but , considered little more than cellular garbage cans acting to discard unwanted molecular components , exosomes remained little studied for the next 10 years . over the past few years , however , evidence has begun to accumulate that the vesicles are like signaling payloads containing cell - specific collections of proteins , lipids , and genetic material that are transported to other cells where they alter function and physiology .
the findings have reignited interest in exosomes , but also more generally in all secreted vesicles , whether they can be called exosomes or not , and the topic has now blossomed into a full - fledged field of research  that of vesicle - modulated cell - cell communication .
two years ago , i began receiving daily emails regarding my articles on exosomes , requesting reprints , details on exosome - related protocols , and advice on the purification and characterization of the vesicles .
having worked in this area for more than 10 years , i thought i knew all the other researchers working on the subject , but the requests were coming in from groups i had n't heard of before .
the flood of inquiries made me realize that the field had been growing , attracting the attention of more and more researchers in a relatively short period of time .
a quick glance at the literature confirmed the trend : while fewer than 20 pubmed - referenced papers using the word  exosomes  were published in early 2003 , and just over 60 in 2007 , nearly 350 exosome studies have been published in the last 3 years alone .
the idea thus came to me and to graa raposo , who discovered exosomes from immune antigen - presenting cells in 1996 , to organize an international workshop on exosomes ( iwe ) .
although a meeting organized by the late rose johnstone , who participated in the first description of exosomes in 1983 , had drawn 25 scientists to montreal , canada , in 2005 , the new meeting , which took place this january at our home institution , the curie institute in paris , attracted nearly 10 times as many attendees .
there , researchers discussed their recent findings on exosomes , and on other types of secreted membrane vesicles , in fields ranging from immunology to neurobiology and tumor biology , as well as potential clinical applications , such as biomarkers or as therapeutic tools . in pluricellular organisms ,
cells communicate with each other via extracellular molecules such as nucleotides , lipids , short peptides , or proteins .
these molecules are released extracellularly by cells and bind to receptors on other cells , thus inducing intracellular signaling and modification of the intracellular physiological state of the recipient cells .
but , in addition to these single molecules , eukaryotic cells also release in their extracellular environment complex structures called membrane vesicles , which contain numerous proteins , lipids , and even nucleic acids , and can affect the cells that encounter these structures in much more complex ways . although known to exist for several decades ( for instance , in blood , where they are generally called  microparticles  , or in seminal fluid , where they are called  prostasomes  ) , membrane vesicles have long been thought of as mere cell debris , signs of cell death , or structures very specific to a unique organ .
the phenomenon of exosomes and their wider involvement in intercellular signaling began to emerge from this picture in the last decade .
exosomes are formed in endosomal compartments called multivesicular endosomes , containing internal vesicles that package and store molecules in membrane - bound structures .
endosomes are generally considered to function as an intermediate compartment between the plasma membrane , where endocytosis of extracellular molecules takes place , and compartments ( lysosomes ) , where these molecules are released and degraded . however , about 25 years ago , the groups of philip stahl   in the united states and rose johnstone in canada   described , using very elegant pulse - chase and electron microscopy experiments , that in reticulocytes undergoing maturation into red blood cells , multivesicular late endosomes could fuse back with the plasma membrane ( instead of with lysosomes ) , and release their contents , including numerous small vesicles , extracellularly . in 1987 ,
the term  exosomes  was proposed to define these extracellularly released intra - endosomal vesicles .
exosomes remained all but forgotten for the next 10 years until raposo , while working with the group of hans geuze in holland in 1996 , showed that epstein 
barr virus ( ebv)-transformed b - lymphocytes secreted exosomes that bore molecules essential for the adaptive immune response : major histocompatibility class ( mhc ) ii dimers bound to antigenic peptides .
these exosomes were also shown to present the mhc  peptide complexes to specific t cells , suggesting that they could play a role in adaptive immune responses .
two years later , the groups of raposo , amigorena , and zitvogel   demonstrated that dendritic cells ( the immune cells that initiate adaptive immune responses by presenting mhc  peptide complexes to nave t cells ) also secrete exosomes bearing functional mhc 
peptide complexes , which could promote induction of antitumor immune responses in mice in vivo .
these results provided the basis for the hypothesis that exosomes could play an active role in intercellular communication , at least in the immune system , and prompted the very first attempt at using them in the clinic , as a new type of anticancer therapy in humans .
( after phase i trials held between 1999 and 2002 , a phase ii trial is currently ongoing at the gustave roussy institute ( with participation of the curie institute ) in france . )
the first major advance of subsequent years was linked to the development of large - scale protein analysis techniques , which allowed us to demonstrate that exosomes represent a specific subcellular compartment , rather than random cell debris .
proteomic analyses of exosome composition were first performed on exosomes secreted by dendritic cells , and then on exosomes from numerous other different cell types , which are compiled in the exocarta compendium .
these studies showed that exosomes did not contain a random array of the intracellular proteins , but a specific set of a few protein families , essentially coming from the plasma membrane , endocytic pathway , and the cytosol , with very limited amounts of proteins from other intracellular compartments ( e.g. , nucleus , endoplasmic reticulum , and golgi apparatus ) , which made them clearly different from membrane vesicles released by apoptotic cells .
these results established that exosomes are actively secreted by live cells , and confirmed their intra - endosomal origin . in parallel ,
studies of exosomes purified in vitro from tissue cultures ( of immune cells but also epithelial and tumor cells ) , started showing that exosomes secreted by one cell could be captured by another cell and transfer information to the latter .
the information transferred could include antigens or mhc  peptide complexes for increasing the range of antigen - presenting cells and the efficiency of induction of immune responses [ 13 - 15 ] , or in some cases negative signals leading to death of the target cell .
these observations provide the basis of the nowadays popular idea that exosomes represent a new type of intercellular messenger . the second real breakthrough in this field came in 2007 , with the convincing description by the group of jan ltvall in sweden of the presence of mrna and microrna ( mirna ) inside these vesicles .
in addition , the authors showed , at least in vitro using large amounts of concentrated exosomes , that some mrna present in exosomes could be translated into proteins in target cells , suggesting that exosomes can transfer genetic information .
this discovery , and the concomitant development of research on mirnas , sparked the recent exponential increase in the numbers of papers on exosomes . in january 2011 , oral and poster presentations at the iwe 2011 covered all the currently explored fields of exosome research spanning immunology and cell biology , their use as biomarkers , and their relationship to tumors .
my four major conclusions from this meeting are that : ( a ) exosomes are not the only secreted membrane vesicles that could act as intercellular messengers , and efforts should be dedicated to combine and compare data obtained on these different vesicles ; ( b ) the next few years should show us whether rna and mirna in circulating vesicles ( exosomes or others ) will prove to be useful biomarkers of various diseases , providing new diagnostic or prognostic tools ; ( c ) although they were originally described in cells of the immune system , exosomes are now known to be secreted by many different cell types , and a large amount of recent data highlight their possible functions as messengers during development of tumors , in some functions of stem cells , in the nervous system , or during interactions of pathogens with their host ; ( d ) although the last 3 decades have expanded our knowledge of exosomes considerably , there is still no definite evidence for their physiological functions in vivo , and demonstrating such functions remains a major challenge of this field .
the term  exosomes  should be used to define secreted membrane vesicles that form inside intracellular multivesicular compartments and that are released upon fusion of these compartments with the plasma membrane ( figure 1 ) .
because of this intracellular origin , exosomes are similar in size to internal vesicles of multivesicular compartments ; that is , around 100 nm in diameter .
exosomes are commonly purified on the basis of size by serial steps of centrifugation , with a final 100,000 g ultracentrifugation to pellet exosomes ; however , this purification is not absolute .
some cells also release other types of membrane vesicles , which can , for instance , directly bud off the plasma membrane towards the outside of the cell .
the size of such vesicles is not as strictly determined as that of exosomes , and they can be anywhere between 501,000 nm in diameter .
various terms have been used for these vesicles , such as  ectosomes  ,  shed vesicles  , or  microvesicles  , but this latter term has also been used more widely for any type of vesicle , whether intra- or extracellular , and regardless of its intracellular origin .
the release of large plasma membrane - derived vesicles is quickly induced after stimuli such as fresh fetal calf serum on tumor cells , complement deposition , or increasing intracellular ca in neutrophils .
vesicles can be separated by size using ultracentrifugation at different speeds , with the larger vesicles pelleting at lower speed than the smaller ones .
but similarly sized vesicles of different intracellular origins ( i.e. , exosomes and small plasma membrane - derived vesicles ) will not be separated by this method .
flotation of vesicles on sucrose gradients can allow some separation of vesicles with different densities , but this separation is not complete .
thus , adequate means to distinguish the different types of vesicles secreted by a cell are still lacking .
proteomic studies of microvesicles or ectosomes have not , so far , been as extensive as for exosomes , but they are underway and they will help to understand the specificity of their secretion . like exosomes , membrane microvesicles and ectosomes
contain various active molecules , such as cytokines , growth factor receptors , and rnas , and also metalloproteases ( which have not been described in exosomes ) , but since studies comparing exosomes and other vesicles released by the same cells are only beginning now , understanding the functional specificity of each type of vesicle should become possible in the next few years .
since the identification of rna in exosomes in 2007 and development of high - throughput techniques for nucleic acid analyses , there have been an ever increasing number of studies reporting sequences of the rna in exosomes .
such studies show that not all messenger rnas present in a cell end up in exosomes , and there is apparently specific targeting of some mrna sequences into the released vesicles .
these last observations refute the idea that mrna in exosomes results from a random contamination of secreted vesicles by mrna released extracellularly by dying cells .
however , it is still unclear from the few published studies whether one can ( as for exosomal proteins ) find a set of exosomal mrna that would be consistently targeted to exosomes in any cell type , possibly in addition to cell type - specific mrna sequences .
understanding the mechanisms of mrna targeting to these vesicles should open the way to understanding the function of rna delivery to secreted vesicles . of note
, mrna has also been described in microvesicles ( or mixed exosome / microvesicle preparations ) released by tumors or embryonic stem cells [ 21 - 23 ] , and some ongoing studies are now comparing sequences of rna targeted to either exosomes or larger vesicles , and determining whether there is a distinct targeting to each of them . the situation is even more complicated for mirna : even though they have also been observed in various exosome preparations , the heterogeneity of techniques used to identify the mirna sequences do not allow one to conclude whether there is specific targeting of mirna to secreted vesicles .
in addition , although extracellular release of mirna can be decreased by some drugs , the literature still describes heterogeneous results concerning the respective contribution of free ( or protein - associated ) mirna , as compared to mirna encapsulated in exosomes or other secreted membrane vesicles . despite the uncertainties about the nature of mrna and mirna - carrying vesicles , the biopharma industry has put a lot of effort into studying the use of secreted vesicles  especially their mrna and mirna content  as biomarkers in clinics .
this is easier than it might seem to be because their encapsulation into membrane vesicles protects extracellular rnas from degradation , and allows efficient recovery from biological fluids , such as plasma , urine , milk , sperm , or saliva .
work in several different laboratories to compare rna ( as well as protein ) sequences in vesicles isolated from normal and cancer cell lines , for instance , and from biological fluids of patients ( cancer or other diseases ) is ongoing .
it is still too early to propose new biomarkers ( either for diagnosis , prognosis , or predictive response to therapies ) from these studies , but given the current effort put in this direction , the next few years should allow us to decide whether such markers exist and are useful .
once secreted , membrane vesicles interact with surrounding cells : many fluorescence microscopy studies show capture of these vesicles and accumulation in internal endocytic or phagocytic compartments , especially in phagocytic cells such as macrophages , but one must keep in mind that vesicles smaller than 200 nm in diameter can not be detected by confocal microscopy techniques , and only electron microscopy allows detailed visualization of exosomes . hence , the way individual vesicles interact with recipient cells is still not known , and has been proposed to involve binding at the cell surface via specific receptors , internalization by endocytosis or micropinocytosis , and/or fusion with plasma membrane or with the limiting membrane of internal compartments .
evidence that fusion does occur in certain cases comes from a recent study showing diffusion of exosomal lipids in the membrane of recipient cells : this observation explains how exosomes could release their internal content ( e.g. , rna ) into the cytosol of recipient cells .
exosomes induce physiological changes in recipient cells upon interaction . because the original description of exosomes was in immune cells
, there is a vast amount of data on the immunological effects of exosomes , and these vary strongly depending on the physiological state of the cells which secrete them ( see   for references ) . for instance , mature dendritic cells secrete exosomes that carry antigens or mhc  peptide complexes , and induce antigen - specific immune responses by other dendritic cells ( especially in the context of anticancer therapies ) ; conversely , exosomes secreted by dendritic cells subjected to immunosuppressive treatments , or modified to express immunosuppressive cytokines , can instead promote tolerogenic immune responses , which could make them useful as a treatment for autoimmune diseases . in a third example , macrophages infected with procaryotic mycobacteria release exosomes that , depending on the mycobacteria strain , bear antigens and promote immune responses ( nonpathogenic strain ) , or inhibit macrophage activation and cytokine secretion , thus dampening immune response ( pathogenic strain ) .
although exosomes secreted by tumor cells carry antigens from the secreting tumors and can be captured and used to present tumor antigens by dendritic cells , they also bear various immunosuppressive molecules , which can inactivate t lymphocytes or natural killer cells , or promote the differentiation of regulatory t lymphocytes or myeloid cells ( which suppress immune responses ) .
the net result of these contradictory effects in vivo is not yet established , and although several groups defend the hypothesis that tumors secrete exosomes to promote their growth by inhibiting antitumor immune responses , or by promoting angiogenesis or migration outside the tumor bed to form metastases , this has not been proved .
still , it has been suggested that depletion of membrane vesicles from the blood circulation of patients could be used as an anticancer treatment , but i would be cautious in using such approaches in the absence of proper understanding of what tumor - derived exosomes or other membrane vesicles do in vivo .
an alternative explanation is that membrane vesicles of tumor origin in the circulation of cancer patients could simply be the result of tumor expansion , rather than a sign of active involvement of these vesicles in tumor progression .
other tissues or cells secrete exosomes bearing immunosuppressive molecules : placenta - derived vesicles , which bear ligands for natural killer lymphocytes , are found in pregnant women 's blood circulation , and extensive analyses of components of these vesicles are underway to identify their role in the mother 's tolerance to the fetus . depending on the state of the host , exosomes ( or vesicles ) present in their bronchoalveolar fluid
can bear tolerizing molecules ( e.g. , in mice tolerized for an allergen ) or , conversely , increase proinflammatory cytokine secretion by airway epithelial cells ( in asthmatic human patients ) .
secretion of exosomes by eukaryotic parasites ( leishmania major ) or pathogens ( such as fungi ) has also been recently described .
it is thought to contribute to tolerance of the host for the parasite by dampening immune response or , conversely , to contribute pathologic inflammatory reactions of the host to the pathogen .
exosomes are secreted by neural , epithelial , muscle , and stem cells , and their range of proposed functions is extensive .
exosomes from mesenchymal stem cells contribute to tissue repair of host cardiac tissue , and , conversely , lung tissue can release exosomes ( or possibly other vesicles ) that modify stem cells to promote differentiation of the lung tissue into lung epithelial cells .
exosomes are secreted by neurons , oligodendroglial cells , and microglia , and exosomes from each source can affect other neural cells [ 28 - 30 ] .
exosomes are involved in the formation / transfer of pathogenic proteins ( such as prions and amyloid deposits ) .
despite the amount of data on what exosomes ( and other secreted membrane vesicles ) can do in vitro , or in vivo when injected into animals , all data discussed above were obtained with vesicles purified and concentrated in vitro from cell culture supernatants or from biological fluids , so it is not clear whether these processes occur under physiological conditions .
the main problem is that the efficiency of the purification and quantification procedures is unknown .
it is likely that ultracentrifugation does not allow 100% recovery of the vesicles secreted at any given time , and that part of the secreted vesicles are even inaccessible to purification because they are recaptured by cells rather than released in the culture medium or fluid , so it is very difficult to know whether the amounts of membrane vesicles used to observe the effects described above correspond to physiological amounts secreted in vivo , or not .
indeed , there remains a nagging question in the exosome field as to whether they actually have any physiological functions in vivo .
answering this question requires tools to inhibit or increase exosome secretion specifically , without affecting secretion of other membrane vesicles , or affecting general secretion of proteins or lipid mediators .
several groups are currently trying to identify such tools , especially by deciphering the molecular mechanisms involved in formation of intracellular vesicles of multivesicular compartments , and in the fusion of these compartments with the plasma membrane .
several molecules have been shown to affect exosome secretion , although in the absence of means to visualize and quantify actual fusion of multivesicular endosomes with the plasma membrane in real time , it is still difficult to know whether they are involved specifically in this final step , or at another stage of the endocytic pathway .
a consensus has not yet been reached on the molecular mechanisms involved . indeed , depending on the cell types ,
different molecules have been described in the biogenesis and secretion of exosomes : rab11 in erythroleukemia cell lines , rab27 in hela cells , and rab35 and neutral sphingomyelinase in oligodendroglial cells .
it remains to be determined whether these discrepancies are due to different machineries used in different cell types , and/or whether the secreted vesicles analyzed in the different studies come from different intracellular compartments ( along the endocytic pathway , or even outside this pathway ) .
recent studies showing that internal vesicles of multivesicular endosomes can , in some circumstances , form without the help of the escrt ( endosomal sorting complex required for transport ) machinery ( described as being responsible for segregation of molecules into internal vesicles of late endosomes in yeast ) suggest that , indeed , more types of multivesicular compartments than initially thought may coexist in the same cells , and may possibly lead to different types of exosomes . in any case , targeting one or the other of these molecules to affect exosome secretion will require careful evaluation of the consequences ( in terms of exosomes , but also other secretions ) in each different model system analyzed . other approaches to address the physiological functions of exosomes in vivo , proposed during the meeting in paris , include generating exosomes with modified expression of a given functional protein and analyzing their functional outcome , or containing bacterial enzymes to identify target cells via measurement of the enzymatic activity . using these approaches in genetically modified mice will be the next step for this challenging field .
finally , similar cell biology studies on the generation of other secreted membrane vesicles are also underway , and hopefully will allow proper discrimination of the roles as messengers of the different types of vesicles .
with the amazing expansion of the number of published studies on exosomes in the last 3 years , it is clear that research on secreted membrane vesicles and their roles as intercellular messengers is now a very exciting field , with new ideas , hypotheses , and questions coming up faster than answers ! the iwe in paris allowed most of the long - term exosome and vesicle aficionados , as well as many of the newcomers , to meet , exchange ideas freely , and to point out current problems with technologies , definitions , and characterization .
the meeting ended with the decision to prolong this very productive workshop by setting up future yearly meetings , virtual spaces to record exchanges and discussions ( such as a website and/or a facebook page ) , and a scientific society dedicated to exosomes and secreted membrane vesicles .
some of these are already underway , with the next iwe tentatively scheduled for april 2012 , a facebook page already up and running , a website for comparison of protein and rna composition of exosomes already set up , and the rest hopefully coming soon .
we strongly encourage the readers of this article to check these web links   to follow the next steps of this emerging scientific world .
 exosomes are small membrane vesicles of endocytic origin secreted by most cell types , and are thought to play important roles in intercellular communications . although exosomes were originally described in 1983 , interest in these vesicles has really increased dramatically in the last 3 years , after the finding that they contain mrna and microrna . 
 this discovery sparked renewed interest for the general field of membrane vesicles involved in intercellular communications , and research on these structures has grown exponentially over the last few years , probing their composition and function , as well as their potential value as biomarkers . 