Purpose
Lineage tracing is key to study the fate of individual cells and their progeny especially in developmental biology. To conduct these studies, we aimed to establish a reproducible model of CDH ...in the most commonly used genetic background strain that is C57BL/6J mice.
Methods
CDH was induced in C57BL/6J dams by maternal administration of nitrofen + bisdiamine at E8.5. Fetuses from olive oil-gavaged mothers served as controls. Lungs from CDH and control fetuses were compared for (1) growth via radial airspace count (RAC), mean linear intercept (MLI) and gene expression for
Fgf10
,
Nrp1
, and
Ctnnb1
; (2) maturation (
Pdpn
,
Spc
,
Ager
,
Abca3
,
Eln
,
Acta2
,
Pdgfra
) via gene and protein expression; (3) vascularization via gene and protein expression (CD31,
Vegfa
,
Vegfr1/2
,
Epas1
,
Enos
). Statistics: unpaired
t
-test or Mann–Whitney test.
Results
Nitrofen + bisdiamine administration resulted in 36% left-sided CDH (31% mortality). CDH fetuses had hypoplastic lungs and impaired growth (lower RAC, higher MLI, lower
Fgf10
,
Nrp1
,
Ctnnb1
), maturation (decreased
Pdpn
,
Ager
,
Eln
gene expression), and vascularization (decreased
Cd31
,
Vegfr1/2
;
Epas1
and
Enos
). Lower protein expression was confirmed for PDPN, ELN and CD31.
Conclusion
Modeling CDH in C57BL/6J mouse fetuses is effective in reproducing the classical CDH hallmarks. This model will be critical for lineage tracing experiments.
The etiology of congenital diaphragmatic hernia (CDH) remains unknown and only 10 to 30% of patients have a genetic cause. Seasonal variation is known to contribute to the development of some ...congenital anomalies. Our aim was to investigate whether CDH births have seasonal variation.
A literature review was conducted for CDH and seasonality. Moreover, data from the CDH International Patient Registry Database were collected for infants with due dates between 2008 and 2014. Due dates were used to determine seasonal distribution of births. Birth rates per month in the United States and Canada were extracted from publicly available databases. Data were analyzed using analysis of variance and contingency tables.
First, the literature review revealed 11 articles, of which 3 were eligible for inclusion. These studies reported conflicting results on seasonality of CDH. Second, we extracted due dates from the CDH International Patient Registry Database (1,259 patients) and found that there were fewer due dates in winter months (12.1 ± 4 patients/month) than in summer (16.7 ± 6 patients/month;
= 0.011) and fall months (16.3 ± 5 patients/month;
= 0.022). Although this trend was similar to that of all births in the United States and Canada, a lower incidence was observed in winter for CDH infants (20.2%) than for the general population (24.1%,
= 0.0012). CDH survival rate did not vary by season.
This study provides evidence for a seasonal variation of CDH births. No causative link was established between CDH development and seasonality. Population-based studies with a focus on exposome data are needed to explain seasonal variation in CDH.
Astrocytes are in constant communication with neurons during the establishment and maturation of functional networks in the developing brain. Astrocytes release extracellular vesicles (EVs) ...containing microRNA (miRNA) cargo that regulates transcript stability in recipient cells. Astrocyte released factors are thought to be involved in neurodevelopmental disorders. Healthy astrocytes partially rescue Rett Syndrome (RTT) neuron function. EVs isolated from stem cell progeny also correct aspects of RTT. EVs cross the blood-brain barrier (BBB) and their cargo is found in peripheral blood which may allow non-invasive detection of EV cargo as biomarkers produced by healthy astrocytes. Here we characterize miRNA cargo and sequence motifs in healthy human astrocyte derived EVs (ADEVs). First, human induced Pluripotent Stem Cells (iPSC) were differentiated into Neural Progenitor Cells (NPCs) and subsequently into astrocytes using a rapid differentiation protocol. iPSC derived astrocytes expressed specific markers, displayed intracellular calcium transients and secreted ADEVs. miRNAs were identified by RNA-Seq on astrocytes and ADEVs and target gene pathway analysis detected brain and immune related terms. The miRNA profile was consistent with astrocyte identity, and included approximately 80 miRNAs found in astrocytes that were relatively depleted in ADEVs suggestive of passive loading. About 120 miRNAs were relatively enriched in ADEVs and motif analysis discovered binding sites for RNA binding proteins FUS, SRSF7 and CELF5. miR-483-5p was the most significantly enriched in ADEVs. This miRNA regulates MECP2 expression in neurons and has been found differentially expressed in blood samples from RTT patients. Our results identify potential miRNA biomarkers selectively sorted into ADEVs and implicate RNA binding protein sequence dependent mechanisms for miRNA cargo loading.
•Astrocytes were derived from human induced Pluripotent Stem Cells.•Extracellular Vesicles were isolated from astrocytes to identify miRNA cargo.•Extracellular Vesicles load miRNA cargo that targets genes expressed in neurons.•RNA motifs for RNA Binding Proteins implicate a miRNA sorting mechanism.•miR483-5p in Extracellular Vesicles is a candidate biomarker for astrocyte function.
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.
Congenital diaphragmatic hernia (CDH) is a devastating disease that still carries a high mortality and morbidity rate. Poor outcomes for fetuses and infants with CDH are mainly related to pulmonary ...hypoplasia (PH) and pulmonary vascular remodeling that leads to pulmonary hypertension (PHTN). Over the last five decades, research efforts have focused on modeling CDH not only to study the pathophysiology of the diaphragmatic defect, pulmonary hypoplasia, and pulmonary hypertension, but also to identify therapies that would promote lung growth and maturation, and correct vascular remodeling. As CDH is a multifactorial condition whose etiology remains unknown, there is not a single model of CDH, rather several ones that replicate different aspects of this disease. While small animals like the mouse and the rat have mainly been used to uncover biological pathways underlying the diaphragmatic defect and poor lung growth, larger animals like the lamb and the rabbit models have been instrumental for pursuing medical and surgical interventions. Overall, the use of animal models has indeed advanced our knowledge on CDH and helped us test innovative therapeutic options. For example, the lamb model of CDH has been the paradigm for testing fetal surgical procedures, including tracheal occlusion, which has been translated to clinical use. In this review, we outline the induction protocols of CDH in animals with the use of chemicals, dietary changes, genetic alterations, and surgical maneuvers, and we describe the studies that have translated experimental results to the bedside.
Background The aim of this study was to quantify mediators of neutrophilic inflammation within airway extracellular vesicles (EVs) of children treated for a cystic fibrosis (CF) pulmonary ...exacerbation (PEx). Methods EVs were isolated from stored sputum samples collected before and after antibiotic therapy for PEx between 2011–2013, and characterised by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Western blot analysis of EV protein extracts was used for EV canonical protein markers CD63, CD9, and flotillin-1 (FLOT1), as well as neutrophil elastase (NE), myeloperoxidase (MPO) and interleukin-8. EV content of NE and MPO was expressed as ratios of NE/FLOT1 and MPO/FLOT1 protein band densities. Results Sputum samples from 21 children aged 13.3 (range 8.0–17.0) years were analysed. NTA showed high concentrations of particles at the size of small EVs (50–200 nm), and typical EV morphology was confirmed by TEM. CD63, CD9 and FLOT1 was detectable in all samples. Median (IQR) NE/FLOT1 increased from 2.46 (1.68–5.25) before to 6.83 (3.89–8.89, p<0.001) after PEx therapy and MPO/FLOT1 from 2.30 (1.38–4.44) to 5.76 (3.45–6.94, p<0.01), while EV size remained unchanged. Improvement in lung function (ppFEV 1 ) with PEx therapy correlated with NE EV content (r=0.657, p=0.001). Conclusions Airways of children with CF contain EVs that carry NE and MPO as cargo. The lower NE and MPO content at the time of PEx compared to after therapy and the correlation with pulmonary function suggest both a functional role of EVs in CF airway inflammation and potential as a biomarker to monitor CF lung disease.
The poor outcomes of babies with congenital diaphragmatic hernia (CDH) are directly related to pulmonary hypoplasia, a condition characterized by impaired lung development. Although the pathogenesis ...of pulmonary hypoplasia is not fully elucidated, there is now evidence that CDH patients have missing or dysregulated microRNAs (miRNAs) that regulate lung development. A prenatal therapy that supplements these missing/dysregulated miRNAs could be a strategy to rescue normal lung development. Extracellular vesicles (EVs), also known as exosomes when of small dimensions, are lipid‐bound nanoparticles that can transfer their heterogeneous cargo (proteins, lipids, small RNAs) to target cells to induce biological responses. Herein, we review all studies that show evidence for stem cell‐derived EVs as a regenerative therapy to rescue normal development in CDH fetal lungs. Particularly, we report studies showing that administration of EVs derived from amniotic fluid stem cells (AFSC‐EVs) to models of pulmonary hypoplasia promotes fetal lung growth and maturation via transfer of miRNAs that are known to regulate lung developmental processes. We also describe that stem cell‐derived EVs exert effects on vascular remodeling, thus possibly preventing postnatal pulmonary hypertension. Finally, we discuss future perspectives and challenges to translate this promising stem cell EV‐based therapy to clinical practice.
Key points
What’s already known about this topic?
The poor outcomes of patients with congenital diaphragmatic hernia (CDH) are directly related to impaired fetal lung development (pulmonary hypoplasia).
There is consensus that the prenatal period is a window of opportunity to treat pulmonary hypoplasia and promote normal lung development.
Several groups have reported that human and experimental CDH lungs have missing or dysregulated microRNAs (miRNAs).
What does this study add?
This review collates all the studies that show evidence for stem cell derived‐extracellular vesicles (EVs) as a therapy to rescue normal development in CDH fetal lungs.
EVs administered to models of pulmonary hypoplasia transfer cargo, including miRNAs that are in part responsible for the EV regenerative effects.
Purpose
Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by impaired epithelial homeostasis. Recently, amniotic fluid stem cells (AFSCs) have been shown to ...promote growth in hypoplastic lungs of rat fetuses with CDH. Herein, we investigated whether CDH hypoplastic lungs mount an endoplasmic reticulum (ER) stress response and whether AFSCs could re-establish pulmonary epithelial homeostasis.
Methods
Primary epithelial cells were isolated from fetal rat lungs at E14.5 from control and nitrofen-exposed dams at E9.5. Nitrofen-exposed epithelial cells were grown in medium alone or co-cultured with AFSCs. Epithelial cell cultures were compared for apoptosis (TUNEL), cytotoxicity (LIVE/DEAD assay), proliferation (5′EdU), and ER stress (CHOP, Bcl-2) using one-way ANOVA (Dunn’s post-test).
Results
Compared to control, nitrofen-exposed epithelial cells had increased cytotoxicity and apoptosis, reduced proliferation, and activated ER stress. AFSCs restored apoptosis, proliferation, and ER stress back to control levels, and significantly reduced cytotoxicity.
Conclusions
This study shows for the first time that ER stress-induced apoptosis is activated in the pulmonary epithelium of hypoplastic lungs from fetuses with CDH. AFSC treatment restores epithelial cellular homeostasis by attenuating the ER stress response and apoptosis, by increasing proliferation and migration ability, and by reducing cytotoxicity.
Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder with a unique, platelet-dependent, gain-of-function defect in fibrinolysis, without systemic fibrinolysis. The hallmark ...feature of QPD is a >100-fold overexpression of PLAU, specifically in megakaryocytes. This overexpression leads to a >100-fold increase in platelet stores of urokinase plasminogen activator (PLAU/uPA); subsequent plasmin-mediated degradation of diverse α-granule proteins; and platelet-dependent, accelerated fibrinolysis. The causative mutation is a 78-kb tandem duplication of PLAU. How this duplication causes megakaryocyte-specific PLAU overexpression is unknown. To investigate the mechanism that causes QPD, we used epigenomic profiling, comparative genomics, and chromatin conformation capture approaches to study PLAU regulation in cultured megakaryocytes from participants with QPD and unaffected controls. QPD duplication led to ectopic interactions between PLAU and a conserved megakaryocyte enhancer found within the same topologically associating domain (TAD). Our results support a unique disease mechanism whereby the reorganization of sub-TAD genome architecture results in a dramatic, cell-type-specific blood disorder phenotype.
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