Exosomes and Microvesicles (MV)
... the content and their physiological function
Societies & Journals:
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
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.
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
a society of Danish researchers with a broad interest in extracellular
vesicles and their biological functions and cargos. http://evsearch.dk
WHY ... EVSEARCH
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.
|Welcome to ExosomesTalk,
a new initiative for scientists in the exciting field of exosome
Join the conversations by responding to questions posted by your fellow
scientists, and post your own questions.
ExosomesTalk is curated by scientists at Life Technologies,
with all content focused on your research needs. Our goal is to help
you achieve faster breakthroughs by building products and services
tailored to your needs.
|Videos on Extracellular
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
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
Part 6: Exosomes - The Next Small Thing
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
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.
-- 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
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
exosome isolation, our understanding to date of exosome composition,
functions, and pathways, and discuss their potential for diagnostic and
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.
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
|As we wait: coping with an imperfect
nomenclature for extracellular vesicles.
Gould SJ and Raposo G
J Extracell Vesicles. 2013 -- eCollection 2013
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
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.
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
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)
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
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
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
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.
-- an integrated database of high-throughput data for systemic analyses
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
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.
-- 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
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.
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 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.
click to enlarge
|Biogenesis and secretion of exosomes.
Kowal J, Tkach M, Théry C
Curr Opin Cell Biol. 2014 Aug;29: 116-125
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
|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
|Biogenesis of extracellular vesicles (EV)
-- exosomes, microvesicles, retrovirus-like vesicles, and apoptotic
Akers JC, Gonda D, Kim R, Carter BS, Chen CC.
J Neurooncol. 2013 May;113(1): 1-11
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.
|Exosomes -- isolation methods and specific
Konstantin Yakimchuk, Karolinska Institutet, Sweden
MATER METHODS 2015;5: 1450
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.
||The method consists of
several centrifugation steps aiming to remove cells, large vesicles and
debris and precipitate exosomes.
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.
||This method combines
ultracentrifugation with sucrose density gradient.
||The method allows
separation of the low-density exosomes from other vesicles, particles
||Very high sensitivity to
the centrifugation time.
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.
are used to separate exosomes from proteins and other macromolecules.
The exosomal population is concentrated on the membrane.
separation of small particles and soluble molecules from exosomes.
During the process the exosomal population is concentrated by the
||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
||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
methods co-isolate non-vesicular contaminants, including lipoproteins.
Also, the presence of the polymer material may not be compatible with
methods are applied. Magnetic beads bound to the specific antibodies
are used to isolate exosomes. Also, ELISA-based separation method was
||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.
||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
|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
|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.
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
|Methods of isolating extracellular vesicles
impact down-stream analyses of their cargoes.
Douglas D. Taylor & Sahil Shah
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,
Methods. 2012 Feb;56(2): 293-304
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)
|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
J Control Release. 2014 200C: 87-96
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,
J Extracell Vesicles. 2014 8;3 -- eCollection 2014.
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
AIM: To develop a single-step protocol to isolate
vesicles from human
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
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
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
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
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
|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
|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.
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
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.
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.
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.
demonstrates that exosomal RNA is protected by RNaseA treatment and
that exosomes provide a consistent source of miRNA for disease
|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
Crescitelli R, Lässer C, Szabó TG, Kittel A, Eldh M,
Dianzani I, Buzás EI, Lötvall J
J Extracell Vesicles. 2013 2 -- eCollection 2013.
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
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
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,
Clin Chem. 2010 56(11): 1733-1741
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.
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.
(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.
Secreted microRNAs -- a new form of
Chen X, Liang H, Zhang J, Zen K, Zhang CY.
Trends Cell Biol. 2012 22(3):125-32
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
Simons M and Raposo G.
Curr Opin Cell Biol. 2009 Aug;21(4): 575-581
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
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.
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.
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
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
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.
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
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).
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.
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.
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
De Jong OG, Van Balkom BW, Schiffelers RM, Bouten CV, Verhaar MC
Front Immunol. 2014 Dec 3;5: 608
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
|Therapeutic potential of extracellular
Merino AM, Hoogduijn MJ, Borras FE, Franquesa M
Front Immunol. 2014 Dec 19;5: 658
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
| ... more
papers in the next months !