Exosomes are small vesicular bodies of 5090nmsize [1] (or 30100 nm according to another reference [2])formed from late endosomes. The formation of the exosome starts from an inner invagination of the endosomalmembrane that results in a luminal vesicle, whereas theendosome becomes a multivesicular body. The fusion oflate endosomes with the cytoplasmic membrane is associated with release of luminal vesicles into the extracellularspace. Thus, exosomes are luminal vesicles deserted fromthe cell [3]. The exosomal cavity has cytoplasmic origin,whereas their membrane is a result of invagination of theendosomal membrane. Theoretically, any cell containingmultivesicles can secret exosomes. In fact, according toexperimental data, exosomes can be produced in vitro bythe majority of differentiated cells: the exosome production by B and Tcells, granulocytes, dendrite cells,platelets, neurons, and epithelial cells is described ratherin detail. Exosomes were also found in vivo, in particular,they are present in many physiological liquids, includingblood serum, bronchoalveolar lavage, urine, and breastmilk [4]. The work published in 1983 on the fate of transferrin receptors during maturation of sheeps reticulocytes is usually considered to be the first scientific

description of exosomes [5]. The authors found that during the maturation of reticulocytes, exosomes could participate in restructuring of membranes by carrying thetransferrin receptors out [6]. Initially researchers considered exosomes as either formations specific for a strictlydefinite type of cells or as byproducts of the cells vitalactivity free of any significant function. The discoverythat antigenpresenting cells can secret exosomes possessing their own immunostimulating activity changedthe attitude of scientific community to these vesicles [7].Valadi et al. described in 2007 a large pool of RNA molecules found in exosomes of mastocytes [8]. It was thispublication that moved the studies in the field on a fundamentally new level.

Each new article now adds surprising examples to theset of supposed functions of exosomes. Exosomes areinvolved in elimination of unwanted proteins from thecell membrane and contribute to initiation and development of the immune response. Leukocytes use the releaseof a huge amount of exosomes during coagulation.Exosomes can be involved in development of some pathological processes, such as growth and spreading of malignant tumors, transfer of infectious agents whole virusparticles or viral RNAs, and also of prions. Exosomesseem to play a special role in the intercellular communication of multicellular organisms as they carry on their

ISSN 00062979, Biochemistry (Moscow), 2013, Vol. 78, No. 1, pp. 17. Pleiades Publishing, Ltd., 2013.Original Russian Text O. N. Gusachenko, M. A. Zenkova, V. V. Vlassov, 2013, published in Biokhimiya, 2013, Vol. 78, No. 1, pp. 513.

REVIEW

1

* To whom correspondence should be addressed.

Nucleic Acids in Exosomes: Disease Markersand Intercellular Communication Molecules

O. N. Gusachenko*, M. A. Zenkova, and V. V. Vlassov

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8,630090 Novosibirsk, Russia; fax: (383) 3635153; Email: niboch@niboch.nsc.ru; oligonucleotide@gmail.com

Received July 2, 2012

AbstractThe term exosomes is currently used to describe specific vesicular structures of endosomal origin produced bythe majority of eukaryotic cells. These natural vesicles have been under study for more than two decades. Nevertheless, areal splash of scientific interest in studies on exosomes took place only during recent years, when the concept of the role andfunctions of exosomes in multicellular organisms was essentially reconsidered. The major role in this was played by the discovery of exosomal mRNA and miRNA in 2007, which stimulated the idea of regulatory and communicative role of exosomes in the organism and also encouraged considering exosomes and other vesicles as potential biomarkers. The presentreview summarizes the up to date knowledge on the composition and probable physiological functions of nucleic acidsreleased by different cells as components of exosomes. We also touch upon the problem of using these data in clinical diagnosis.

DOI: 10.1134/S000629791301001X

Key words: exosomes, microvesicles, extracellular nucleic acids, biomarkers, intercellular communication, miRNA, mRNA

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surface various protein and lipid ligands and also delivernew proteins and nucleic acids into recipient cells [9].Exosomes have been found to contain different RNAs,including messenger RNA (mRNA), microRNA(miRNA), and long noncoding RNA. The main function of mRNA in the organism is the transmission ofinformation about the amino acid sequence of a proteinto the synthetic machinery of the cell. The transfer ofintact mRNAs as parts of exosomes can serve forexchanging phenotypic features between cells, becauserecipient cells get mRNAs of proteins not expressed inthem initially. The miRNAs are short noncoding RNAsof 2125 nucleotides length. They are involved in regulation of gene expression on posttranscriptional level bytriggering mechanisms of translation blocking or destruction of mRNAs containing complementary or partiallycomplementary sequences. In some cases, miRNAs canregulate gene expression also on the level of transcription,influencing the modification of histones and methylationof promoter regions of DNA [10]. Supposedly, themiRNA targets are present in approximately 60% of allmammalian genes. In any cell of the body, there are largeamounts of miRNAs, and each miRNA is able to influence hundreds of mRNA targets. The miRNAs areknown to be involved in regulation of various processesincluding the cell differentiation, division, apoptosis,endocrine system functioning, hemopoiesis, and morphogenesis of different organs [10]; the role of miRNAsin development of various pathological processes, such ascancer, viral diseases, hereditary diseases, etc. is alsounder active investigation.

The work of Ratajczak et al. on cultivation of embryonic stem cells [11] was one of the first demonstrations ofhorizontal mRNA transfer between cells [11]. Theauthors found that vesicles produced by these cells couldtransfer specific mRNAs into precursors of hematopoietic cells, which resulted in changes of cells phenotype.Later the presence of extracellular miRNAs in variousbiological fluids of the body was shown [12]. Based on theavailable experimental data, it was supposed that nucleicacids transferred in the exosomal cavity should have anepigenetic influence on the organisms cells. Thishypothesis not only changed ideas about intercellularcommunication mechanisms, but it also stimulated studies on possible use of exosomal RNAs as biomarkers.

METHODS FOR ISOLATION OF EXOSOMES

In the literature the terms exosome andmicrovesicle often overlap, and sometimes they areeven used as synonyms. In the first place, this is due topreferential use by authors of the more generalized termmicrovesicles that also includes larger membrane formations (more than 1 m), instead of a strict determination of the type of vesicles under study. Differences in the

initial biological materials and also different approachesand goals of studies have resulted in a rather confused terminology, and within its limits vesicular particles produced by cells can be designated in various ways, e.g.microparticles, microvesicles, ectosomes or sheddingmicrovesicle, nanovesicles (nanoparticles), exosomes,exosomelike particles, apoptotic blebs, promininosomes, prostatosomes, dexosomes (dex), texosomes(tex), epididimosomes, argosomes, archaeosomes, oroncosomes [13]. Most strictly, the term exosome designates the membrane particles of endocytotic origin withsize less than 100 nm that are produced as a result offusion of multivesicular bodies with the plasma membrane.

The classical protocol for isolation of exosomesincludes a number of successive centrifugations thatallows to get rid of cells and larger particles, and then exosomes are precipitated by ultracentrifugation at 100,000g[14]. In this case, the desired particles are subtracted froma sample by their size. Thus, this approach is not specificfor vesicles of strictly endosomal origin. A very simpleadditional procedure of filtration through smallporousfilters (0.10.2 m) can free the final precipitate fromlarger particles (microvesicles) [15], but it cannot separate exosomes from other particles with similar size (protein aggregations). Exosomes can be additionally purifiedby isolation in a sucrose gradient or on a sucrose cushion.Exosomes are known to float at the density range from1.13 g/ml (products of Bcells) to 1.19 g/ml (products ofintestine cells) [14]. This purification results in a sufficiently effective separation of exosomes from unassociated protein complexes and fragments of nucleosomesreleased by apoptotic bodies.

The use of successive centrifugation for isolation of arelatively small quantity of exosomes is a rather inconvenient and laborious procedure. Lamparski et al. worked outa protocol for isolation of clinical grade exosomes inwhich the precipitation stage was replaced by an ultrafiltration procedure with subsequent purification of thepreparation on a sucrose cushion. The authors proposedthis method as more rapid, productive, and reproduciblethan the classic one [16]. In work [17], Cheruvanky et al.used a commercial nanomembrane concentrator to prepare exosomes suitable for clinical analysis, and this alsoallowed them to avoid the ultracentrifugation. In additionto filtration, exosomes can be isolated from specimensunder analysis by precipitation in the presence of different polymers (as done for isolation of viral particles) witha subsequent filtration or centrifugation. Supposedly, thisprinciple is used in ExoQuick product (SystemBiosciences, USA; http://www.systembio.com) designedfor exosomes isolation from urine or blood serum.However, despite the declared efficiency, according to theusers references this preparation can precipitate not onlyexosomes but also other particles, and this creates difficulties in its use.

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Using antibodies against exosomal markers or proteins specific for definite cell types seems to be a promising approach for selective isolation of exosomes of knownnature. Depending on the purpose and methodology ofthe study, antibodies can be immobilized on magneticbeads, chromatographic matrices, plates, or other working surfaces. This approach for isolation results in preparation of exosomes with a known set of antigens, but thesubsequent washing of exosomes from the antibodies canbe rather inefficient, and this limits their further application. Flow cytometry of exosomes on beads is a convenient method for detecting the known protein markers inthe preparation and serves as additional method of quality control. Currently other affinity approaches for isolation of exosomes including filtration systems or captureon affinity columns are under development [18]. Usinglectins capable of binding sugar residues exposed on thesurface of vesicles seems to be a new interesting approachin this field (http://www.aethlonmedical.com).

Vesicles can be characterized as exosomes based on acombination of morphological, biochemical, and physical properties. Because of their small size, exosomes canbe observed only by electron microscopy [19]. The quality of an exosome preparation can be also assessed by separation in polyacrylamide gel with subsequent specificand nonspecific staining of proteins. Many proteins areaccumulated in exosomes selectively (e.g. flotillin, Alix,CD63) and can be used as markers of the exosomal fraction. Exosomes are also characterized by saturation oftheir membrane with cholesterol, sphingolipids, andglycerolipids that makes them similar to lipid rafts.Exosomes of some cells contain not only protein andmembrane components, but also different types of RNA,which can be easily isolated and identified by standardanalytical methods of molecular biology. It is rather probable, that the presence of nucleic acids in exosomes canalso be used for detection of the exosomal fraction in apreparation and for identification of exosomes with a definite histological origin.

RNA AS A COMPONENT OF EXOSOMES

In the work published by Valadi et al. in 2007 about1300 different mRNAs and 121 miRNAs were mentioned(according to the database http://mirbase.org there areabout 1500 different miRNA known in human to thedate). Later, RNAs were found in exosomes of variouscells (mast cells, tracheobronchial cells, dendrite cells, Band Tlymphocytes, cells of intestine, lung, and stomachcancers, and of pancreatic adenocarcinoma [2023]) andalso in exosomes isolated from different bodily fluids(blood, saliva, breast milk, pleural fluid, ascites [24, 25]).By the initiative of Simpson and Mathivanan, the electronic database ExoCarta (http://exocarta.org) was created, which includes information about proteins, lipids,

mRNAs, and miRNAs of exosomes with different origin.According to the data of ExoCarta, in the majority of thepublished works new RNAs were identified usingmicroarray methods. Now several thousands of differentRNAs are described as presumable components of mammalian exosomes (more than 1600 mRNAs and morethan 700 miRNAs according to ExoCarta).

On appearance of the first data about exosomalRNAs, their presence in the exosome preparations wasinitially supposed to be due to contamination of the specimens by molecules of nucleic acids released from dyingcells. On the other hand, different RNAs could beenclosed in an exosome during its formation due to occasional capture of molecules occurring in the cytoplasm ofthe cells. According to data published by Valadi et al. [8]and also by several other groups, concentration ratios ofmany mRNAs found in the exosome preparations significantly differ from the ratios in the cells cytoplasm. Theresults of Ohshima et al. revealed a selectivity of miRNAspackage in exosomes of metastasizing intestinal cancer,whereas the initial poorly metastasizing cell line did notdisplay a similar selectivity. The authors concluded that inthis case the secretion of miRNAs by the exosomal pathway could play a role in oncogenesis [21]. The existenceof a specific mechanism of RNA sorting for directedsecretion was suggested. An attempt to perform computeraided analysis of exosomal RNAs was performed byBatagov et al. [26]. They reported the presence of somespecific repeats in sequences of excreted RNAs that couldact as ciselements under selective conditions. In the caseof experimental confirmation of the data, this study canat least partially dispel the mystery of the mechanism ofRNA excretion via exosomal pathway. In any case, nodata was actually found to the date in favor of existence ofspecific exosomal RNA sequences. On the contrary, itwas shown for some tumors that exosomes can containsets of miRNAs with the composition similar to that inthe initial cancer cells [27, 28]. Although some data indicate differences in the pools of intracellular and excretedRNAs, it is still unclear whether such sorting is selective.

But how the sorting of RNAs into exosome is realized? It was found that multivesicular bodies and effectorcomplexes of miRNAs are functionally related [29, 30].Such colocalization seems to indicate that multivesicular bodies are places for assembly of miRNAproteincomplexes, and during this process miRNAs also can beselected for secretion in exosomes. Kosaka et al. [31]showed that the release of exosomal miRNAs can be controlled by sphingomyelinase 2 and realized via aceramidedependent pathway. This corresponds with theobservations of Mittelbrunn et al., showing that inhibition of sphingomyelinase expression leads to disturbancein the miRNA transfer into antigenpresenting cells andthat ceramide is involved in triggering miRNA secretionby the cells [23]. Montecalvo et al. [32] studied the mechanism of exosomal miRNA transfer between dendritic

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cells. Exosomes isolated from the culture of dendriticcells contained 200 miRNAs, and five of them were present only in exosomes of immature dendritic cells, whereas 58 miRNAs were found only in exosomes of maturedendritic cells. Thus, the authors showed that dendriticcells can produce exosomes with different RNA composition depending on the stage of their developmental. Theset of miRNA present in exosomes was also different fromthat in the initial cells. The functional activity of twomiRNAs transferred by exosomes was confirmed in thisstudy using the transfection of treated cells with luciferaseconjugated target.

The fact that nucleic acids can be captured into exosomes and that their presence inside the vesicles does notseem to be accidental suggests the supposition on theprobable role of exosomes in the horizontal transfer ofgenetic information between cells of a multicellularorganism. In particular, exosomes can be unique transducers of signaling information between distant cells andtissues. Thus, mRNA inside exosomes of mastocytes wasshown to be translationally active, i.e. on entrance intorecipient cells this RNA could participate in the synthesisof new proteins [8]. The authors also found that suchtransfer of information can occur in vitro between thecells of two different organisms mouse and human:upon incubation of a culture of human mastocytes in thepresence of mouse exosomes, mouse proteins weredetected in the human cells, and three of these proteinswere absent in the exosomes themselves, but theirmRNAs were found in the vesicles. The mechanism ofinteraction between cells and exosomes is still underinvestigation. The difficulty of these studies is associatedin particular with the extremely small size of the exosome,which prevents observations with a fluorescence microscope. The transfer of material through exosomesbetween cells can also vary depending on the type andstate of the cells. Thus, if an interaction of an exosomewith a cell occurs with involvement of surface receptors,we may speak about a specific directed delivery of theexosomal material into strictly definite types of cells. Thepossible existence of such mechanism was also confirmedby data of Valadi et al. [8]. They found that exosomalRNA could be transferred between mastocytes, but it didnot penetrate into CD4 lymphocytes.

The transport of nucleic acids inside the exosomescan play an important role in functioning of a multicellular organism; moreover, the supposed specificity of thisprocess can, on necessity, contribute to the directedtransfection of an exogenous material. AlvaresErviti etal. [33] used exosomes of mice dendritic cells as a transfection vector for short interfering RNAs. It was supposedthat the use of exosomes produced by animals own cellswill lead to the decreased immunogenicity of this vector,whereas inclusion into exosomes of a hybrid protein consisting of Lamp2b (a membrane protein of the exosomalfraction) and a neuronspecific peptide RVG should

result in a directed delivery of RNA into the cells of nervous tissue. The intravenous injection of exosomes electroporated with delivered RNAs resulted in a specific delivery of biologically active RNAs into neurons, microgliacells, and brain oligodendrocytes, but not into other tissues (liver, spleen, kidneys, etc.). The exosomemediatedtransfection allowed the authors to obtain significantdecrease of the levels of the target mRNA and of the protein encoded by it (by 60 and 62%, respectively). In thiscase the authors showed not only the possibility of usingshort RNAs inside exosomes as a transfecting systemcapable of inhibiting the target gene expression in braintissues (i.e. of penetrating across the bloodbrain barrier), but the principle of in vivo functioning of exosomeswas demonstrated, which is of no less importance.

What possible biological role the exosomal RNAs, orshuttle RNAs (shRNAs) as they are often referred to,might play? According to analysis performed with theIngenuity system software (http://www.ingenuity.com),mRNAtranscripts from exosomes of mastocytes arefunctionally related to the processes of cytogenesis, protein synthesis, and posttranscriptional modification ofRNA [8]. Analysis of miRNAs found in exosomes of thesecells is more complicated, first of all because of multiplicity of miRNA functions. The noticeable differencebetween the miRNA pool in the exosomes of mastocytesand in the cytoplasm probably indicates a certain directed regulatory function of these RNAs inside the vesicles.The identified miRNAs include let7, lin4, miR1, miR15, miR16, miR17, miR18, miR181, miR375, etc.These miRNAs are involved in the regulation of celldevelopment, angiogenesis, hematopoiesis, exocytosis,and oncogenesis. The released miRNAs are now considered to represent a new class of paracrine regulation molecules. A possible example of this function might be aninvolvement of exosomal miRNAs in the regulation ofcardiomyocyte growth. It was shown that the miRNAsfrom fibroblasts can penetrate into cardiomyocytes andinduce the development of hypertrophy [34].

Regulation of the immune system can be an important function of released miRNAs. Both T and Blymphocytes and also dendritic cells of the immune systemare known to secrete miRNAs as components of exosomes [23]. It was also shown that the interaction of Tlymphocytes with antigenpresenting cells could be associated with antigeninduced directed transfer ofmiRNAs. Exosomal miRNAs are transferred into antigenpresenting cells during formation of the immunesynapse with Tlymphocyte, and they can function in therecipient cell. The involvement of exosomes in the transfer of biologically active RNAs from mother to child during pregnancy and breastfeeding might represent aunique example of immunological functions of exosomes.Specific placental miRNAs were detected in exosomes ofthe villiferous trophoblast [35]. The exchange of exosomal contents between the fetus and mothers tissues is

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supposed to provide adaptation of the two organisms toone another during the progression of pregnancy. Breastmilk also contains a significant amount of immunorelevant miRNAs, and at least some of them are inside theexosomes [36]. These miRNAs might play an importantrole in development of the childs immune system.

In addition to involvement in normal functioning ofthe body, exosomal RNAs can also play a crucial role indevelopment of health disorders. Progression of oncological diseases directly depends on the efficiency of communication between the malignant and healthy cells.Primary glioblastoma cells were shown to secrete microvesicles and exosomes containing mRNAs and miRNAs[37]. It was found that a culture of multivesicular braincells is capable of taking up these vesicles, which resultedin translation of the transported mRNAs. Thus, using theexosomal pathway, tumor cells could modify the translation profiles of surrounding healthy cells. Oshima et al.[21] suggested that exosomal RNAs could have an oncogenetic function. They found that miRNAs of the let7family were intensively excreted inside exosomes of theAZP7a cell line from metastasizing intestinal cancer.Because miRNAs of the let7 family usually mediate thesuppression of tumor growth, the authors supposed thatsecretion of these RNAs inside the exosomes allowed thetumor to decrease the antioncogenic activity of the cellsand increase the probability of the tumor metastasis.

Exosomes are also known to promote distributionand maintain the populations of some pathogens including certain viruses and prions [38]. Type4 herpes virus, orEpsteinBarr virus, is considered to be one of the mostwidespread human viruses. On the grounds of exosomalRNA profiles analysis for infected cells, Pegtel et al.detected in the vesicles virusencoded miRNAs [39]. Thiswork was performed on a coincubation model usinginfected Blymphoid cells with a culture of dendriticcells. The results revealed an ability of the viral miRNAsto be transferred into noninfected cells and display theretheir biological activity. While the viral DNA was foundonly in the Blymphocyte population, miRNAs of theEpsteinBarr virus were detected not only in the infectedBlymphocytes, but also in other (noninfected) cells ofpatients with an increased level of the virus in the body.These data on transduction of viral miRNA into noninfected cells confirm the hypothesis about the evolutionof herpes viruses in the direction of exploitation of thehosts cells interference machinery.

EXOSOMAL NUCLEIC ACIDS AS BIOMARKERS

The cells in the organism release exosomes into theextracellular space normally and also on development ofdiseases. These vesicles contain macromolecules of proteins, lipids, and nucleic acids that can be used as biomarkers of the producing cell state. The presence of a sig

nificant amount of exosomes in physiological fluids of thebody (blood plasma, urine, breast milk, sperm, saliva,etc.) provides the opportunity to use noninvasive or lowlyinvasive approaches for sampling; therefore, these particles are a desirable object for development of approachesfor routine clinical analyses. Exosomal nucleic acids possess many features of good biomarkers. The excretednucleic acids are highly stable in biological fluids (thisobservation is often used to indirectly confirm the releaseof nucleic acids as parts of special protective complexes).The levels of RNAs in preparations can be easily determined by standard methods of molecular biology based onquantitative PCR, which possess high sensitivity and efficiency. Strictly speaking, RNAs can be found in physiological fluids not only in exosomes, but also in other vesicles, as well as in complexes with proteins. Comparison ofmRNA pools in exosomes and microvesicles of similarorigin (or in combined specimens of exosomes andmicrovesicles) might be of particular interest [29]. At present, there is no uniform opinion on the main form of RNAtransfer in intercellular fluids: for example, the results ofTurchinovich et al. [40] show that the major part of extracellular RNA is vesiclesfree. In our opinion, it is reasonable to suggest that the release of RNA can exploit different mechanisms and depends on the secretion conditions(developmental state of the cell, macro and microenvironment, presence of pathology, general state of theorganism) and on the type of RNA targets (i.e. what formof the transported RNA can be taken up by the recipientcell). However, the lack of clear knowledge on this subjectdoes not prevent many medicobiological companies fromthe initiation of new projects, which include the use ofexosomal nucleic acids in diagnostics. Exosomes havebeen shown to contain tissue or tumorspecific sets ofmiRNAs that seems to be promising for diagnosis. Beforethe discovery of exosomal RNAs, the analysis of tumormiRNA profile usually required biopsy an invasive procedure, which might be strongly undesirable or evenimpossible in certain types of cancer. Application of RNAprofiles analysis can be preferable in diagnostics of thetumors which do not possess any known and reliablemolecular markers, e.g. in ovarian cancer [37]. Skog et al.were first to demonstrate that secreted miRNAs can beused for diagnosis of glioblastoma and determining thestage of desease [37]. It was found that the level of exosomes in blood serum of patients with ovarian cancer correlated with the disease stage; moreover, the total numberof exosomes in the patients blood was higher than in thecontrol group [27]. The authors also reported that miRNAprofiles in specimens of the exosomes were close to themiRNA set of the tumors. Similar results were obtained ina study on RNA composition of exosomes of healthydonors and patients with lung cancer [28]. The knowledgeon the presence of viral RNAs in the exosomes (e.g. exosomes of type4 herpes virus) can also be applied for theinfection detection in the organism.

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Sjogrens syndrome is an autoimmune systemic disease manifesting itself in particular by chronic influenceon the salivary and lachrymal glands. Michael et al. isolated exosomes from the saliva of patients with this syndromeand showed that these vesicles contain significant amountsof miRNA. On the grounds of obtained data, the authorssuggested the possibility of new diagnostic method elaboration for the asymptomatic patients [41]. In this case, thenoninvasive and rapid analysis of the patients saliva canbe a good alternative for the routinely used biopsy.

Urine is another easily accessible fluid naturallyreleased from the body. Tests using exosomes in urine arenow actively developed for diagnosis of urogenital diseases, in particular, of prostate cancer [42]. Two specificmRNAs (PCA3 and a product of specific chromosomeaberration TMPRSS2:ERG), which are hyperexpressedon prostate carcinoma, were found in exosomes of thepatients urine.

Other prospects of using exosomal RNAs as biomarkers include diagnosis of probable traumas of thebrain by the presence of specific vesicular miRNAs in thecerebrospinal fluid, the early detection of vascular diseases, and also diagnosis in the case of asymptomatic diabetes based on specific profiles of blood miRNAs. Finally,the diagnostic potential of exosomal biomarkersembraces not only pathological processes. Thus, comparison of miRNA levels in blood plasma of pregnant andnonpregnant women revealed the correlation with theplacentaassociated miRNAs and the presence of pregnancy and also with its stage [43].

The concept of intercellular communication viadirected and selective transfer of genetic material by exosomes is very prospective for modern biology and medicine. It should be emphasized that the overwhelmingmajority of experimental data describing various processes involving exosomes in vitro and in vivo has beenobtained with microparticles isolated from cell cultures.The correlation between the conclusions based on thesedata and the real principles of exosome functioning underconditions of a living body still relates to one of the mostsignificant issues in this field. Nevertheless, this does notdiminish the value of the obtained results, as they directly indicate the practical potential of exosomes application. Although current knowledge fails to give a completeidea about the origin and nature of RNAcontaining vesicles, many pharmacological companies are intensivelyworking to create test systems based on exosomes. Insome laboratories, the contents of exosomes (and also ofother extracellular vesicles) are comparatively analyzed inspecimens obtained from healthy donors and patientswith different diseases. There are many prerequisitesfavoring the application of exosomal RNAs as biomarkers. Among the problems, which are to be solved by thetest system developers, lies the necessity to elaborate aneffective and economical isolation method of exosomes

(or their components, e.g. RNA) from small volumes ofbiological fluids. Another issue is standardization ofmethods for RNA analysis in specimens, including thesearch for stable control biomarkers with low variability inthe population and high reproducibility in standard analytical procedures. Considering the amount and intensityof modern studies in this field, it will be possible to verifythe efficiency of currently developing approaches within12 years. In which areas of diagnostics the exosomesbased methods will be actually applicable practice andfurther studies will show.

The work was financially supported by RFFI (grantNo. 110401429a), program Molecular and CellularBiology of the Presidium of the Russian Academy ofSciences, and Siberian Branch of the Russian Academyof Sciences (project No. 84).

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