Identification of storage conditions stabilizing extracellular vesicles pre

Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS-HAT). We report that PBS-HAT buffer facilitates clearly improved short-term and long-term EV preservation for samples stored at -80°C, stability throughout several freeze-thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications

Alveolar macrophage- derived extracellular vesicles inhibit endosomal fusion of influenza virus

Alveolar macrophages (AMs) and epithelial cells (ECs) are the lone resident lung cells positioned to respond to pathogens at early stages of infection. Extracellular vesicles (EVs) are important vectors of paracrine signaling implicated in a range of (patho)physiologic contexts. Here we demonstrate that AMs, but not ECs, constitutively secrete paracrine activity localized to EVs which inhibits influenza infection of ECs in vitro and in vivo. AMs exposed to cigarette smoke extract lost the inhibitory activity of their secreted EVs. Influenza strains varied in their susceptibility to inhibition by AM- EVs. Only those exhibiting early endosomal escape and high pH of fusion were inhibited via a reduction in endosomal pH. By contrast, strains exhibiting later endosomal escape and lower fusion pH proved resistant to inhibition. These results extend our understanding of how resident AMs participate in host defense and have broader implications in the defense and treatment of pathogens internalized within endosomes.SynopsisExtracellular vesicles are emerging as homeostatic vectors, but poorly understood in influenza infection. Here, alveolar macrophage- derived extracellular vesicles inhibit influenza- endosome fusion in a strain- specific, and pH- dependent manner.Following initial infection of epithelial cells, the influenza virus traffics within host cell endosomes which undergo progressive acidification.Prior to gaining entry into the nucleus for its replication, influenza virus must fuse with endosome membranes- an event initiated at a strain- specific pH.Alveolar macrophages secrete extracellular vesicles which, when internalized by epithelial cells, lead to accelerated acidification of endosomes.Infection of epithelial cells by influenza strains which preferentially fuse with endosome membranes at high pH is inhibited by extracellular vesicles. Infection by influenza strains which fuse at low pH is unaffected by extracellular vesicles.Extracellular vesicles secreted from alveolar macrophages can promote acidification of endosomes in influenza virus- infected epithelial cells to inhibit viral replication.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156477/5/embj2020105057-sup-0002-EVFigs.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156477/4/embj2020105057_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156477/3/embj2020105057.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156477/2/embj2020105057-sup-0001-Appendix.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156477/1/embj2020105057.reviewer_comments.pd

Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Extracellular vesicles (EV) convey biological information by transmitting macromolecules between cells and tissues and are of great promise as pharmaceutical nanocarriers, and as therapeutic per se. Strategies for customizing the EV surface and cargo are being developed to enable their tracking, visualization, loading with pharmaceutical agents and decoration of the surface with tissue targeting ligands. While much progress has been made in the engineering of EVs, an exhaustive comparative analysis of the most commonly exploited EV-associated proteins, as well as a quantification at the molecular level are lacking. Here, we selected 12 EV-related proteins based on MS-proteomics data for comparative quantification of their EV engineering potential. All proteins were expressed with fluorescent protein (FP) tags in EV-producing cells; both parent cells as well as the recovered vesicles were characterized biochemically and biophysically. Using Fluorescence Correlation Spectroscopy (FCS) we quantified the number of FP-tagged molecules per vesicle. We observed different loading efficiencies and specificities for the different proteins into EVs. For the candidates showing the highest loading efficiency in terms of engineering, the molecular levels in the vesicles did not exceed ca 40–60 fluorescent proteins per vesicle upon transient overexpression in the cells. Some of the GFP-tagged EV reporters showed quenched fluorescence and were either non-vesicular, despite co-purification with EVs, or comprised a significant fraction of truncated GFP. The co-expression of each target protein with CD63 was further quantified by widefield and confocal imaging of single vesicles after double transfection of parent cells. In summary, we provide a quantitative comparison for the most commonly used sorting proteins for bioengineering of EVs and introduce a set of biophysical techniques for straightforward quantitative and qualitative characterization of fluorescent EVs to link single vesicle analysis with single molecule quantification

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications

Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome

Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49–96%) corresponded to rRNA-, coding- and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics

Engineered extracellular vesicle decoy receptor-mediated modulation of the IL6 trans-signalling pathway in muscle

The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two main mechanisms, the classical and trans-signalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention. Extracellular vesicles (EVs) are natural, lipid-bound nanoparticles, with potential as targeted delivery vehicles for therapeutic macromolecules. Here, we engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signalling pathway. The potency of the IL6ST decoy receptor EVs was optimized by inclusion of a GCN4 dimerization domain and a peptide sequence derived from syntenin-1 which targets the decoy receptor to EVs. The resulting engineered EVs were able to efficiently inhibit activation of the IL6 trans-signalling pathway in reporter cells, while having no effect on the IL6 classical signalling. IL6ST decoy receptor EVs, were also capable of blocking the IL6 trans-signalling pathway in C2C12 myoblasts and myotubes, thereby inhibiting the phosphorylation of STAT3 and partially reversing the anti-differentiation effects observed when treating cells with IL6/IL6R complexes. Treatment of a Duchenne muscular dystrophy mouse model with IL6ST decoy receptor EVs resulted in a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of these mice, thereby demonstrating in vivo activity of the decoy receptor EVs as a potential therapy. Taken together, this study reveals the IL6 trans-signalling pathway as a promising therapeutic target in DMD, and demonstrates the therapeutic potential of IL6ST decoy receptor EVs