeca Lopes Figueira ◽

Lina Antounians ◽

Sree Gandhi ◽

Matthew Wales ◽

...

Keyword(s):

Amniotic Fluid ◽

Congenital Diaphragmatic Hernia ◽

Extracellular Vesicles ◽

Diaphragmatic Hernia ◽

Lung Development ◽

Pulmonary Hypoplasia ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Neuroendocrine Cells ◽

Beta Catenin

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.

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The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques

Scientific Reports ◽

10.1038/s41598-018-38320-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 18

Author(s):

Lina Antounians ◽

Areti Tzanetakis ◽

Ornella Pellerito ◽

Vincenzo D. Catania ◽

Adrienne Sulistyo ◽

...

Keyword(s):

Stem Cell ◽

Amniotic Fluid ◽

Extracellular Vesicles ◽

Lessons Learned ◽

Isolation Techniques ◽

Amniotic Fluid Stem Cell

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Transamniotic Stem Cell Therapy for Experimental Congenital Diaphragmatic Hernia: Structural, Transcriptional, and Cell Kinetics Analyses in the Nitrofen Model

Fetal Diagnosis and Therapy ◽

10.1159/000515277 ◽

2021 ◽

pp. 1-11

Author(s):

Alexander V. Chalphin ◽

Stefanie P. Lazow ◽

Daniel F. Labuz ◽

Sarah A. Tracy ◽

Ina Kycia ◽

...

Keyword(s):

Stem Cell ◽

Cell Therapy ◽

Congenital Diaphragmatic Hernia ◽

Stem Cell Therapy ◽

Diaphragmatic Hernia ◽

Cell Kinetics ◽

Donor Cell ◽

Wald Test ◽

Saline Group ◽

Pulmonary Vasculature

Purpose: We examined select pulmonary effects and donor cell kinetics after transamniotic stem cell therapy (TRASCET) in a model of congenital diaphragmatic hernia (CDH). Methods: Pregnant dams (n = 58) received nitrofen on gestational day 9.5 (E9) to induce fetal CDH. Fetuses (n = 681) were divided into 4 groups: untreated (n = 99) and 3 groups receiving volume-matched intra-amniotic injections on E17 of either saline (n = 142), luciferase-labeled amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 299), or acellular recombinant luciferase (n = 141). Pulmonary morphometry, quantitative gene expression of pulmonary vascular tone mediators, or screening for labeled afMSCs were performed at term (E22). Statistical comparisons were by Mann-Whitney U-test, nested ANOVA, and Wald test. Results: TRASCET led to significant downregulation of endothelial nitric oxide synthase and endothelin receptor-A expressions compared to both untreated and saline groups (both p < 0.001). TRASCET also led to a significant decrease in arteriole wall thickness compared to the untreated group (p < 0.001) but not the saline group (p = 0.180). Donor afMSCs were identified in the bone marrow and umbilical cord (p = 0.035 and 0.015, respectively, vs. plain luciferase controls). Conclusions: The effects of TRASCET in experimental CDH appear to be centered on the pulmonary vasculature and to derive from circulating donor cells.

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Increased c-kit and stem cell factor expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia

Journal of Pediatric Surgery ◽

10.1016/j.jpedsurg.2016.02.007 ◽

2016 ◽

Vol 51 (5) ◽

pp. 706-709 ◽

Cited By ~ 4

Author(s):

Toshiaki Takahashi ◽

Florian Friedmacher ◽

Julia Zimmer ◽

Prem Puri

Keyword(s):

Stem Cell ◽

Congenital Diaphragmatic Hernia ◽

Diaphragmatic Hernia ◽

Stem Cell Factor ◽

Pulmonary Vasculature ◽

Factor Expression

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First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential

Stem Cells Translational Medicine ◽

10.1002/sctm.16-0297 ◽

2017 ◽

Vol 6 (5) ◽

pp. 1340-1355 ◽

Cited By ~ 49

Author(s):

Carolina Balbi ◽

Martina Piccoli ◽

Lucio Barile ◽

Andrea Papait ◽

Andrea Armirotti ◽

...

Keyword(s):

Stem Cell ◽

Amniotic Fluid ◽

Extracellular Vesicles ◽

Human Amniotic Fluid ◽

Amniotic Fluid Stem Cell

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Amniotic Fluid Stem Cell-Derived Extracellular Vesicles Counteract Steroid-Induced Osteoporosis In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms22010038 ◽

2020 ◽

Vol 22 (1) ◽

pp. 38

Author(s):

Martina Gatti ◽

Francesca Beretti ◽

Manuela Zavatti ◽

Emma Bertucci ◽

Soraia Ribeiro Luz ◽

...

Keyword(s):

Amniotic Fluid ◽

Extracellular Vesicles ◽

Culture Medium ◽

Cell Potential ◽

Osteoblast Cell Line ◽

Local Bone ◽

Current Therapies ◽

Amniotic Fluid Stem Cell ◽

Related Proteins

Background—Osteoporosis is characterized by defects in both quality and quantity of bone tissue, which imply high susceptibility to fractures with limitations of autonomy. Current therapies for osteoporosis are mostly concentrated on how to inhibit bone resorption but give serious adverse effects. Therefore, more effective and safer therapies are needed that even encourage bone formation. Here we examined the effect of extracellular vesicles secreted by human amniotic fluid stem cells (AFSC) (AFSC-EV) on a model of osteoporosis in vitro. Methods—human AFSC-EV were added to the culture medium of a human pre-osteoblast cell line (HOB) induced to differentiate, and then treated with dexamethasone as osteoporosis inducer. Aspects of differentiation and viability were assessed by immunofluorescence, Western blot, mass spectrometry, and histological assays. Since steroids induce oxidative stress, the levels of reactive oxygen species and of redox related proteins were evaluated. Results—AFSC-EV were able to ameliorate the differentiation ability of HOB both in the case of pre-osteoblasts and when the differentiation process was affected by dexamethasone. Moreover, the viability was increased and parallelly apoptotic markers were reduced. The presence of EV positively modulated the redox unbalance due to dexamethasone. Conclusion—these findings demonstrated that EV from hAFSC have the ability to recover precursor cell potential and delay local bone loss in steroid-related osteoporosis.

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