flow-cytometric-analysis-extracellular-vesicles-from-cell-conditioned

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Cardiocentro Ticino Foundation, Switzerland

Recent evidence suggests that cardiac progenitor cells (CPCs) may improve cardiac function after injury. The underlying mechanisms are indirect, but their mediators remain unidentified. Exosomes and other secreted membrane vesicles, hereafter collectively referred to as extracellular vesicles (EVs), act as paracrine signalling mediators. Here, we report that EVs secreted by human CPCs are crucial cardioprotective agents.

Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction.

Virtually all cells in the organism secrete extracellular vesicles (EVs), a heterogeneous population of lipid bilayer membrane-enclosed vesicles that transport and deliver payloads of proteins and nucleic acids to recipient cells, thus playing central roles in cell-cell communications. Exosomes, nanosized EVs of endosomal origin, regulate many pathophysiological processes including immune responses and inflammation, tumour growth, and infection. Healthy subjects and patients with different diseases release exosomes with different RNA and protein contents into the circulation, which can be measured as biomarkers. The discovery of exosomes as natural carriers of functional small RNA and proteins has raised great interest in the drug delivery field, as it may be possible to harness these vesicles for therapeutic delivery of miRNA, siRNA, mRNA, lncRNA, peptides, and synthetic drugs. However, systemically delivered exosomes accumulate in liver, kidney, and spleen. Targeted exosomes can be obtained by displaying targeting molecules, such as peptides or antibody fragments recognizing target antigens, on the outer surface of exosomes. Display of glycosylphosphatidylinositol (GPI)-anchored nanobodies on EVs is a novel technique that enables EV display of a variety of proteins including antibodies, reporter proteins, and signaling molecules. However, naturally secreted exosomes show limited pharmaceutical acceptability. Engineered exosome mimetics that incorporate desirable components of natural exosomes into synthetic liposomes or nanoparticles, and are assembled using controllable procedures may be more acceptable pharmaceutically. In this communication, we review the current understanding of physiological and pathophysiological roles of exosomes, their potential applications as diagnostic markers, and current efforts to develop improved exosome-based drug delivery systems.

Exosomes: Therapy delivery tools and biomarkers of diseases.

Cell therapy has been evaluated to enhance heart function after injury. Delivered cells mostly act via paracrine mechanisms, including secreted growth factors, cytokines, and vesicles, such as exosomes (Exo). Intramyocardial injection of cardiac-resident progenitor cells (CPC)-derived Exo reduced scarring and improved cardiac function after myocardial infarction in rats. Here, we explore a clinically relevant approach to enhance the homing process to cardiomyocytes (CM), which is crucial for therapeutic efficacy upon systemic delivery of Exo. By overexpressing exosomal CXCR4, we increased the efficacy of plasmatic injection of cardioprotective Exo-CPC by increasing their bioavailability to ischemic hearts. Intravenous injection of Exo significantly reduced infarct size and improved left ventricle ejection fraction at 4 weeks compared to Exo ( < 0.01). Hemodynamic measurements showed that Exo improved dp/dt min, as compared to Exo and PBS group. In vitro, Exo was more bioactive than Exo in preventing CM death. This in vitro effect was independent from SDF-1α, as shown by using AMD3100 as specific CXCR4 antagonist. We showed, for the first time, that systemic administration of Exo derived from CXCR4-overexpressing CPC improves heart function in a rat model of ischemia reperfusion injury These data represent a substantial step toward clinical application of Exo-based therapeutics in cardiovascular disease.

Lucio Barile

Cellular and Molecular Cardiology Laboratory

Cardiocentro Ticino Foundation, Switzerland

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Lucio Barile

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Cellular and Molecular Cardiology Laboratory

Cardiocentro Ticino Foundation, Switzerland

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Cellular and Molecular Cardiology Laboratory