iPSC-derived healthy human astrocytes selectively load miRNAs targeting neuronal genes into extracellular vesicles

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.</p

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

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, CDH babies 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. Herein, we describe a stem cell-based approach 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 promotes branching morphogenesis and alveolarization, rescues tissue homeostasis, and stimulates epithelial cell and fibroblast differentiation. We confirmed this regenerative ability in human models of lung injury, where human AFSC-EVs obtained following good manufacturing practices restored pulmonary epithelial homeostasis. Investigating EV mechanism of action by tracking AFSC-EV cargo transfer, profiling EV protein and RNA content, and sequencing target lung epithelial cells, we found that AFSC-EV beneficial effects were exerted via the release of RNA cargo. Particularly, miRNAs that regulate the expression of genes involved in lung development, such as the miR17~92 cluster and its paralogues, were highly enriched in AFSCEVs 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

An Inter-vascular Bed and Inter-species Investigation of Epigenetic Regulatory Elements in Endothelial Cells

A major challenge in human genetics is to understand the mechanisms that control gene expression. To identify gene regulatory regions required for vascular homeostasis, we performed chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) for a variety of histone modifications and the JUN transcription factor in primary aortic endothelial cells (ECs) isolated from human, rat (Rattus norvegicus) and bovine (Bos taurus). We generated a chromatin state map for human aortic ECs and found that the vast majority of regulatory regions in aortic ECs were also active in venous ECs. By comparing the genomic occupancy of JUN and a histone modification indicative of active enhancers (H3K27ac) between species, we identified a set of conserved regulatory regions that were enriched for EC-specific pathways and human regulatory disease variants. Overall, we demonstrate that comparative epigenomics is a viable strategy to identify functionally important vascular gene regulatory elements.M.Sc.2018-11-09 00:00:0

Animal models of necrotizing enterocolitis: review of the literature and state of the art

Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal surgical emergency in preterm neonates. Over the last five decades, a variety of experimental models have been developed to study the pathophysiology of this disease and to test the effectiveness of novel therapeutic strategies. Experimental NEC is mainly modeled in neonatal rats, mice and piglets. In this review, we focus on these experimental models and discuss the major advantages and disadvantages of each. We also briefly discuss other models that are not as widely used but have contributed to our current knowledge of NEC

Leveraging cell type specific regulatory regions to detect SNPs associated with tissue factor pathway inhibitor plasma levels

International audienceTissue factor pathway inhibitor (TFPI) regulates the formation of intravascular blood clots, which manifest clinically as ischemic heart disease, ischemic stroke, and venous thromboembolism (VTE). TFPI plasma levels are heritable, but the genetics underlying TFPI plasma level variability are poorly understood. Herein we report the first genome‐wide association scan (GWAS) of TFPI plasma levels, conducted in 251 individuals from five extended French‐Canadian Families ascertained on VTE. To improve discovery, we also applied a hypothesis‐driven (HD) GWAS approach that prioritized single nucleotide polymorphisms (SNPs) in (1) hemostasis pathway genes, and (2) vascular endothelial cell (EC) regulatory regions, which are among the highest expressers of TFPI. Our GWAS identified 131 SNPs with suggestive evidence of association (P‐value < 5 × 10 −8), but no SNPs reached the genome‐wide threshold for statistical significance. Hemostasis pathway genes were not enriched for TFPI plasma level associated SNPs (global hypothesis test P‐ value = 0.147), but EC regulatory regions contained more TFPI plasma level associated SNPs than expected by chance (global hypothesis test P‐value = 0.046). We therefore stratified our genome‐wide SNPs, prioritizing those in EC regulatory regions via stratified false discovery rate (sFDR) control, and reranked the SNPs by q‐value. The minimum q‐value was 0.27, and the top‐ranked SNPs did not show association evidence in the MARTHA replication sample of 1,033 unrelated VTE cases. Although this study did not result in new loci for TFPI, our work lays out a strategy to utilize epigenomic data in prioritization schemes for future GWAS studies

Experimental necrotizing enterocolitis induces neuroinflammation in the neonatal brain

Abstract Background Necrotizing enterocolitis (NEC) is an inflammatory gastrointestinal disease primarily affecting preterm neonates. Neonates with NEC suffer from a degree of neurodevelopmental delay that is not explained by prematurity alone. There is a need to understand the pathogenesis of neurodevelopmental delay in NEC. In this study, we assessed the macroscopic and microscopic changes that occur to brain cell populations in specific brain regions in a neonatal mouse model of NEC. Moreover, we investigated the role of intestinal inflammation as part of the mechanism responsible for the changes observed in the brain of pups with NEC. Methods Brains of mice were assessed for gross morphology and cerebral cortex thickness (using histology). Markers for mature neurons, oligodendrocytes, neural progenitor cells, microglia, and astrocytes were used to quantify their cell populations in different regions of the brain. Levels of cell apoptosis in the brain were measured by Western blotting and immunohistochemistry. Endoplasmic reticulum (ER) stress markers and levels of pro-inflammatory cytokines (in the ileum and brain) were measured by RT-qPCR and Western blotting. A Pearson test was used to correlate the levels of cytokines (ELISA) in the brain and ileum and to correlate activated microglia and astrocyte populations to the severity of NEC. Results NEC pups had smaller brain weights, higher brain-to-body weight ratios, and thinner cortices compared to control pups. NEC pups had increased levels of apoptosis and ER stress. In addition, NEC was associated with a reduction in the number of neurons, oligodendrocytes, and neural progenitors in specific regions of the brain. Levels of pro-inflammatory cytokines and the density of activated microglia and astrocytes were increased in the brain and positively correlated with the increase in the levels pro-inflammatory cytokines in the gut and the severity of NEC damage respectively. Conclusions NEC is associated with severe changes in brain morphology, a pro-inflammatory response in the brain that alters cell homeostasis and density of brain cell populations in specific cerebral regions. We show that the severity of neuroinflammation is associated with the severity of NEC. Our findings suggest that early intervention during NEC may reduce the chance of acute neuroinflammation and cerebral damage

Genetic variation 25.1 Mb upstream of tissue factor pathway inhibitor is associated with TFPI plasma levels and venous thromboembolism

International audienceBackgroundTissue factor pathway inhibitor (TFPI) regulates fibrin clot formation, and low TFPI plasma levels increase the risk of arterial and venous thromboembolism (VTE). TFPI plasma levels are also heritable, and a previous linkage scan implicated the chromosome 2q region, but no specific genes.ObjectivesWe sought to replicate the linkage region in an independent sample and to identify the causal locus. MethodsWe first ran a linkage analysis of microsatellite markers and TFPI plasma levels in 251 individuals from the F5L Family Study and replicated the linkage peak on chromosome 2q (LOD=3.06). We next defined a follow-up region that included 112603 SNPs under the linkage peak, and meta-analyzed associations between these SNPs and TFPI plasma levels across the F5L Family Study and MARTHA, a study of 1033 unrelated VTE patients. SNPs with FDR q<0.10 were tested for association with TFPI plasma levels in 892 patients with coronary artery disease in the AtheroGene study.Results and ConclusionsOne SNP, rs62187992, was associated with TFPI plasma levels in all three samples (β=+0.14 P=4.23x10-6 combined; β=+0.16, P=0.02 in F5L Family Study; β=+0.13, P=6.3x10-4 in MARTHA; β=+0.17, P=0.03 in AtheroGene) and contributed to the linkage peak in the F5L Family Study. rs62187992 was also associated with clinical VTE (odds ratio=0.90, P=0.03) in the INVENT consortium of over 7000 cases and their controls and was marginally associated with TFPI expression (β=+0.19, P=0.08) in human aortic endothelial cells, a primary site of TFPI synthesis. The biological mechanisms underlying these associations remain to be elucidated