This paper explores the biocompatibility and immunotoxicity of bovine milk-derived extracellular vesicles (EVs), a potentially important scalable resource for the production of therapeutic EVs. Using a new method coupling acid treatment and ultracentrifugation, recovered milk EVs are found not to trigger activation of Raw264.7 macrophages and, when introduced intravenously to mice, did not elicit systemic toxicity or, upon repeated introduction, anaphylaxis. However, certain cytokines (IL6 and GCSF) were found to be slightly induced shortly after EV introduction but returned to normal levels after 14 days. Cosmo Bio antibody (anti-human CD81: clone 12C4, developed by Shionogi Pharmaceutical) was used to monitor EV recovery. (Somiya, M., Yoshioka, Y., Ochiya, T. (2018). Biocompatibility of highly purified bovine milk-derived extracellular vesicles Journal of Extracellular Vesicles 7(1), 1440132.)
This paper addresses the functional interaction between murine macrophages and HDL particles from healthy and coronary atherosclerosis patients. It finds that HDL(healthy) particles act by a clatherin dependent endocytosis pathway to elevate phagocytosis and attenuate LTB4 production whereas HDL(athero) particles de novo synthesize LTB4 that blocks endocytic uptake and attenuation of cellular LTB4 production. The authors speculate that neutrophilic exosomes could be the source of the LTB4 synthetic enzymes carried by HDL (athero) particles. Cosmo Bio ExoTrap™ Exosome Isolation Spin Column Kit and anti-human CD9 antibody (clone 12A12, developed by Shionogi Pharmaceutical) were used to isolate and monitor exosome preparations. Cosmo Bio antibody (anti-human CD81: clone 12C4, developed by Shionogi Pharmaceutical) was used to monitor EV recovery. (Tsuda, S., Shinohara, M., Oshita, T., Nagao, M., Tanaka, N., Mori, T., Hara, T., Irino, Y., Toh, R., Ishida, T., Hirata, K. (2017). Novel mechanism of regulation of the 5-lipoxygenase/leukotriene B4 pathway by high-density lipoprotein in macrophages Scientific Reports 7(1), 12989.)
This report explores the effect of exosome secretion on exosome-secreting cells. The authors find that exosome secretion in senescent and pre-senescent human diploid fibroblasts plays a critical role in maintaining cellular homeostasis. Inhibition of exosome secretion by RNAi and pharmacologic approaches led to the accumulation of nuclear double stranded DNA in the cytoplasm that activated the STING-dependent innate cytoplasmic DNA sensor pathway. This activation lead to the production of type I interferon and the elevation of intracellular ROS which in turn caused DNA damage and the activation of the DNA damage response pathway. Cosmo Bio antibody (anti-human CD81: clone 12C4, developed by Shionogi Pharmaceutical) and other exosomal marker antibodies were used to monitor exosome recovery from culture supernatants and liver tissue. (Takahashi, A., Okada, R., Nagao, K., Kawamata, Y., Hanyu, A., Yoshimoto, S., Takasugi, M., Watanabe, S., Kanemaki, M., Obuse, C., Hara, E. (2017). Exosomes maintain cellular homeostasis by excreting harmful DNA from cells Nature Communications 8(1), 15287.)
This critically important paper reveals results of new and improved exosome purification techniques [high resolution density gradient fractionation and direct immuno-affinity capture (DIC)], revealing that: 1) CD9+CD63+CD81+ small extracellular vesicles (classical-exosome phenotype) lack the proteins GAPDH and HSP90; 2) lack cytoskeletal proteins; 3) lack glycolytic enzymes; 4) lack membrane-bound annexins A1 and A2 (of which A1 is shown, instead, to be a specific marker of microvesicles); 5) lack Ago proteins 1-4 and all RISC and other miRNA-associated enzymes; and 6) surprisingly, lack double-stranded DNA. The lack of these elements suggests that the process of exosome loading must be a highly regulated process and that many previous studies reporting the association of exosomes with DNA, and a variety of proteins (including RNA binding proteins) are likely to have resulted from incomplete purification. (Jeppesen, D., Fenix, A., Franklin, J., Higginbotham, J., Zhang, Q., Zimmerman, L., Liebler, D., Ping, J., Liu, Q., Evans, R., Fissell, W., Patton, J., Rome, L., Burnette, D., Coffey, R. (2019). Reassessment of Exosome Composition Cell 177(2), 428-445.e18.)
This Leading Edge Preview of a recent Cell paper from Jeppesen’s lab covers the highlights of the results of a new exosome purification technique that revealed CD9+CD63+CD81+ small vesicles (classical exosome phenotype) lack the proteins GAPDH and HSP90; 2) lack cytoskeletal proteins; 3) lack glycolytic enzymes; 4) lack membrane-bound annexins A1 and A2, shown instead to be specific markers of microvesicles; 5) lack Ago proteins 1-4 and all RISC and other miRNA-associated enzymes; and 6) surprisingly, lack DNA. The lack of these elements suggests that the process of exosome loading must be a highly regulated process. (Pluchino, S., Smith, J. (2019). Explicating Exosomes: Reclassifying the Rising Stars of Intercellular Communication Cell 177(2), 225-227.)
Currently, there is no consensus on the best method to isolate exosomes. This paper compares differential soluble factor (in particular, cytokine) contamination of exosome preparations purified from the conditioned media of 2 different melanoma cell lines (2183-Her4 and 888-mel) by 3 different methods: 1) ultracentrifugation; 2) Rapid Exosome Isolation Using Size exclusion chromatography (REIUS); and 3) ExoQuick ULTRA (EqU, System Biosciences). Exosome isolates from both melanoma cell lines reveal cytokines are isolated along with exosomes but their expression profiles depend on the cell line and exosome isolation method. In particular, ultracentrifugation can lead to significant co-isolation of contaminating cytokines. Thus, many factors reported as exosome payloads may instead be co-elutes specific to the isolation technique used. (Shu, S., Yang, Y., Allen, C., Hurley, E., Tung, K., Minderman, H., Wu, Y., Ernstoff, M. (2019). Purity and yield of melanoma exosomes are dependent on isolation method Journal of Extracellular Vesicles 9(1), 1692401.)
This paper introduces a new technology (Single Particle Interferomtric Reflectance Imaging Sensor: SP-IRIS) that can multiplex phenotype and digitally count various populations of individual exosomes captured on a microarray-based solid phase chip. Exosomes are captured on the surface of silicon chips through antibodies targeting exosomal markers. Interference of light reflected from the sensor surface is modified by the presence of particles producing a distinct signal that correlates to the size of the particle. This is the first technology where a single instrument provides size and multi-phenotype information from a single exosome preparation. Validation was performed using 20ul of human cerebrospinal fluid and benchmark comparisons were made with Nanoparticle Tracking Analysis, atomic force microscopy and scanning electron microscopy. (Daaboul, G., Gagni, P., Benussi, L., Bettotti, P., Ciani, M., Cretich, M., Freedman, D., Ghidoni, R., Ozkumur, A., Piotto, C., Prosperi, D., Santini, B., Ünlü, M., Chiari, M. (2016). Digital Detection of Exosomes by Interferometric Imaging Scientific Reports 6(1), 37246.)
This paper compares extracellular vesicle (EV) isolation methods (ExoQuick™ Exosome Precipitation Kit, System Biosciences; Total Exosome Isolation Reagent, Life Technologies; Urine Exosome RNA Isolation Kit, Norgen Biotek; and differential ultracentrifugation) and RNA extraction methods (Total RNA Purification Kit and Urine Exosome RNA Isolation Kit, Norgen Biotek; SeraMir™ Exosome RNA Purification Column kit, System Biosciences; miRNeasy Micro kit and exoRNeasy Serum/Plasma kit, Qiagen; mirVana™ miRNA Isolation Kit, Ambion; and Total Exosome RNA & Protein Isolation kit, Invitrogen) from human serum or urine samples and sought to identify suitable endogenous normalization controls (ECs) for qRTPCR analysis. The study identified RNU48 and HY3 for urine and U6 and HY3 for serum to be optimal ECs for EV analysis. (Crossland, R., Norden, J., Bibby, L., Davis, J., Dickinson, A. (2016). Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine Journal of Immunological Methods 429(Kidney Int. 82 2012), 39-49.)
This is a review of the clinical applications of extracellular vesicle (EV) research by one of the leaders in the field. After describing the still unresolved issue of a standardized clinically applicable EV isolation methodology, the focus moves to the latest developments in: 1) EVs as drug delivery/vaccination vehicles; 2) therapeutically targeting EV biogenesis and uptake as a strategy to address cancer; and 3) EVs as a minimally invasive source of diagnostic and prognostic biomarkers for diseases like cancer. (Yamamoto, T., Kosaka, N., Ochiya, T. (2019). Latest advances in extracellular vesicles: from bench to bedside Science and Technology of Advanced Materials 20(1), 746-757.)
