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1. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

Author

Clotilde Théry, Kenneth W Witwer, Elena Aikawa, Maria Jose Alcaraz, Johnathon D Anderson, Ramaroson Andriantsitohaina, Anna Antoniou, Tanina Arab, Fabienne Archer, Georgia K Atkin-Smith, D Craig Ayre, Jean-Marie Bach, Daniel Bachurski, Hossein Baharvand, Leonora Balaj, Shawn Baldacchino, Natalie N Bauer, Amy A Baxter, Mary Bebawy, Carla Beckham, Apolonija Bedina Zavec, Abderrahim Benmoussa, Anna C Berardi, Paolo Bergese, Ewa Bielska, Cherie Blenkiron, Sylwia Bobis-Wozowicz, Eric Boilard, Wilfrid Boireau, Antonella Bongiovanni, Francesc E Borràs, Steffi Bosch, Chantal M Boulanger, Xandra Breakefield, Andrew M Breglio, Meadhbh Á Brennan, David R Brigstock, Alain Brisson, Marike LD Broekman, Jacqueline F Bromberg, Paulina Bryl-Górecka, Shilpa Buch, Amy H Buck, Dylan Burger, Sara Busatto, Dominik Buschmann, Benedetta Bussolati, Edit I Buzás, James Bryan Byrd, Giovanni Camussi, David RF Carter, Sarah Caruso, Lawrence W Chamley, Yu-Ting Chang, Chihchen Chen, Shuai Chen, Lesley Cheng, Andrew R Chin, Aled Clayton, Stefano P Clerici, Alex Cocks, Emanuele Cocucci, Robert J Coffey, Anabela Cordeiro-da-Silva, Yvonne Couch, Frank AW Coumans, Beth Coyle, Rossella Crescitelli, Miria Ferreira Criado, Crislyn D’Souza-Schorey, Saumya Das, Amrita Datta Chaudhuri, Paola de Candia, Eliezer F De Santana, Olivier De Wever, Hernando A del Portillo, Tanguy Demaret, Sarah Deville, Andrew Devitt, Bert Dhondt, Dolores Di Vizio, Lothar C Dieterich, Vincenza Dolo, Ana Paula Dominguez Rubio, Massimo Dominici, Mauricio R Dourado, Tom AP Driedonks, Filipe V Duarte, Heather M Duncan, Ramon M Eichenberger, Karin Ekström, Samir EL Andaloussi, Celine Elie-Caille, Uta Erdbrügger, Juan M Falcón-Pérez, Farah Fatima, Jason E Fish, Miguel Flores-Bellver, András Försönits, Annie Frelet-Barrand, Fabia Fricke, Gregor Fuhrmann, Susanne Gabrielsson, Ana Gámez-Valero, Chris Gardiner, Kathrin Gärtner, Raphael Gaudin, Yong Song Gho, Bernd Giebel, Caroline Gilbert, Mario Gimona, Ilaria Giusti, Deborah CI Goberdhan, André Görgens, Sharon M Gorski, David W Greening, Julia Christina Gross, Alice Gualerzi, Gopal N Gupta, Dakota Gustafson, Aase Handberg, Reka A Haraszti, Paul Harrison, Hargita Hegyesi, An Hendrix, Andrew F Hill, Fred H Hochberg, Karl F Hoffmann, Beth Holder, Harry Holthofer, Baharak Hosseinkhani, Guoku Hu, Yiyao Huang, Veronica Huber, Stuart Hunt, Ahmed Gamal-Eldin Ibrahim, Tsuneya Ikezu, Jameel M Inal, Mustafa Isin, Alena Ivanova, Hannah K Jackson, Soren Jacobsen, Steven M Jay, Muthuvel Jayachandran, Guido Jenster, Lanzhou Jiang, Suzanne M Johnson, Jennifer C Jones, Ambrose Jong, Tijana Jovanovic-Talisman, Stephanie Jung, Raghu Kalluri, Shin-ichi Kano, Sukhbir Kaur, Yumi Kawamura, Evan T Keller, Delaram Khamari, Elena Khomyakova, Anastasia Khvorova, Peter Kierulf, Kwang Pyo Kim, Thomas Kislinger, Mikael Klingeborn, David J Klinke, Miroslaw Kornek, Maja M Kosanović, Árpád Ferenc Kovács, Eva-Maria Krämer-Albers, Susanne Krasemann, Mirja Krause, Igor V Kurochkin, Gina D Kusuma, Sören Kuypers, Saara Laitinen, Scott M Langevin, Lucia R Languino, Joanne Lannigan, Cecilia Lässer, Louise C Laurent, Gregory Lavieu, Elisa Lázaro-Ibáñez, Soazig Le Lay, Myung-Shin Lee, Yi Xin Fiona Lee, Debora S Lemos, Metka Lenassi, Aleksandra Leszczynska, Isaac TS Li, Ke Liao, Sten F Libregts, Erzsebet Ligeti, Rebecca Lim, Sai Kiang Lim, Aija Linē, Karen Linnemannstöns, Alicia Llorente, Catherine A Lombard, Magdalena J Lorenowicz, Ákos M Lörincz, Jan Lötvall, Jason Lovett, Michelle C Lowry, Xavier Loyer, Quan Lu, Barbara Lukomska, Taral R Lunavat, Sybren LN Maas, Harmeet Malhi, Antonio Marcilla, Jacopo Mariani, Javier Mariscal, Elena S Martens-Uzunova, Lorena Martin-Jaular, M Carmen Martinez, Vilma Regina Martins, Mathilde Mathieu, Suresh Mathivanan, Marco Maugeri, Lynda K McGinnis, Mark J McVey, David G Meckes, Katie L Meehan, Inge Mertens, Valentina R Minciacchi, Andreas Möller, Malene Møller Jørgensen, Aizea Morales-Kastresana, Jess Morhayim, François Mullier, Maurizio Muraca, Luca Musante, Veronika Mussack, Dillon C Muth, Kathryn H Myburgh, Tanbir Najrana, Muhammad Nawaz, Irina Nazarenko, Peter Nejsum, Christian Neri, Tommaso Neri, Rienk Nieuwland, Leonardo Nimrichter, John P Nolan, Esther NM Nolte-’t Hoen, Nicole Noren Hooten, Lorraine O’Driscoll, Tina O’Grady, Ana O’Loghlen, Takahiro Ochiya, Martin Olivier, Alberto Ortiz, Luis A Ortiz, Xabier Osteikoetxea, Ole Østergaard, Matias Ostrowski, Jaesung Park, D. Michiel Pegtel, Hector Peinado, Francesca Perut, Michael W Pfaffl, Donald G Phinney, Bartijn CH Pieters, Ryan C Pink, David S Pisetsky, Elke Pogge von Strandmann, Iva Polakovicova, Ivan KH Poon, Bonita H Powell, Ilaria Prada, Lynn Pulliam, Peter Quesenberry, Annalisa Radeghieri, Robert L Raffai, Stefania Raimondo, Janusz Rak, Marcel I Ramirez, Graça Raposo, Morsi S Rayyan, Neta Regev-Rudzki, Franz L Ricklefs, Paul D Robbins, David D Roberts, Silvia C Rodrigues, Eva Rohde, Sophie Rome, Kasper MA Rouschop, Aurelia Rughetti, Ashley E Russell, Paula Saá, Susmita Sahoo, Edison Salas-Huenuleo, Catherine Sánchez, Julie A Saugstad, Meike J Saul, Raymond M Schiffelers, Raphael Schneider, Tine Hiorth Schøyen, Aaron Scott, Eriomina Shahaj, Shivani Sharma, Olga Shatnyeva, Faezeh Shekari, Ganesh Vilas Shelke, Ashok K Shetty, Kiyotaka Shiba, Pia R-M Siljander, Andreia M Silva, Agata Skowronek, Orman L Snyder, Rodrigo Pedro Soares, Barbara W Sódar, Carolina Soekmadji, Javier Sotillo, Philip D Stahl, Willem Stoorvogel, Shannon L Stott, Erwin F Strasser, Simon Swift, Hidetoshi Tahara, Muneesh Tewari, Kate Timms, Swasti Tiwari, Rochelle Tixeira, Mercedes Tkach, Wei Seong Toh, Richard Tomasini, Ana Claudia Torrecilhas, Juan Pablo Tosar, Vasilis Toxavidis, Lorena Urbanelli, Pieter Vader, Bas WM van Balkom, Susanne G van der Grein, Jan Van Deun, Martijn JC van Herwijnen, Kendall Van Keuren-Jensen, Guillaume van Niel, Martin E van Royen, Andre J van Wijnen, M Helena Vasconcelos, Ivan J Vechetti, Tiago D Veit, Laura J Vella, Émilie Velot, Frederik J Verweij, Beate Vestad, Jose L Viñas, Tamás Visnovitz, Krisztina V Vukman, Jessica Wahlgren, Dionysios C Watson, Marca HM Wauben, Alissa Weaver, Jason P Webber, Viktoria Weber, Ann M Wehman, Daniel J Weiss, Joshua A Welsh, Sebastian Wendt, Asa M Wheelock, Zoltán Wiener, Leonie Witte, Joy Wolfram, Angeliki Xagorari, Patricia Xander, Jing Xu, Xiaomei Yan, María Yáñez-Mó, Hang Yin, Yuana Yuana, Valentina Zappulli, Jana Zarubova, Vytautas Žėkas, Jian-ye Zhang, Zezhou Zhao, Lei Zheng, Alexander R Zheutlin, Antje M Zickler, Pascale Zimmermann, Angela M Zivkovic, Davide Zocco, and Ewa K Zuba-Surma

Subjects
extracellular vesicles, exosomes, ectosomes, microvesicles, minimal information requirements, guidelines, standardization, microparticles, rigor, reproducibility, Cytology, QH573-671
Abstract

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Published
2018
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2. Analysis of the regenerative capacity of human serum exosomes after a simple multistep separation from lipoproteins

Author

Nicholas J. Woudberg, Deon Bezuidenhout, Jason Lovett, Paul Human, Candice de Boer, K. H. Myburgh, Bridget Calder, Dee Blackhurst, Martina Steinmaurer, David Marais, Neil H. Davies, Jonathan M. Blackburn, and Sandrine Lecour

Subjects
Serum, Apolipoprotein B, Lipoproteins, 0206 medical engineering, Biomedical Engineering, Neovascularization, Physiologic, Medicine (miscellaneous), Nanoparticle tracking analysis, 02 engineering and technology, Exosomes, Exosome, Biomaterials, 03 medical and health sciences, In vivo, Animals, Humans, 030304 developmental biology, 0303 health sciences, biology, Chemistry, Dermis, 020601 biomedical engineering, Blood proteins, Microvesicles, Rats, Biochemistry, biology.protein, Density gradient ultracentrifugation, Lipoprotein
Abstract

Due to the abundance of lipoproteins in blood, it is challenging to characterize the biological functions and components of blood-derived extracellular vesicles. The aim of this study was to develop a multiple-step purification protocol to separate serum exosomes from serum proteins and lipoproteins and assess their regenerative potential. Exosomes were isolated by concentrating them in human serum using ultracentrifugation (UC), followed sequentially by density gradient (DG) UC and size exclusion chromatography (SEC). Purity and characterization were assessed by western blots, Lipoprint®, enzyme-linked immunosorbent assay, electron microscopy, mass spectrometry, and nanoparticle tracking analysis. Functionality was assessed by cell proliferation analysis and with an in vivo subcutaneous angiogenesis model. SEC alone isolated nano-sized vesicles possessing vesicle markers TSG101 and CD9, but there was a substantial presence of apolipoprotein B, predominantly derived from very-low- and intermediate-density lipoprotein particles. This was reduced to an undetectable level using the combined UC DG SEC approach. Mass spectrometry identified 224 proteins in UC DG SEC isolates relative to the 135 from SEC, with considerable increases in exosome-related proteins and reductions in lipoproteins. A consistent but limited increase in human dermal fibroblast proliferation and evidence of neovascularization enhancement were observed after exposure to UC DG SEC exosomes. An UC DG SEC purification protocol considerably improved the removal of lipoproteins during isolation of serum exosomes. The purified exosomes stimulated cell proliferation and potentially increased an in vivo angiogenic response. This multistep purification allows for more accurate identification of serum exosome functional activity and composition.

Published
2020

3. Investigation of Circulating Extracellular Vesicle MicroRNA Following Two Consecutive Bouts of Muscle-Damaging Exercise

Author

Peter J. Durcan, Kathryn H. Myburgh, and Jason Lovett

Subjects
0301 basic medicine, medicine.medical_specialty, Physiology, Nanoparticle tracking analysis, exosomes, medicine.disease_cause, Exosome, lcsh:Physiology, 03 medical and health sciences, 0302 clinical medicine, Jumping, Physiology (medical), Internal medicine, microRNA, medicine, blood samples, skeletal muscle, Original Research, exercise, lcsh:QP1-981, Chemistry, myomiR, Skeletal muscle, Extracellular vesicle, eccentric contraction, Microvesicles, microRNAs, 030104 developmental biology, Endocrinology, medicine.anatomical_structure, extracellular vesicles, 030217 neurology & neurosurgery, Intracellular
Abstract

Background: Extracellular vesicles (EVs) are nano-sized vesicles that are known to be powerful mediators of intercellular communication via their microRNA (miR) content. A paucity of information on EV-mediated communication arising from skeletal muscle (SkM) in response to exercise-induced muscle damage is present in the published literature. Lack of such information inhibits our understanding of muscle injury and repair processes. Aims: To assess circulating EV levels and selected miR content within them, in response to two consecutive bouts of muscle-damaging exercise. Methods: Serum creatine kinase activity (CK) and EVs were analyzed from the blood of 9 healthy, untrained males at baseline, and at 2 and 24 h post-exercise. The exercise regimen consisted of a combination of plyometric jumping and downhill running. Perceived muscle pain (PMP) was assessed on a scale from 1 to 10. Plasma EVs were isolated using size exclusion columns and visualized with transmission electron microscopy (TEM). EV size and number were quantified using nanoparticle tracking analysis (NTA). miR expression was quantified using qPCR, with normalization to an exogenous control (cel-miR-39). Results: PMP and CK were significantly elevated post-exercise compared to baseline levels, providing indirect evidence for muscle damage. EV visualization using TEM revealed an abundant and heterogeneously sized pool of intact particles within the exosome size range (30–150 nm). No significant change in mean EV size or number was seen over time. The SkM-specific miR-206 in EVs was found to be variable among participants and no significant change occurred in SkM-important miRs; 1, 133a, 133b, 486, and 499a. However, EV miR-31 decreased from baseline to 24 h post-exercise (p = 0.027). Conclusion: Mild to moderate exercise-induced muscle damage altered the miR-31 profile of circulating EVs within the first 24 h post-exercise, but not that of myomiRs in EVs. These data demonstrate that EVs carry selectively packaged cargo which can be affected by exercise. Future research into the total miR content of EVs in response to exercise-induced muscle damage may reveal other miRs responsive to this relatively mild perturbation. More time points post-muscle-damaging exercise would provide a better understanding of the temporal EV myomiR response.

Published
2018

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