Most of the current exosome analysis strategies are time-consuming and largely dependent on commercial extraction kit based pre-isolation step, which requires extensive sample manipulations, costly isolation kits, reagents, tedious procedures, and sophisticated equipment, and is prone to bias/artifacts. Herein we introduce a simple method for direct isolation and subsequent detection of a specific population of exosomes using an engineered superparamagnetic material with multi-functional properties, namely gold-loaded ferric oxide nanocubes (Au-NPFe2O3NC). In this method, the Au-NPFe2O3NC were initially functionalized with a generic tetraspanin (exosomes associated) antibody (i.e., CD63) and dispersed in sample fluids where they work as 'dispersible nanocarriers' to capture the bulk population of exosomes. After magnetic collection and purification, Au-NPFe2O3NC-bound exosomes were transferred to the tissue-specific antibody-modified screen-printed electrode. As a proof of principle, we used a specific placental marker, placenta alkaline phosphatase (PLAP), to detect exosomes secreted from placental cells. The peroxidase-like activity of Au-NPFe2O3NC was then used to accomplish an ELISA-based sensing protocol for naked-eye observation along with UV-visible and electrochemical detection of PLAP-specific exosomes present in placental cell-conditioned media. We demonstrated excellent agreement in analytical performance for the detection of placental cell-derived exosomes (i.e., linear dynamic range, 10 3-10 7 exosomes/mL; limit of detection, 10 3 exosomes/mL; relative standard deviation (%RSD) of < 5.5 % for n=3) using with and without commercial "total exosome isolation kit" based preisolation step. We envisage that this highly sensitive, rapid and inexpensive assay could be useful in quantifying specific populations of exosomes for various clinical applications, focusing on pregnancy complications.
There is increasing evidence that miRNAs, which are enriched in nanovesicles called exosomes, are important regulators of gene expression. When compared with normal pregnancies, pregnancies with gestational diabetes mellitus (GDM) are associated with skeletal muscle insulin resistance as well as increased levels of circulating placental exosomes. Here we investigated whether placental exosomes in GDM carry a specific set of miRNAs associated with skeletal muscle insulin sensitivity. Exosomes were isolated from chorionic villous (CV) explants from both women with Normal Glucose Tolerant (NGT) and GDM pregnancies. Using miRNA sequencing, we identified a specific set of miRNAs selectively enriched with exosomes and compared with their cells of origin indicating a specific packaging of miRNAs into exosomes. Gene target and ontology analysis of miRNA differentially expressed in exosomes secreted in GDM compared with NGT are associated with pathways regulating cell migration and carbohydrate metabolism. We determined the expression of a selected set of miRNAs in placenta, plasma, and skeletal muscle biopsies from NGT and GDM. Interestingly, the expression of these miRNAs varied in a consistent pattern in the placenta, in circulating exosomes, and in skeletal muscle in GDM. Placental exosomes from GDM pregnancies decreased insulin-stimulated migration and glucose uptake in primary skeletal muscle cells obtained from patients with normal insulin sensitivity. Interestingly, placental exosomes from NGT increase migration and glucose uptake in response to insulin in skeletal muscle from diabetic subjects. These findings suggest that placental exosomes might have a role in the changes on insulin sensitivity in normal and GDM pregnancies.
Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.
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