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Journal ArticleDOI

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

07 May 2007-Nature Cell Biology (Nature Publishing Group)-Vol. 9, Iss: 6, pp 654-659
TL;DR: It is shown that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location, and it is proposed that this RNA is called “exosomal shuttle RNA” (esRNA).
Abstract: Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).

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Citations
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Journal ArticleDOI
TL;DR: This comprehensive review summarizes current knowledge of EV uptake mechanisms and seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route.
Abstract: Extracellular vesicles (EVs) are small vesicles released by donor cells that can be taken up by recipient cells. Despite their discovery decades ago, it has only recently become apparent that EVs play an important role in cell-to-cell communication. EVs can carry a range of nucleic acids and proteins which can have a significant impact on the phenotype of the recipient. For this phenotypic effect to occur, EVs need to fuse with target cell membranes, either directly with the plasma membrane or with the endosomal membrane after endocytic uptake. EVs are of therapeutic interest because they are deregulated in diseases such as cancer and they could be harnessed to deliver drugs to target cells. It is therefore important to understand the molecular mechanisms by which EVs are taken up into cells. This comprehensive review summarizes current knowledge of EV uptake mechanisms. Cells appear to take up EVs by a variety of endocytic pathways, including clathrin-dependent endocytosis, and clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft–mediated internalization. Indeed, it seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route. The uptake mechanism used by a given EV may depend on proteins and glycoproteins found on the surface of both the vesicle and the target cell. Further research is needed to understand the precise rules that underpin EV entry into cells. Keywords: extracellular vesicles; EV uptake; EV internalization; cell–EV interaction; endocytosis; cell communication; exosomes (Published: 4 August 2014) Citation: Journal of Extracellular Vesicles 2014, 3 : 24641 - http://dx.doi.org/10.3402/jev.v3.24641

1,809 citations


Cites background from "Exosome-mediated transfer of mRNAs ..."

  • ...and post-translational regulation of target mRNAs (3)....

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  • ...EVs have been shown to transfer functional mRNA and miRNA from mouse to human mast cells where mouse proteins were identified in the recipient human cells (3)....

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Journal ArticleDOI
TL;DR: It is shown that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAAs are transferable and functional in the recipient cells and that a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipients cells, thereby leading to cell growth inhibition.

1,751 citations

Journal ArticleDOI
TL;DR: A comprehensive overview of extracellular vesicles is given in this article, where the authors compare results from meta-analyses of published proteomic studies on membrane Vesicles.
Abstract: Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy.

1,737 citations


Cites background from "Exosome-mediated transfer of mRNAs ..."

  • ...Exosomes may horizontally transfer mRNA and miRNA [18]....

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  • ...diseases [18]: eMVs may contribute to vascular injury and they are capable of inducing endothelial cell activation, impairing vasorelaxation [154], and promoting arterial...

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Journal ArticleDOI
TL;DR: This is the first study to show that extracellular miRNAs are predominantly exosomes/microvesicles free and are associated with Ago proteins, and hypothesize that ext racellular miRNA are in the most part by-products of dead cells that remain in extrace cellular space due to the high stability of the Ago2 protein and Ago2-miRNA complex.
Abstract: MicroRNAs (miRNAs), a class of post-transcriptional gene expression regulators, have recently been detected in human body fluids, including peripheral blood plasma as extracellular nuclease resistant entities. However, the origin and function of extracellular circulating miRNA remain essentially unknown. Here, we confirmed that circulating mature miRNA in contrast to mRNA or snRNA is strikingly stable in blood plasma and cell culture media. Furthermore, we found that most miRNA in plasma and cell culture media completely passed through 0.22 µm filters but remained in the supernatant after ultracentrifugation at 110 000g indicating the non-vesicular origin of the extracellular miRNA. Furthermore, western blot immunoassay revealed that extracellular miRNA ultrafiltrated together with the 96 kDa Ago2 protein, a part of RNA-induced silencing complex. Moreover, miRNAs in both blood plasma and cell culture media co-immunoprecipited with anti-Ago2 antibody in a detergent free environment. This is the first study to show that extracellular miRNAs are predominantly exosomes/microvesicles free and are associated with Ago proteins. We hypothesize that extracellular miRNAs are in the most part by-products of dead cells that remain in extracellular space due to the high stability of the Ago2 protein and Ago2-miRNA complex. Nevertheless, our data does not reject the possibility that some miRNAs can be associated with exosomes.

1,712 citations


Cites background or methods from "Exosome-mediated transfer of mRNAs ..."

  • ...Recently, it has been suggested that extracellular miRNA entrapped in exosomes to be involved in intercellular communication (12,14,15)....

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  • ...To further confirm the hypothesis of exosome-free extracellular miRNA, we fractionated blood plasma and conditioned media of MCF7 cells according to well-developed methods for exosomes isolation (12,18)....

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  • ...Although, exosomal miRNA has been hypothesized to be involved in intercellular communication (12,15) it remains unclear whether all extracellular miRNAs are associated with exosomes and whether extracellular miRNA are present in physiologically relevant amounts for cell-to-cell signaling....

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  • ...Extracellular miRNAs have recently been detected in exosomes isolated from peripheral blood and culture media of several cell lines (12,16)....

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Journal ArticleDOI
TL;DR: Strategies for exosome isolation, the understanding to date of exosomes composition, functions, and pathways, and their potential for diagnostic and therapeutic applications are summarized.

1,639 citations


Cites background from "Exosome-mediated transfer of mRNAs ..."

  • ...Although protein transport by exosomes was accepted by the research community, the transport of RNA was not shown until much later — Valadi et al. 2007 [10] is the first publication to definitively show that RNA was carried in exosomes as well....

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References
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Journal ArticleDOI
TL;DR: PicTar, a computational method for identifying common targets of micro RNAs, is presented and widespread coordinate control executed by microRNAs is suggested, thus providing evidence for coordinate microRNA control in mammals.
Abstract: MicroRNAs are small noncoding RNAs that recognize and bind to partially complementary sites in the 3' untranslated regions of target genes in animals and, by unknown mechanisms, regulate protein production of the target transcript. Different combinations of microRNAs are expressed in different cell types and may coordinately regulate cell-specific target genes. Here, we present PicTar, a computational method for identifying common targets of microRNAs. Statistical tests using genome-wide alignments of eight vertebrate genomes, PicTar's ability to specifically recover published microRNA targets, and experimental validation of seven predicted targets suggest that PicTar has an excellent success rate in predicting targets for single microRNAs and for combinations of microRNAs. We find that vertebrate microRNAs target, on average, roughly 200 transcripts each. Furthermore, our results suggest widespread coordinate control executed by microRNAs. In particular, we experimentally validate common regulation of Mtpn by miR-375, miR-124 and let-7b and thus provide evidence for coordinate microRNA control in mammals.

4,660 citations


"Exosome-mediated transfer of mRNAs ..." refers background in this paper

  • ...Hypothetically, the 121 miRNAs that we have found in the mast cell exosomes may interfere with 24,000 mRNAs as it has been suggested that each species may interact with up to 200 mRNA...

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Journal ArticleDOI
TL;DR: It is demonstrated by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles, suggesting a role for exosomes in antigen presentation in vivo.
Abstract: Antigen-presenting cells contain a specialized late endocytic compartment, MIIC (major histocompatibility complex [MHC] class II-enriched compartment), that harbors newly synthesized MHC class II molecules in transit to the plasma membrane. MIICs have a limiting membrane enclosing characteristic internal membrane vesicles. Both the limiting membrane and the internal vesicles contain MHC class II. In this study on B lymphoblastoid cells, we demonstrate by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles. These secreted vesicles, named exosomes, were isolated from the cell culture media by differential centrifugation followed by flotation on sucrose density gradients. The overall surface protein composition of exosomes differed significantly from that of the plasma membrane. Exosome-bound MHC class II was in a compact, peptide-bound conformation. Metabolically labeled MHC class II was released into the extracellular medium with relatively slow kinetics, 10 +/- 4% in 24 h, indicating that direct fusion of MIICs with the plasma membrane is not the major pathway by which MHC class II reaches the plasma membrane. Exosomes derived from both human and murine B lymphocytes induced antigen-specific MHC class II-restricted T cell responses. These data suggest a role for exosomes in antigen presentation in vivo.

2,978 citations

Journal ArticleDOI
TL;DR: The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine and identify numerous protein components of MVBs and of the endosomal pathway in general.
Abstract: Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented "cytoplasmic-side inward," consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.

1,941 citations

Journal ArticleDOI
01 Jul 1983-Cell
TL;DR: The fate of the transferrin receptor during in vitro maturation of sheep reticulocytes has been followed using FITC- and 125I-labeled anti-transferrin-receptor antibodies and it can be shown that at 0 degree C or in phosphate-buffered saline the rate of vesicle release is less than that at 37 degrees C in culture medium.

1,543 citations


"Exosome-mediated transfer of mRNAs ..." refers background in this paper

  • ...Many cells have the capacity to release exosomes, including reticulocyte...

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Journal ArticleDOI
01 May 2006-Leukemia
TL;DR: ES-MV isolated from murine ES cells in serum-free cultures significantly enhanced survival and improved expansion of murine HPC, and upregulated the expression of early pluripotent and early hematopoietic stem cells in these cells.
Abstract: Membrane-derived vesicles (MV) are released from the surface of activated eucaryotic cells and exert pleiotropic effects on surrounding cells. Since the maintenance of pluripotency and undifferentiated propagation of embryonic stem (ES) cells in vitro requires tight cell to cell contacts and effective intercellular signaling, we hypothesize that MV derived from ES cells (ES-MV) express stem cell-specific molecules that may also support self-renewal and expansion of adult stem cells. To address this hypothesis, we employed expansion of hematopoietic progenitor cells (HPC) as a model. We found that ES-MV (10 microg/ml) isolated from murine ES cells (ES-D3) in serum-free cultures significantly (i) enhanced survival and improved expansion of murine HPC, (ii) upregulated the expression of early pluripotent (Oct-4, Nanog and Rex-1) and early hematopoietic stem cells (Scl, HoxB4 and GATA 2) markers in these cells, and (iii) induced phosphorylation of MAPK p42/44 and serine-threonine kinase AKT. Furthermore, molecular analysis revealed that ES-MV express Wnt-3 protein and are selectively highly enriched in mRNA for several pluripotent transcription factors as compared to parental ES cells. More important, this mRNA could be delivered by ES-MV to target cells and translated into the corresponding proteins. The biological effects of ES-MV were inhibited after heat inactivation or pretreatment with RNAse, indicating a major involvement of protein and mRNA components of ES-MV in the observed phenomena. We postulate that ES-MV may efficiently expand HPC by stimulating them with ES-MV expressed ligands (e.g., Wnt-3) as well as increase their pluripotency after horizontal transfer of ES-derived mRNA.

1,464 citations