Exosomes and Extracellular-Vesicles (EV)
... ... the content and physiological function

Interesting links:
Leadling Exosome Societies & Journals:

extracellular RNA  ( exRNA )

Ribonucleic acid (RNA) was once thought to exist in a stable form only inside cells, where it served as an intermediate in the translation from genes to proteins. However, recent research has indicated that RNAs can play a role in a variety of complex cellular functions, including newly discovered mechanisms of cell-to-cell communication. RNA can be exported from cells in extracellular vesicles or bound to lipids or proteins, to circulate through the body and affect cells at a great distance. These extracellular RNAs, or “exRNAs,” may also be absorbed from food, the microbes that live in our bodies, or the environment, potentially eliciting a variety of biological responses. However, the actual impact of these exRNAs is not known. An opportunity exists to establish entirely new paradigms of intercellular and inter-species information exchange based on the release, transport, uptake, and regulatory role of exRNAs.

Learn About Exosomes
Scientists’ Overview of Exosomes -- What, Why, How?

Researchers from academia and biotechnology firms summarize the basics of exosomes and how they are viewed as crucial to many advancements in medicine and the bio-sciences.


The launch of Journal of Extracellular Vesicles (JEV), the official journal of the International Society for Extracellular Vesicles about microvesicles, exosomes, ectosomes and other extracellular vesicles
Jan Lötvall, Lawrence Rajendran, Yong Song Gho, Clotilde Thery, Marca Wauben, Graca Raposo, Margareta Sjöstrand, Douglas Taylor, Esbjörn Telemo and Xandra O. Breakefield.
Journal of Extracellular Vesicles 2012, 1: 18514

EVSEARCH -- Extracellular Vesicle Research Center Denmark
EVSearch is a society of Danish researchers with a broad interest in extracellular vesicles and their biological functions and cargos. http://evsearch.dk

During the past few years intense and exciting research in extra-cellular vesicles has generated evidence for a new system for the exchange of information between tissues. Extracellular vesicles display a variable and abundant spectrum of bio-active substances and receptors on their surface, and harbor a concentrated set of cytokines, signaling proteins and various forms of RNA, allowing specific interaction and cross-talk with various target tissues. Thus, extracellular vesicles may be considered as veritable vectors for the intercellular exchange and biological signals and information, and may transfer part of their components and content to selected target cells, thus mediating cell activation, phenotypic modification, and reprogramming of cell function.

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Videos on Extracellular RNA

Learn more about Unlocking the Mysteries of Extracellular RNA Communication here
Watch a mini documentary series on Exosomes by Life TechnologiesCorp, featuring several ExRNA Communication grantees and Working Group members!
Part 1: What is an Exosome? Exit Disclaimer
Part 2: The History and Promise of Exosomes
Part 3: Exosomes in Cancer Research
Part 4: Curiosity and a Passion for Science
Part 5: Collaboration - The Key to Scientific Success
Part 6: Exosomes - The Next Small Thing

Introduction & Overview
Exosomes are cell-derived vesicles that are present in many tissues and (perhaps) in all biological fluids, including blood, milk, urine, sweat and cell culture supernatant. The reported size of exosomes is between 30 and 100 nm in diameter. There are a lot of release mechanisms proposed. Exosomes are either released from the cell when cytoplasmic multivesicular bodies fuse with the plasma membrane or they are released directly from the plasma membrane. You will find interesting papers about the exosome biogenesis and the exosome release below!
It is becoming increasingly clear that exosomes have specialized functions and play a key role in inter-cellular communication, e.g. in the immune system, in cancer progression, in coagulation, in intercellular signalling, and in cellular waste management. Consequently, there is a growing interest in molecular diagnostic and in clinical application of exosomes. Exosomes can potentially be used for prognosis, therapy, and biomarkers for health or disease, especially in cancer progression and metastasis.
A lot of research is done in exosome purification and isolation, their size and content characterization, by quantifying surface- and intra-luminal proteins, membrane fatty acids, and the high concentrated regulative small RNA.

REVIEW -- Exosomes -- Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials.
Vlassov AV, Magdaleno S, Setterquist R, Conrad R.
Biochim Biophys Acta. 2012 1820(7): 940-948

BACKGROUND: Cells continuously secrete a large number of microvesicles, macromolecular complexes, and small molecules into the extracellular space. Of the secreted microvesicles, the nanoparticles called exosomes are currently undergoing intense scrutiny. These are small vesicles (30-120 nm) containing nucleic acid and protein, perceived to be carriers of this cargo between diverse locations in the body. They are distinguished in their genesis by being budded into endosomes to form multivesicular bodies (MVBs) in the cytoplasm. The exosomes are released to extracellular fluids by fusion of these multivesicular bodies with the cell surface, resulting in secretion in bursts. Exosomes are secreted by all types of cells in culture, and also found in abundance in body fluids including blood, saliva, urine, and breast milk.
SCOPE OF REVIEW: In this review, we summarize strategies for exosome isolation, our understanding to date of exosome composition, functions, and pathways, and discuss their potential for diagnostic and therapeutic applications.
MAJOR CONCLUSIONS: Currently, the control of exosome formation, the makeup of the "cargo", biological pathways and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication--exosomes are thought to function as the messengers, delivering various effectors or signaling macromolecules between supposedly very specific cells.
GENERAL SIGNIFICANCE: Both seasoned and newer investigators of nanovesicles have presented various viewpoints on what exosomes are, with some differences but a large common area. It would be useful to develop a codified definition of exosomes in both descriptive and practical terms. We hope this in turns leads to a consistent set of practices for their isolation, characterization and manipulation.
Extracellular Vesicles -- Exosomes, Microvesicles, and friends.
Raposo G and Stoorvogel W
J Cell Biol. 2013 200(4): 373-383

Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.

Exosome Nomenclature
As we wait: coping with an imperfect nomenclature for extracellular vesicles.
Gould SJ and Raposo G
J Extracell Vesicles. 2013 -- eCollection 2013

There is increasing evidence that secreted vesicles play important roles in numerous aspects of biology (e.g. intercellular vesicle traffic, immunity, development, neurobiology and microbiology), contribute to many human diseases (e.g. cancer, neurodegenerative disorders and HIV/AIDS) and have significant biotechnological potential. This expanding interest in extracellular vesicles has also highlighted some vexing problems related to their nomenclature. At the first meeting of the International Society for Extracellular Vesicles (ISEV) in Gothenburg, Sweden (April 2012), the authors chaired a session on the issue of vesicle nomenclature. Although it was not possible to reach a broad agreement on vesicle nomenclature, members of the session did reach consensus on 2 points. First, ISEV should strive to protect the scientific independence of its members on this issue. Second, that we (S.J.G. and G.R) should articulate some of the relevant points of concern in the Journal of Extracellular Vesicles.
Classification, functions, and clinical relevance of extracellular vesicles.
van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R.
Pharmacol Rev. 2012 64(3): 676-705

Both eukaryotic and prokaryotic cells release small, phospholipid-enclosed vesicles into their environment. Why do cells release vesicles? Initial studies showed that eukaryotic vesicles are used to remove obsolete cellular molecules. Although this release of vesicles is beneficial to the cell, the vesicles can also be a danger to their environment, for instance in blood, where vesicles can provide a surface supporting coagulation. Evidence is accumulating that vesicles are cargo containers used by eukaryotic cells to exchange biomolecules as transmembrane receptors and genetic information. Because also bacteria communicate to each other via extracellular vesicles, the intercellular communication via extracellular cargo carriers seems to be conserved throughout evolution, and therefore vesicles are likely to be a highly efficient, robust, and economic manner of exchanging information between cells. Furthermore, vesicles protect cells from accumulation of waste or drugs, they contribute to physiology and pathology, and they have a myriad of potential clinical applications, ranging from biomarkers to anticancer therapy. Because vesicles may pass the blood-brain barrier, they can perhaps even be considered naturally occurring liposomes. Unfortunately, pathways of vesicle release and vesicles themselves are also being used by tumors and infectious diseases to facilitate spreading, and to escape from immune surveillance. In this review, the different types, nomenclature, functions, and clinical relevance of vesicles will be discussed.

Ectosomes and exosomes: shedding the confusion between extracellular vesicles.
Cocucci E & Meldolesi J
Trends Cell Biol. 2015 Feb 12.

Long- and short-distance communication can take multiple forms. Among them are exosomes and ectosomes, extracellular vesicles (EVs) released from the cell to deliver signals to target cells. While most of our understanding of how these vesicles are assembled and work comes from mechanistic studies performed on exosomes, recent studies have begun to shift their focus to ectosomes. Unlike exosomes, which are released on the exocytosis of multivesicular bodies (MVBs), ectosomes are ubiquitous vesicles assembled at and released from the plasma membrane. Here we review the similarities and differences between these two classes of vesicle, suggesting that, despite their considerable differences, the functions of ectosomes may be largely analogous to those of exosomes. Both vesicles appear to be promising targets in the diagnosis and therapy of diseases, especially cancer.
ExoCarta 2012 -- database of exosomal proteins, RNA and lipids.
Mathivanan S, Fahner CJ, Reid GE, Simpson RJ.
Nucleic Acids Res. 2012 40 (Database issue): D1241-1244

Exosomes are membraneous nanovesicles of endocytic origin released by most cell types from diverse organisms; they play a critical role in cell-cell communication. ExoCarta (http://www.exocarta.org) is a manually curated database of exosomal proteins, RNA and lipids. The database catalogs information from both published and unpublished exosomal studies. The mode of exosomal purification and characterization, the biophysical and molecular properties are listed in the database aiding biomedical scientists in assessing the quality of the exosomal preparation and the corresponding data obtained. Currently, ExoCarta (Version 3.1) contains information on 11,261 protein entries, 2375 mRNA entries and 764 miRNA entries that were obtained from 134 exosomal studies. In addition to the data update, as a new feature, lipids identified in exosomes are added to ExoCarta. We believe that this free web-based community resource will aid researchers in identifying molecular signatures (proteins/RNA/lipids) that are specific to certain tissue/cell type derived exosomes and trigger new exosomal studies.
ExoCarta -- as a resource for exosomal research.
Simpson RJ, Kalra H, Mathivanan S.
J Extracell Vesicles. 2012 Apr 16: 1

Exosomes are a class of extracellular vesicles that are secreted by various cell types. Unlike other extracellular vesicles (ectosomes and apoptotic blebs), exosomes are of endocytic origin. The roles of exosomes in vaccine/drug delivery, intercellular communication and as a possible source of disease biomarkers have sparked immense interest in them, resulting in a plethora of studies. Whilst multidimensional datasets are continuously generated, it is difficult to harness the true potential of the data until they are compiled and made accessible to the biomedical researchers. Here, we describe ExoCarta (www.exocarta.org), a manually curated database of exosomal proteins, RNA and lipids. Datasets currently present in ExoCarta are integrated from both published and unpublished exosomal studies. Since its launch in 2009, ExoCarta has been accessed by more than 16,000 unique users. In this article, we discuss the utility of ExoCarta for exosomal research and urge biomedical researchers in the field to deposit their datasets directly to ExoCarta.

Vesiclepedia -- a compendium for extracellular vesicles with continuous community annotation.
Kalra H, Simpson RJ, Ji H, Aikawa E, Altevogt P, Askenase P, Bond VC, Borràs FE, Breakefield X, Budnik V, Buzas E, Camussi G, Clayton A, Cocucci E, Falcon-Perez JM, Gabrielsson S, Gho YS, Gupta D, Harsha HC, Hendrix A, Hill AF, Inal JM, Jenster G, Krämer-Albers EM, Lim SK, Llorente A, Lötvall J, Marcilla A, Mincheva-Nilsson L, Nazarenko I, Nieuwland R, Nolte-'t Hoen EN, Pandey A, Patel T, Piper MG, Pluchino S, Prasad TS, Rajendran L, Raposo G, Record M, Reid GE, Sánchez-Madrid F, Schiffelers RM, Siljander P, Stensballe A, Stoorvogel W, Taylor D, Thery C, Valadi H, van Balkom BW, Vázquez J, Vidal M, Wauben MH, Yáñez-Mó M, Zoeller M, Mathivanan S.
PLoS Biol. 2012;10(12): e1001450

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.
EVpedia -- an integrated database of high-throughput data for systemic analyses of extracellular vesicles.
Kim DK, Kang B, Kim OY, Choi DS, Lee J, Kim SR, Go G, Yoon YJ, Kim JH, Jang SC, Park KS, Choi EJ, Kim KP, Desiderio DM, Kim YK, Lötvall J, Hwang D, Gho YS.
J Extracell Vesicles. 2013: 2 -- eCollection 2013

Secretion of extracellular vesicles is a general cellular activity that spans the range from simple unicellular organisms (e.g. archaea; Gram-positive and Gram-negative bacteria) to complex multicellular ones, suggesting that this extracellular vesicle-mediated communication is evolutionarily conserved. Extracellular vesicles are spherical bilayered proteolipids with a mean diameter of 20-1,000 nm, which are known to contain various bioactive molecules including proteins, lipids, and nucleic acids. Here, we present EVpedia, which is an integrated database of high-throughput datasets from prokaryotic and eukaryotic extracellular vesicles. EVpedia provides high-throughput datasets of vesicular components (proteins, mRNAs, miRNAs, and lipids) present on prokaryotic, non-mammalian eukaryotic, and mammalian extracellular vesicles. In addition, EVpedia also provides an array of tools, such as the search and browse of vesicular components, Gene Ontology enrichment analysis, network analysis of vesicular proteins and mRNAs, and a comparison of vesicular datasets by ortholog identification. Moreover, publications on extracellular vesicle studies are listed in the database. This free web-based database of EVpedia (http://evpedia.info) might serve as a fundamental repository to stimulate the advancement of extracellular vesicle studies and to elucidate the novel functions of these complex extracellular organelles.
EVpedia -- a community web portal for extracellular vesicles research.
Kim DK1, Lee J1, Kim SR1, Choi DS1, Yoon YJ1, Kim JH1, Go G1, Nhung D1, Hong K1, Jang SC1, Kim SH1, Park KS1, Kim OY1, Park HT1, Seo JH1, Aikawa E1, Baj-Krzyworzeka M1, van Balkom BW1, Belting M1, Blanc L1, Bond V1, Bongiovanni A1, Borràs FE1, Buée L1, Buzás EI1, Cheng L1, Clayton A1, Cocucci E1, Dela Cruz CS1, Desiderio DM1, Di Vizio D1, Ekström K2, Falcon-Perez JM1, Gardiner C1, Giebel B1, Greening DW1, Gross JC1, Gupta D1, Hendrix A1, Hill AF1, Hill MM1, Nolte-'t Hoen E1, Hwang DW1, Inal J1, Jagannadham MV1, Jayachandran M1, Jee YK1, Jørgensen M1, Kim KP1, Kim YK1, Kislinger T1, Lässer C1, Lee DS1, Lee H1, van Leeuwen J1, Lener T2, Liu ML2, Lötvall J1, Marcilla A1, Mathivanan S1, Möller A1, Morhayim J1, Mullier F2, Nazarenko I1, Nieuwland R1, Nunes DN1, Pang K2, Park J1, Patel T1, Pocsfalvi G1, Del Portillo H1, Putz U1, Ramirez MI1, Rodrigues ML2, Roh TY2, Royo F1, Sahoo S1, Schiffelers R1, Sharma S1, Siljander P1, Simpson RJ1, Soekmadji C1, Stahl P1, Stensballe A1, Stępień E1, Tahara H1, Trummer A1, Valadi H1, Vella LJ1, Wai SN1, Witwer K1, Yáñez-Mó M1, Youn H1, Zeidler R1, Gho YS1.
Bioinformatics. 2014 Nov 10. pii: btu741

MOTIVATION: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging.
RESULTS: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. Availability and implementation: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at www.evpedia.info

Exosome biogenesis and release
Exosome Explosion
By Clotilde Théry -- The Scientist 1st July 2011
These small membrane vesicles do much more than clean up a cell’s trash—they also carry signals to distant parts of the body, where they can impact multiple dimensions of cellular life.

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Biogenesis and secretion of exosomes.
Kowal J, Tkach M, Théry C
Curr Opin Cell Biol. 2014 Aug;29: 116-125

Although observed for several decades, the release of membrane-enclosed vesicles by cells into their surrounding environment has been the subject of increasing interest in the past few years, which led to the creation, in 2012, of a scientific society dedicated to the subject: the International Society for Extracellular Vesicles. Convincing evidence that vesicles allow exchange of complex information fuelled this rise in interest. But it has also become clear that different types of secreted vesicles co-exist, with different intracellular origins and modes of formation, and thus probably different compositions and functions. Exosomes are one sub-type of secreted vesicles. They form inside eukaryotic cells in multivesicular compartments, and are secreted when these compartments fuse with the plasma membrane. Interestingly, different families of molecules have been shown to allow intracellular formation of exosomes and their subsequent secretion, which suggests that even among exosomes different sub-types exist.
Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.
Colombo M, Raposo G, Théry C.
Annu Rev Cell Dev Biol. 2014;30: 255-289

In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.
Biogenesis of extracellular vesicles (EV) -- exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies.
Akers JC, Gonda D, Kim R, Carter BS, Chen CC.
J Neurooncol. 2013 May;113(1): 1-11

Recent studies suggest both normal and cancerous cells secrete vesicles into the extracellular space. These extracellular vesicles (EVs) contain materials that mirror the genetic and proteomic content of the secreting cell. The identification of cancer-specific material in EVs isolated from the biofluids (e.g., serum, cerebrospinal fluid, urine) of cancer patients suggests EVs as an attractive platform for biomarker development. It is important to recognize that the EVs derived from clinical samples are likely highly heterogeneous in make-up and arose from diverse sets of biologic processes. This article aims to review the biologic processes that give rise to various types of EVs, including exosomes, microvesicles, retrovirus like particles, and apoptotic bodies. Clinical pertinence of these EVs to neuro-oncology will also be discussed.

Purification and Isolation

Exosomes -- isolation methods and specific markers
Konstantin Yakimchuk, Karolinska Institutet, Sweden
MATER METHODS 2015;5: 1450

Various methods for isolation of exosomes from biological fluids have been developed. They include centrifugation, chromatography, filtration, polymer-based precipitation and immunological separation. Recent technical improvements in these methods have made the isolation process faster and easier. Contamination of isolated exosome with non-exosomal particles can cause wrong conclusions about biological activities of obtained exosomes and therefore should be avoided. Exosomes from different specimens can possess different protein/lipid and luminal contents and different sedimentation characteristics.
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Exosomes isolation methods
Isolation methods Mechanism Advantages Disadvantages
Differential centrifugation The method consists of several centrifugation steps aiming to remove cells, large vesicles and debris and precipitate exosomes. Differential centrifugation is the standard and very common method used to isolate exosomes from biological fluids and media. The efficiency of the method is lower when viscous biological fluids such as plasma and serum are used for analysis.
Density gradient centrifugation This method combines ultracentrifugation with sucrose density gradient. The method allows separation of the low-density exosomes from other vesicles, particles and contaminants. Very high sensitivity to the centrifugation time.
Size exclusion chromatography Size-exclusion chromatography separates macromolecules on the base of their size. It applies a column packed with porous polymeric beads. The method allows precise separation of large and small molecules and application of various solutions. Compared to centrifugation methods, the structure of exosomes isolated by chromatography is not affected by shearing force. The method requires a long running time, which limits applications of chromatographical isolation for processing multiple biological samples.
Filtration Ultrafiltration membranes are used to separate exosomes from proteins and other macromolecules. The exosomal population is concentrated on the membrane. Filtration allows separation of small particles and soluble molecules from exosomes. During the process the exosomal population is concentrated by the filtration membrane. Exosomes can adhere to the filtration membranes and become lost for the following analysis. Also, since the additional force is applied to pass the analyzed liquid through the membranes, the exosomes can potentially be deformed or damaged.
Polymer-based precipitation The technique includes mixing the biological fluid with polymer-containing precipitation solution, incubation step and centrifugation at low speed. The advantages of precipitation include the mild effect on isolated exosomes and usage of neutral pH. Polymer-based precipitation methods co-isolate non-vesicular contaminants, including lipoproteins. Also, the presence of the polymer material may not be compatible with down-stream analysis.
Immunological separation Various immunological methods are applied. Magnetic beads bound to the specific antibodies are used to isolate exosomes. Also, ELISA-based separation method was developed. The method allows isolation of all exosomes or selective subtypes of exosomes. Also, it may be applied for characterization and quantitation of exosomal proteins. The method is not applicable for large sample volumes. Also, the isolated vesicles may lose the functional activity.
Isolation by sieving This technique isolates exosomes by sieving them via a membrane and performing filtration by pressure or electrophoresis. Relatively short separation time and gives high purity of isolated exosomes. Low recovery of isolated exosomes.
Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
Witwer KW, Buzás EI, Bemis LT, Bora A, Lässer C, Lötvall J, Nolte-'t Hoen EN, Piper MG, Sivaraman S, Skog J, Théry C, Wauben MH, Hochberg F.
J Extracell Vesicles. 2013 May 27;2

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the "ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)", 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments.
The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.
Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A.
J Extracell Vesicles. 2014 Sep 18;3 -- eCollection 2014.

Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.

Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.
Douglas D. Taylor & Sahil Shah
Methods (2015)

Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10 years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term ‘‘exosomes’’ was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use ‘‘exosomes’’ as synonymous with ‘‘extracellular vesicles.’’ While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle’s membrane structure and non-standardized parameters leading to qualitative and quantitative variability. The down-stream analyses of these resulting varying exosomes can yield misleading results and conclusions.
Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.
Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ.
Methods. 2012 Feb;56(2): 293-304

Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling.
Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics.
Maas SL, de Vrij J, van der Vlist EJ, Geragousian B, van Bloois L, Mastrobattista E, Schiffelers RM, Wauben MH, Broekman ML, Nolte-'t Hoen EN
J Control Release. 2014 200C: 87-96

Nano-sized extracelullar vesicles (EVs) released by various cell types play important roles in a plethora of (patho)physiological processes and are increasingly recognized as biomarkers for disease. In addition, engineered EV and EV-inspired liposomes hold great potential as drug delivery systems. Major technologies developed for high-throughput analysis of individual EV include nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (tRPS) and high-resolution flow cytometry (hFC). Currently, there is a need for comparative studies on the available technologies to improve standardization of vesicle analysis in diagnostic or therapeutic settings. We investigated the possibilities, limitations and comparability of NTA, tRPS and hFC for analysis of tumor cell-derived EVs and synthetic mimics (i.e. differently sized liposomes). NTA and tRPS instrument settings were identified that significantly affected the quantification of these particles. Furthermore, we detailed the differences in absolute quantification of EVs and liposomes using the three technologies. This study increases our understanding of possibilities and pitfalls of NTA, tRPS and hFC, which will benefit standardized and large-scale clinical application of (engineered) EVs and EV-mimics in the future.
Single-step isolation of extracellular vesicles by size-exclusion chromatography.
Böing AN, van der Pol E, Grootemaat AE, Coumans FA, Sturk A, Nieuwland R
J Extracell Vesicles. 2014 8;3 -- eCollection 2014.

BACKGROUND: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively.
AIM: To develop a single-step protocol to isolate vesicles from human body fluids.
METHODS: Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction.
RESULTS: Fractions 9-12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18-20 (32%±2 of total), and protein in fractions 19-21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9-12, with an 8-fold and 70-fold enrichment compared to HDL and protein.
CONCLUSIONS: SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles.
Current methods for the isolation of extracellular vesicles.
Momen-Heravi F, Balaj L, Alian S, Mantel PY, Halleck AE, Trachtenberg AJ, Soria CE, Oquin S, Bonebreak CM, Saracoglu E, Skog J, Kuo WP.
Biol Chem. 2013 Oct;394(10): 1253-1262

Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.
Methods for extracellular vesicles isolation in a hospital setting.
Sáenz-Cuesta M, Arbelaiz A, Oregi A, Irizar H, Osorio-Querejeta I, Muñoz-Culla M, Banales JM, Falcón-Pérez JM, Olascoaga J, Otaegui D.
Front Immunol. 2015 Feb 13;6: 50

The research in extracellular vesicles (EVs) has been rising during the last decade. However, there is no clear consensus on the most accurate protocol to isolate and analyze them. Besides, most of the current protocols are difficult to implement in a hospital setting due to being very time-consuming or to requirements of specific infrastructure. Thus, our aim is to compare five different protocols (comprising two different medium-speed differential centrifugation protocols; commercially polymeric precipitation - exoquick - acid precipitation; and ultracentrifugation) for blood and urine samples to determine the most suitable one for the isolation of EVs. Nanoparticle tracking analysis, flow cytometry, western blot (WB), electronic microscopy, and spectrophotometry were used to characterize basic aspects of EVs such as concentration, size distribution, cell-origin and transmembrane markers, and RNA concentration. The highest EV concentrations were obtained using the exoquick protocol, followed by both differential centrifugation protocols, while the ultracentrifugation and acid-precipitation protocols yielded considerably lower EV concentrations. The five protocols isolated EVs of similar characteristics regarding markers and RNA concentration; however, standard protocol recovered only small EVs. EV isolated with exoquick presented difficult to be analyzed with WB. The RNA concentrations obtained from urine-derived EVs were similar to those obtained from blood-derived ones, despite the urine EV concentration being 10-20 times lower. We consider that a medium-speed differential centrifugation could be suitable to be applied in a hospital setting as it requires the simplest infrastructure and recovers higher concentration of EV than standard protocol. A workflow from sampling to characterization of EVs is proposed.

The exosomal content
Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis.
Gardiner C, Ferreira YJ, Dragovic RA, Redman CW, Sargent IL.
J Extracell Vesicles. 2013 -- eCollection 2013

Nanoparticle tracking analysis (NTA) is a light-scattering technique that is useful for the rapid sizing and enumeration of extracellular vesicles (EVs). As a relatively new method, NTA has been criticised for a lack of standardisation. We propose the use of silica microspheres for the calibration of NTA measurements and describe in detail a protocol for the analysis of EVs by NTA which should minimise many of the sources of variability and imprecision associated with this technique.

Importance of RNA isolation methods for analysis of exosomal RNA -- evaluation of different methods.
Eldh M, Lötvall J, Malmhäll C, Ekström K.
Mol Immunol. 2012 Apr;50(4): 278-86.

Exosomes are small RNA containing vesicles of endocytic origin, which can take part in cell-to-cell communication partly by the transfer of exosomal RNA between cells. Exosomes are released by many cells and can also be found in several biological fluids including blood plasma and breast milk. Exosomes differ compared to their donor cells not only in size but also in RNA, protein and lipid composition. The aim of the current study was to determine the optimal RNA extraction method for analysis of exosomal RNA, to support future studies determining the biological roles of the exosomal RNA. Different methods were used to extract exosomal and cellular RNA. All methods evaluated extracted high quality and purity RNA as determined by RNA integrity number (RIN) and OD values for cellular RNA using capillary electrophoresis and spectrophotometer. Interestingly, the exosomal RNA yield differed substantially between the different RNA isolation methods. There was also a difference in the exosomal RNA patterns in the electropherograms, indicating that the tested methods extract exosomal RNA with different size distribution. A pure column based approach resulted in the highest RNA yield and the broadest RNA size distribution, whereas phenol and combined phenol and column based approaches lost primarily large RNAs. Moreover, the use of phenol and combined techniques resulted in reduced yield of exosomal RNA, with a more narrow size distribution pattern resulting in an enrichment of small RNA including microRNA. In conclusion, the current study presents a unique comparison of seven different methods for extraction of exosomal RNA. As the different isolation methods give extensive variation in exosomal RNA yield and patterns, it is crucial to select an isolation approach depending on the research question at hand.

Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood.
Cheng L, Sharples RA, Scicluna BJ, Hill AF
J Extracell Vesicles. 2014 Mar 26;3 -- eCollection 2014

INTRODUCTION: microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed in the host cell and released extracellularly into the bloodstream. Normally involved in post-transcriptional gene silencing, the deregulation of miRNA has been shown to influence pathogenesis of a number of diseases.
BACKGROUND: Next-generation deep sequencing (NGS) has provided the ability to profile miRNA in biological fluids making this approach a viable screening tool to detect miRNA biomarkers. However, collection and handling procedures of blood needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays.
METHODS: Here, we use NGS to profile miRNA in various blood components and identify differences in profiles within peripheral blood compared to cell-free plasma or serum and extracellular vesicles known as exosomes. We also analyse and compare the miRNA content in exosomes prepared by ultracentrifugation methods and commercial exosome isolation kits including treating samples with RNaseA.
CONCLUSION: This study demonstrates that exosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection.

The majority of microRNAs detectable in serum and saliva is concentrated in exosomes.
Gallo A, Tandon M, Alevizos I, Illei GG
PLoS One. 2012; 7(3): e30679

There is an increasing interest in using microRNAs (miRNA) as biomarkers in autoimmune diseases. They are easily accessible in many body fluids but it is controversial if they are circulating freely or are encapsulated in microvesicles, particularly exosomes. We investigated if the majority of miRNas in serum and saliva are free-circulating or concentrated in exosomes. Exosomes were isolated by ultracentrifugation from fresh and frozen human serum and saliva. The amount of selected miRNAs extracted from the exosomal pellet and the exosome-depleted serum and saliva was compared by quantitative RT-PCR. Some miRNAs tested are ubiquitously expressed, others were previously reported as biomarkers. We included miRNAs previously reported to be free circulating and some thought to be exosome specific. The purity of exosome fraction was confirmed by electronmicroscopy and western blot. The concentration of miRNAs was consistently higher in the exosome pellet compared to the exosome-depleted supernatant. We obtained the same results using an equal volume or equal amount of total RNA as input of the RT-qPCR. The concentration of miRNA in whole, unfractionated serum, was between the exosomal pellet and the exosome-depleted supernatant. Selected miRNAs, which were detectable in exosomes, were undetectable in whole serum and the exosome-depleted supernantant. Exosome isolation improves the sensitivity of miRNA amplification from human biologic fluids. Exosomal miRNA should be the starting point for early biomarker studies to reduce the probability of false negative results involving low abundance miRNAs that may be missed by using unfractionated serum or saliva.

Distinct RNA profiles in subpopulations of extracellular vesicles:  apoptotic bodies, microvesicles and exosomes.
Crescitelli R, Lässer C, Szabó TG, Kittel A, Eldh M, Dianzani I, Buzás EI, Lötvall J
J Extracell Vesicles. 2013 2 -- eCollection 2013.

INTRODUCTION: In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination.
METHOD: EVS RELEASED FROM THREE DIFFERENT KINDS OF CELL LINES: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer(®).
RESULTS: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as these cells do not express CD9.
CONCLUSIONS: Our results demonstrate that centrifugation-based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63-coated beads for flow cytometry analysis.
Quantitative and stoichiometric analysis of the microRNA content of exosomes.
Chevillet JR, Kang Q, Ruf IK, Briggs HA, Vojtech LN, Hughes SM, Cheng HH, Arroyo JD, Meredith EK, Gallichotte EN, Pogosova-Agadjanyan EL, Morrissey C, Stirewalt DL, Hladik F, Yu EY, Higano CS, Tewari M
Proc Natl Acad Sci U S A. 2014 111(41): 14888-14893

Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.
The microRNA spectrum in 12 body fluids.
Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, Galas DJ, Wang K.
Clin Chem. 2010 56(11): 1733-1741

BACKGROUND: MicroRNAs (miRNAs) are small, noncoding RNAs that play an important role in regulating various biological processes through their interaction with cellular messenger RNAs. Extracellular miRNAs in serum, plasma, saliva, and urine have recently been shown to be associated with various pathological conditions including cancer.
METHODS: With the goal of assessing the distribution of miRNAs and demonstrating the potential use of miRNAs as biomarkers, we examined the presence of miRNAs in 12 human body fluids and urine samples from women in different stages of pregnancy or patients with different urothelial cancers. Using quantitative PCR, we conducted a global survey of the miRNA distribution in these fluids.
RESULTS: miRNAs were present in all fluids tested and showed distinct compositions in different fluid types. Several of the highly abundant miRNAs in these fluids were common among multiple fluid types, and some of the miRNAs were enriched in specific fluids. We also observed distinct miRNA patterns in the urine samples obtained from individuals with different physiopathological conditions.
CONCLUSIONS: MicroRNAs are ubiquitous in all the body fluid types tested. Fluid type-specific miRNAs may have functional roles associated with the surrounding tissues. In addition, the changes in miRNA spectra observed in the urine samples from patients with different urothelial conditions demonstrates the potential for using concentrations of specific miRNAs in body fluids as biomarkers for detecting and monitoring various physiopathological conditions.

Deep sequencing of RNA from three different extracellular vesicle (EV) subtypes released from the human LIM1863 colon cancer cell line uncovers distinct miRNA-enrichment signatures.
Ji H, Chen M, Greening DW, He W, Rai A, Zhang W, Simpson RJ
PLoS One. 2014 9(10): e110314 -- eCollection 2014.

Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863--shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs--miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.

Inter-cellular Communication
Secreted microRNAs -- a new form of intercellular communication.
Chen X, Liang H, Zhang J, Zen K, Zhang CY.
Trends Cell Biol. 2012 22(3):125-32

In multicellular organisms, cell-to-cell communication is of particular importance for the proper development and function of the organism as a whole. Intensive studies over the past three years suggesting horizontal transfer of secreted microRNAs (miRNAs) between cells point to a potentially novel role for these molecules in intercellular communication. Using a microvesicle-dependent, or RNA-binding protein-associated, active trafficking system, secreted miRNAs can be delivered into recipient cells where they function as endogenous miRNAs, simultaneously regulating multiple target genes or signaling events. In this Opinion, we summarize recent literature on the biogenesis and uptake of secreted miRNAs, propose a possible working model for how secreted miRNAs might be sorted and transferred between cells and speculate on their biological significance.
Microvesicles as mediators of intercellular communication in cancer -- the emerging science of cellular 'debris'.
Lee TH, D'Asti E, Magnus N, Al-Nedawi K, Meehan B, Rak J.
Semin Immunopathol. 2011 Sep;33(5): 455-467

Cancer cells emit a heterogeneous mixture of vesicular, organelle-like structures (microvesicles, MVs) into their surroundings including blood and body fluids. MVs are generated via diverse biological mechanisms triggered by pathways involved in oncogenic transformation, microenvironmental stimulation, cellular activation, stress, or death. Vesiculation events occur either at the plasma membrane (ectosomes, shed vesicles) or within endosomal structures (exosomes). MVs are increasingly recognized as mediators of intercellular communication due to their capacity to merge with and transfer a repertoire of bioactive molecular content (cargo) to recipient cells. Such processes may occur both locally and systemically, contributing to the formation of microenvironmental fields and niches. The bioactive cargo of MVs may include growth factors and their receptors, proteases, adhesion molecules, signalling molecules, as well as DNA, mRNA, and microRNA (miRs) sequences. Tumour cells emit large quantities of MVs containing procoagulant, growth regulatory and oncogenic cargo (oncosomes), which can be transferred throughout the cancer cell population and to non-transformed stromal cells, endothelial cells and possibly to the inflammatory infiltrates (oncogenic field effect). These events likely impact tumour invasion, angiogenesis, metastasis, drug resistance, and cancer stem cell hierarchy. Ongoing studies explore the molecular mechanisms and mediators of MV-based intercellular communication (cancer vesiculome) with the hope of using this information as a possible source of therapeutic targets and disease biomarkers in cancer.
Exosomes--vesicular carriers for intercellular communication.
Simons M and Raposo G.
Curr Opin Cell Biol. 2009 Aug;21(4): 575-581

Cells release different types of vesicular carriers of membrane and cytosolic components into the extracellular space. These vesicles are generated within the endosomal system or at the plasma membrane. Among the various kinds of secreted membrane vesicles, exosomes are vesicles with a diameter of 40-100 nm that are secreted upon fusion of multivesicular endosomes with the cell surface. Exosomes transfer not only membrane components but also nucleic acid between different cells, emphasizing their role in intercellular communication. This ability is likely to underlie the different physiological and pathological events, in which exosomes from different cell origins have been implicated. Only recently light have been shed on the subcellular compartments and mechanisms involved in their biogenesis and secretion opening new avenues to understand their functions.

Characterization of mRNA and microRNA in human mast cell-derived exosomes and their transfer to other mast cells and blood CD34 progenitor cells.
Ekström K, Valadi H, Sjöstrand M, Malmhäll C, Bossios A, Eldh M, Lötvall J.
J Extracell Vesicles. 2012; 1 -- eCollection 2012

BACKGROUND: Exosomes are nanosized vesicles of endocytic origin that are released into the extracellular environment by many different cells. It has been shown that exosomes from various cellular origins contain a substantial amount of RNA (mainly mRNA and microRNA). More importantly, exosomes are capable of delivering their RNA content to target cells, which is a novel way of cell-to-cell communication. The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34(+) progenitor cells.
METHODS: The mRNA and microRNA content of exosomes from a human mast cell line, HMC-1, was analysed by using microarray technology. Co-culture experiments followed by flow cytometry analysis and confocal microscopy as well as radioactive labeling experiments were performed to examine the uptake of these exosomes and the shuttle of the RNA to other mast cells and CD34(+) progenitor cells.
RESULTS: In this study, we show that human mast cells release RNA-containing exosomes, with the capacity to shuttle RNA between cells. Interestingly, by using microRNA microarray analysis, 116 microRNAs could be identified in the exosomes and 134 microRNAs in the donor mast cells. Furthermore, DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. In addition, transfer experiments revealed that exosomes can shuttle RNA between human mast cells and to CD34(+) hematopoietic progenitor cells.
CONCLUSION: These findings suggest that exosomal shuttle RNA (esRNA) can play a role in the communication between cells, including mast cells and CD34(+) progenitor cells, implying a role in cells maturation process.
Exosomes and other extracellular vesicles in host-pathogen interactions.
Schorey JS, Cheng Y, Singh PP, Smith VL.
EMBO Rep. 2015 16(1): 34-43

An effective immune response requires the engagement of host receptors by pathogen-derived molecules and the stimulation of an appropriate cellular response. Therefore, a crucial factor in our ability to control an infection is the accessibility of our immune cells to the foreign material. Exosomes-which are extracellular vesicles that function in intercellular communication may play a key role in the dissemination of pathogen- as well as host-derived molecules during infection. In this review, we highlight the composition and function of exosomes and other extracellular vesicles produced during viral, parasitic, fungal and bacterial infections and describe how these vesicles could function to either promote or inhibit host immunity.


Exosome Function & Physiology

Extracellular vesicle-depleted fetal bovine and human sera have reduced capacity to support cell growth.
Eitan E, Zhang S, Witwer KW, Mattson MP
J Extracell Vesicles. 2015 Mar 26;4: 26373 -- eCollection 2015

BACKGROUND: Fetal bovine serum (FBS) is the most widely used serum supplement for mammalian cell culture. It supports cell growth by providing nutrients, growth signals, and protection from stress. Attempts to develop serum-free media that support cell expansion to the same extent as serum-supplemented media have not yet succeeded, suggesting that FBS contains one or more as-yet-undefined growth factors. One potential vehicle for the delivery of growth factors from serum to cultured cells is extracellular vesicles (EVs).
METHODS: EV-depleted FBS and human serum were generated by 120,000g centrifugation, and its cell growth-supporting activity was measured. Isolated EVs from FBS were quantified and characterized by nanoparticle tracking analysis, electron microscopy, and protein assay. EV internalization into cells was quantified using fluorescent plate reader analysis and microscopy.
RESULTS: Most cell types cultured with EV-depleted FBS showed a reduced growth rate but not an increased sensitivity to the DNA-damaging agent etoposide and the endoplasmic reticulum stress-inducing chemical tunicamycin. Supplying cells with isolated FBS-derived EVs enhanced their growth. FBS-derived EVs were internalized by mouse and human cells wherein 65±26% of them interacted with the lysosomes. EV-depleted human serum also exhibited reduced cell growth-promoting activity.
CONCLUSIONS: EVs play a role in the cell growth and survival-promoting effects of FBS and human serum. Thus, it is important to take the effect of EV depletion under consideration when planning EV extraction experiments and while attempting to develop serum-free media that support rapid cell expansion. In addition, these findings suggest roles for circulating EVs in supporting cell growth and survival in vivo.
Extracellular vesicles -- potential roles in regenerative medicine.
De Jong OG, Van Balkom BW, Schiffelers RM, Bouten CV, Verhaar MC
Front Immunol. 2014 Dec 3;5: 608

Extracellular vesicles (EV) consist of exosomes, which are released upon fusion of the multivesicular body with the cell membrane, and microvesicles, which are released directly from the cell membrane. EV can mediate cell-cell communication and are involved in many processes, including immune signaling, angiogenesis, stress response, senescence, proliferation, and cell differentiation. The vast amount of processes that EV are involved in and the versatility of manner in which they can influence the behavior of recipient cells make EV an interesting source for both therapeutic and diagnostic applications. Successes in the fields of tumor biology and immunology sparked the exploration of the potential of EV in the field of regenerative medicine. Indeed, EV are involved in restoring tissue and organ damage, and may partially explain the paracrine effects observed in stem cell-based therapeutic approaches. The function and content of EV may also harbor information that can be used in tissue engineering, in which paracrine signaling is employed to modulate cell recruitment, differentiation, and proliferation. In this review, we discuss the function and role of EV in regenerative medicine and elaborate on potential applications in tissue engineering.
Therapeutic potential of extracellular vesicles.
Merino AM, Hoogduijn MJ, Borras FE, Franquesa M
Front Immunol. 2014 Dec 19;5: 658

Extracellular vesicles (EV) have emerged as important mediators of intercellular communication. By their origin, we can find vesicles derived from plasmamembrane such as microvesicles, ectosomes, and membrane particles or exosomes, which originate in the endosomal membrane compartment. They contain numerous proteins, lipids, and even nucleic acids like mRNA and miRNA that can affect the cell sthat encounter these structures in complex ways. The EV have recently gained interest for their therapeutic potential both as a treatment itself and as a biomarker of several pathologies. There searchlines involving EV cover a wide range of aspects from basic research on the EV biology to the manipulation or monitoring of EV for therapeutic purposes.

The Trojan exosome hypothesis.
Gould SJ, Booth AM, Hildreth JE.
Proc Natl Acad Sci U S A. 2003 100(19): 10592-10297

We propose that retroviruses exploit a cell-encoded pathway of intercellular vesicle traffic, exosome exchange, for both the biogenesis of retroviral particles and a low-efficiency but mechanistically important mode of infection. This Trojan exosome hypothesis reconciles current paradigms of retrovirus-directed transmission with the unique lipid composition of retroviral particles, the host cell proteins present in retroviral particles, the complex cell biology of retroviral release, and the ability of retroviruses to infect cells independently of Envelope protein-receptor interactions. An exosomal origin also predicts that retroviruses pose an unsolvable paradox for adaptive immune responses, that retroviral antigen vaccines are unlikely to provide prophylactic protection, and that alloimmunity is a central component of antiretroviral immunity. Finally, the Trojan exosome hypothesis has important implications for the fight against HIV and AIDS, including how to develop new antiretroviral therapies, assess the risk of retroviral infection, and generate effective antiretroviral vaccines.

Exosomes and cancer
Extracellular Vesicles in Cancer:  Exosomes, Microvesicles and the Emerging Role of Large Oncosomes.
Minciacchi VR, Freeman MR, Di Vizio D
Semin Cell Dev Biol. 2015 Feb 23

Since their first description, extracellular vesicles (EVs) have been the topic of avid study in a variety of physiologic contexts and are now thought to play an important role in cancer. The state of knowledge on biogenesis, molecular content and horizontal communication of diverse types of cancer EVs has expanded considerably in recent years. As a consequence, a plethora of information about EV composition and molecular function has emerged, along with the notion that cancer cells rely on these particles to invade tissues and propagate oncogenic signals at distance. The number of in vivo studies, designed to achieve a deeper understanding of the extent to which EV biology can be applied to clinically relevant settings, is rapidly growing. This review summarizes recent studies on cancer-derived EV functions, with an overview about biogenesis and molecular cargo of exosomes, microvesicles and large oncosomes. We also discuss current challenges and emerging technologies that might improve EV detection in various biological systems. Further studies on the functional role of EVs in specific steps of cancer formation and progression will expand our understanding of the diversity of paracrine signaling mechanisms in malignant growth.
 ... more papers in the next months !

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