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

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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Fetal lung underdevelopment is rescued by administration of amniotic fluid stem cell extracellular vesicles in rodents

Science Translational Medicine ◽

10.1126/scitranslmed.aax5941 ◽

2021 ◽

Vol 13 (590) ◽

pp. eaax5941

Author(s):

Lina Antounians ◽

Vincenzo D. Catania ◽

Louise Montalva ◽

Benjamin D. Liu ◽

Huayun Hou ◽

...

Keyword(s):

Stem Cell ◽

Epithelial Cells ◽

Amniotic Fluid ◽

Extracellular Vesicles ◽

Lung Development ◽

Pulmonary Hypoplasia ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Lung Growth ◽

Regenerative Ability

Fetal lung underdevelopment, also known as pulmonary hypoplasia, is characterized by decreased lung growth and maturation. The most common birth defect found in babies with pulmonary hypoplasia is congenital diaphragmatic hernia (CDH). Despite research and clinical advances, babies with CDH still have high morbidity and mortality rates, which are directly related to the severity of lung underdevelopment. To date, there is no effective treatment that promotes fetal lung growth and maturation. Here, we describe a stem cell–based approach in rodents that enhances fetal lung development via the administration of extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs). Using fetal rodent models of pulmonary hypoplasia (primary epithelial cells, organoids, explants, and in vivo), we demonstrated that AFSC-EV administration promoted branching morphogenesis and alveolarization, rescued tissue homeostasis, and stimulated epithelial cell and fibroblast differentiation. We confirmed this regenerative ability in in vitro models of lung injury using human material, where human AFSC-EVs obtained following good manufacturing practices restored pulmonary epithelial homeostasis. Investigating EV mechanism of action, we found that AFSC-EV beneficial effects were exerted via the release of RNA cargo. MicroRNAs regulating the expression of genes involved in lung development, such as the miR17–92 cluster and its paralogs, were highly enriched in AFSC-EVs and were increased in AFSC-EV–treated primary lung epithelial cells compared to untreated cells. Our findings suggest that AFSC-EVs hold regenerative ability for underdeveloped fetal lungs, demonstrating potential for therapeutic application in patients with pulmonary hypoplasia.

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Impaired Fetal Lung Development can be Rescued by Administration of Extracellular Vesicles Derived from Amniotic Fluid Stem Cells

10.1101/2020.08.07.240408 ◽

2020 ◽

Author(s):

Lina Antounians ◽

Vincenzo D. Catania ◽

Louise Montalva ◽

Benjamin D. Liu ◽

Huayun Hou ◽

...

Keyword(s):

Stem Cells ◽

Amniotic Fluid ◽

Extracellular Vesicles ◽

Lung Development ◽

Pulmonary Hypoplasia ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Amniotic Fluid Stem Cells ◽

Fetal Lung Development ◽

Regenerative Ability

AbstractIncomplete lung development, also known as pulmonary hypoplasia, is a recognized cause of neonatal death and poor outcome for survivors. To date, there is no effective treatment that promotes fetal lung growth and maturation. Herein, we describe a novel stem cell-based approach that enhances fetal lung development via the administration of extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs). In experimental models of pulmonary hypoplasia, administration of AFSC-EVs promoted lung branching morphogenesis and alveolarization, and stimulated pulmonary epithelial cell and fibroblast differentiation. This regenerative ability was confirmed in two models of injured human lung cells, where human AFSC-EVs obtained following good manufacturing practices restored pulmonary epithelial homeostasis. AFSC-EV beneficial effects were exerted via the release of RNA cargo, primarily miRNAs, that regulate the expression of genes involved in fetal lung development. Our findings suggest that AFSC-EVs hold regenerative ability for underdeveloped fetal lungs, demonstrating potential for therapeutic application.One Sentence SummaryFetal lung regeneration via administration of extracellular vesicles derived from amniotic fluid stem cells

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Autophagy is impaired in fetal hypoplastic lungs and rescued by administration of amniotic fluid stem cell extracellular vesicles

10.1101/2022.01.12.476078 ◽

2022 ◽

Author(s):

Kasra Khalaj ◽

Lina Antounians ◽

Rebeca Lopes Figueira ◽

Martin Post ◽

Augusto Zani

Keyword(s):

Stem Cell ◽

Amniotic Fluid ◽

Extracellular Vesicles ◽

Pulmonary Hypoplasia ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Lung Epithelial Cells ◽

Normal Lung ◽

Amniotic Fluid Stem Cell ◽

Human Fetal Lung

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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STATs in Lung Development: Distinct Early and Late Expression, Growth Modulation and Signaling Dysregulation in Congenital Diaphragmatic Hernia

Cellular Physiology and Biochemistry ◽

10.1159/000486218 ◽

2017 ◽

Vol 45 (1) ◽

pp. 1-14 ◽

Cited By ~ 5

Author(s):

Paulina Piairo ◽

Rute S. Moura ◽

Maria João Baptista ◽

Jorge Correia-Pinto ◽

Cristina Nogueira-Silva

Keyword(s):

Congenital Diaphragmatic Hernia ◽

Diaphragmatic Hernia ◽

Lung Development ◽

Pulmonary Hypoplasia ◽

Fetal Lung ◽

Developmental Expression ◽

Lung Growth ◽

Growth Modulation ◽

Late Expression

Background: Congenital diaphragmatic hernia (CDH) is a life-threatening developmental anomaly, intrinsically combining severe pulmonary hypoplasia and hypertension. During development, signal transducers and activators of transcription (STAT) are utilized to elicit cell growth, differentiation, and survival. Methods: We used the nitrofen-induced CDH rat model. At selected gestational time points, lungs were divided into two experimental groups, i.e., control or CDH. We performed immunohistochemistry and western blotting analysis to investigate the developmental expression profile of the complete family of STATs (STAT1-6), plus specific STATs activation (p-STAT3, p-STAT6) and regulation by SOCS (SOCS3) in normal lungs against those of diseased lungs. The normal fetal lung explants were treated with piceatannol (STAT3 inhibitor) in vitro followed by morphometrical analysis. Results: Molecular profiling of STATs during the lung development revealed distinct early and late expression signatures. Experimental CDH altered the STATs expression, activation, and regulation in the fetal lungs. In particular, STAT3 and STAT6 were persistently over-expressed and early over-activated. Piceatannol treatment dose-dependently stimulated the fetal lung growth. Conclusion: These findings suggest that STATs play an important role during normal fetal lung development and CDH pathogenesis. Moreover, functionally targeting STAT signaling modulates fetal lung growth, which highlights that STAT3 and STAT6 signaling might be promising therapeutic targets in reducing or preventing pulmonary hypoplasia in CDH.

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Analysis of embryonic pulmonary blood-flow: potential influence on airway branching morphogenesis and development of pulmonary hypoplasia in congenital diaphragmatic hernia (CDH)

Journal of the American College of Surgeons ◽

10.1016/j.jamcollsurg.2008.06.122 ◽

2008 ◽

Vol 207 (3) ◽

pp. S56-S57

Author(s):

Marcus M. Malek ◽

Sohail R. Shah ◽

Adam M. Jakub ◽

Maria F. Branca ◽

Xiaojun Huang ◽

...

Keyword(s):

Blood Flow ◽

Congenital Diaphragmatic Hernia ◽

Diaphragmatic Hernia ◽

Pulmonary Blood Flow ◽

Pulmonary Hypoplasia ◽

Branching Morphogenesis ◽

Potential Influence ◽

Flow Potential ◽

Airway Branching

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Care of the fetus/newborn with congenital diaphragmatic hernia

Fetal and Maternal Medicine Review ◽

10.1017/s0965539500001017 ◽

1994 ◽

Vol 6 (2) ◽

pp. 81-94 ◽

Cited By ~ 1

Author(s):

Duncan T Wilcox ◽

Hratch L Karamanoukian ◽

Philip L Glick

Keyword(s):

Congenital Diaphragmatic Hernia ◽

Diaphragmatic Hernia ◽

Lung Development ◽

Pulmonary Hypoplasia ◽

Thoracic Cavity ◽

Live Births ◽

Surfactant System ◽

Vascular Branching

Congenital diaphragmatic hernia (CDH) is a simple anatomical defect. The reported incidence is between 1:2000 and 1:5000 live births, with the majority affecting the left side. A defect in the diaphragm allows abdominal viscera to herniate into and stay within the thoracic cavity during crucial stages of lung development. This results in pulmonary hypoplasia, a reduction in pulmonary vascular branching and an alteration in the surfactant system.

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ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

Respiratory Research ◽

10.1186/s12931-020-01568-w ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Ana N. Gonçalves ◽

Jorge Correia-Pinto ◽

Cristina Nogueira-Silva

Keyword(s):

Western Blot ◽

Congenital Diaphragmatic Hernia ◽

Progenitor Cells ◽

Expression Profile ◽

Diaphragmatic Hernia ◽

Ex Vivo ◽

Expression Profiles ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Lung Growth

Abstract Background Characterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance. Methods The expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and β-Catenin were quantified by Western blot. Results Experimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho β-Catenin, and BMP4. Conclusions These studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

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