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Discipline
Biological
Keywords
Exosomes
Extracellular Vesicles
DNA
Uptake
Observation Type
Standalone
Nature
Standard Data
Submitted
Nov 6th, 2015
Published
Feb 21st, 2016
  • Abstract

    Extracellular vesicles (EVs) carry multiple bioactive molecules, including proteins and nucleic acids. Cell-free, extracellular DNA has been reported to be present in cell cultures and in multiple body fluids, but its relative location in relation to EVs has not been described. This study demonstrates that DNase-sensitive nucleic acids are present on the surface of EV isolates. Association of EV-DNA was revealed by increase of EV zeta potential and particle number upon DNase treatment. Additionally, cells exposed to EVs with associated DNA show the presence of cytoplasmic DNA traces intracellularly. In conclusion, we suggest that DNA can be associated to the surface of EVs and can be taken up by recipient cells. DNA on EV surfaces may influence their function in recipient cells.

  • Figure
  • Introduction

    Extracellular vesicles (EVs) are membrane bound structures released by cells, and found in all body fluids. The populations of EVs are diverse, and the nomenclature includes terms such as exosomes, ectosomes and microvesicles. Previously, it has been shown that the subpopulations of EVs called exosomes can carry both mRNA and microRNA, which can mediate function in recipient cells. Additionally, dsDNA has previously been proposed to be associated with EVs, but its relative location to/in EVs has not been well described. This study reports for the first time that cell-free DNA can be associated with the outside of EVs, which may influence aggregation of these EVs.

  • Objective

    This work aims to determine the relative location of cell-free DNA in relation to extracellular vesicles.

  • Results & Discussion

    On a nucleic acid-stained gel, clear DNA bands were visualized, and these were present not in samples pretreated with DNase-I but in samples treated with RNAse (Fig. 1A). The EVs fraction isolated from the density gradients had a charge of -70 mV, which was increased to -50 mV by DNase-I treatment (Fig. 1B). This result indicates that DNA is present on the outside of the EVs, and this DNA contributes to the negative charge of the vesicles. Further, DNase treatment increased the number of particles measured by nanoparticle tracking analysis, suggesting that the DNA to a certain degree contributes to aggregation of the isolated EVs (Fig. 1C). As a biological readout, isolated EVs associated with extracellular DNA were taken up by human mesenchymal stem cell in a time-dependent manner (Fig. 1D and E).

    EVs carry multiple bioactive molecules, including proteins, various RNA species, and according to the present study, DNA. In our case, the DNA was observed in EV isolates from a human mast cell line. Specifically, our study argues that the DNase-sensitive nucleic acids are present on the outside of the EVs. Furthermore, the EV-associated DNA can be taken up by recipient cells, which could alter cellular responses.

    It is known that EVs can mediate an array of biological messages to recipient cells- including surface-to-surface antigen presentation and receptors activation- and can deliver RNA (e.g. mRNA, miRNA) cargo to recipient cells. However, the presence and function of DNA as a cargo on the outside of EVs is less explored. EVs have previously been associated with cell-free DNA that carries retrotransposon elements and oncogenes, but overall EV-associated DNA has been extensively characterized. A recent report emphasizes the presence of dsDNA inside of the EVs, whereas we find that a majority of DNA from the human mast cell line is associated with outer perimeter of EVs, since it is sensitive to DNase treatment without lysing the EVs. Our study also indicates, for the first time, that DNA covering floated EVs can lead to an increase in the net negative charge of the vesicles. This was confirmed by reduction of net negative charge from EVs by DNase treatment. We were also able to monitor the increase in particle numbers after DNase treatment, indirectly suggesting that EV-DNA may lead to aggregation of EVs. These results are in line with previous observation made in various electron micrographs, showing clustering of EVs, which could have occurred because of DNA on the surface of these vesicles. The aggregation of EVs may be secondary to nonspecific aggregation of EVs during ultracentrifugation, but our study suggests that EV-related DNA can contribute to this observation. As we observed that the extracellular DNA floated in the density gradient, we argue strongly that it is associated with the floating vesicles with relatively low density. Also, it is has been shown that exogenous plasmids DNA if associated with EVs are taken up more efficiently in recipient cells than free DNA, again arguing that association to EVs could be involved in DNA uptake. Similarly, we also observed a time-dependent increase in cytoplasmic DNA foci in EV treated recipient human mesenchymal stem cells. Overall, this study highlights the need to define the EV-associated DNA in delivering biological function in cells that take up these EVs.

  • Conclusions

    We conclude that cell-free DNA can be associated with the outside of EVs, which can cause aggregation of these EVs, possibly influencing the effects of EVs in recipient cells.

  • Limitations

    This work is based on cell-line-produced EVs, which may not reflect a biological function in vivo.

  • Alt. Explanations

    It is not known whether the EVs leave the producing cells with DNA on their surface, or whether that occurs extracellularly. DNA from the cells or supernatant of the cell culture could have been sticking to the released EVs at an early stage of the different isolation steps, which in that case putatively could be considered to be a contamination to the EVs.

  • Conjectures

    In the future, the detailed nature of the EV-associated DNA needs to be determined, in relation to sequences and intracellular origin. Also, the role of the EV-associated DNA in recipient cells may be studied.

  • Methods

    EVs were isolated from conditional medium from HMC1.2 cells cultured in medium supplemented with exosomes-depleted FBS. Cells used had a viability exceeding 99%. We used differential centrifugations to remove cells (300 x g, 10 min), apoptotic bodies and microvesicles together (16,000 x g, 20 min), with a final pelleting of remaining EVs at 120,000 x g for 3 h.

    The EVs were further separated from any other pelleted material using a density gradient (at the 20–24% iodixanol concentration). EVs were treated with DNase against double stranded DNA (DNase-I; Turbo-DNase) and RNase, without lysing the EVs. Later, DNA was isolated from the EVs using Qiamp DNA isolation kits, and visualized by nucleic acid stain after agarose gel electrophoresis. Zeta potential on EVs and total particle numbers were measured per unit of protein using a nanoparticle tracking analysis system (ZetaViewer®, Particle Metrix, Germany). Uptake of DNase treated EVs was visualized by DAPI in recipient human mesenchymal stem cells, using fluorescent microscopy.

  • Funding statement

    This work was funded by the Swedish Cancer Foundation, the Swedish Research Council, Assar Gabrielssons Fond, the Swedish Heart and Lung Foundation as well as VBG-group Centre for allergy and asthma research.

  • Ethics statement

    Ethical approval: Not applicable.

    No animal was used in the experiments. Therefore, no ethical approval is required.

  • References
  • 1
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    Matters11.5/20

    Human mast cells release extracellular vesicle-associated DNA

    Affiliation listing not available.
    Abstractlink

    Extracellular vesicles (EVs) carry multiple bioactive molecules, including proteins and nucleic acids. Cell-free, extracellular DNA has been reported to be present in cell cultures and in multiple body fluids, but its relative location in relation to EVs has not been described. This study demonstrates that DNase-sensitive nucleic acids are present on the surface of EV isolates. Association of EV-DNA was revealed by increase of EV zeta potential and particle number upon DNase treatment. Additionally, cells exposed to EVs with associated DNA show the presence of cytoplasmic DNA traces intracellularly. In conclusion, we suggest that DNA can be associated to the surface of EVs and can be taken up by recipient cells. DNA on EV surfaces may influence their function in recipient cells.

    Figurelink

    Figure 1. DNA is associated with extracellular vesicles.

    (A) Isolated EVs, and EVs were floated on an optiprep gradient and treated with DNase-I or RNAse prior to DNA isolation (apoptotic bodies and microvesicles had been removed at 16000 g). Nucleic acids were visualized with agarose gel electrophoresis labeled with nucleic acid stain (GelStar). The isolated EVs were also analyzed with NTA (Zetaviewer®) to determine the zeta potential (B) on EVs, and the particle number (C) with or without DNase treatment. Isolated EVs were incubated with human mesenchymal stem cells for 1 or 24 h, and DNA was labeled with DAPI and visualized by fluorescent microscopy at 24 h (D), and cytoplasmic DAPI staining foci per cell were counted (E). Student's T-test was used to determine significant differences between groups (*p <0.05. N = 3).

    Introductionlink

    Extracellular vesicles (EVs) are membrane bound structures released by cells, and found in all body fluids. The populations of EVs are diverse, and the nomenclature includes terms such as exosomes, ectosomes and microvesicles. Previously, it has been shown that the subpopulations of EVs called exosomes can carry both mRNA and microRNA, which can mediate function in recipient cells[1]. Additionally, dsDNA has previously been proposed to be associated with EVs[2][3][4][5][6], but its relative location to/in EVs has not been well described. This study reports for the first time that cell-free DNA can be associated with the outside of EVs, which may influence aggregation of these EVs.

    Objectivelink

    This work aims to determine the relative location of cell-free DNA in relation to extracellular vesicles.

    Results & Discussionlink

    On a nucleic acid-stained gel, clear DNA bands were visualized, and these were present not in samples pretreated with DNase-I but in samples treated with RNAse (Fig. 1A). The EVs fraction isolated from the density gradients had a charge of -70 mV, which was increased to -50 mV by DNase-I treatment (Fig. 1B). This result indicates that DNA is present on the outside of the EVs, and this DNA contributes to the negative charge of the vesicles. Further, DNase treatment increased the number of particles measured by nanoparticle tracking analysis, suggesting that the DNA to a certain degree contributes to aggregation of the isolated EVs (Fig. 1C). As a biological readout, isolated EVs associated with extracellular DNA were taken up by human mesenchymal stem cell in a time-dependent manner (Fig. 1D and E).

    EVs carry multiple bioactive molecules, including proteins, various RNA species, and according to the present study, DNA. In our case, the DNA was observed in EV isolates from a human mast cell line. Specifically, our study argues that the DNase-sensitive nucleic acids are present on the outside of the EVs. Furthermore, the EV-associated DNA can be taken up by recipient cells, which could alter cellular responses.

    It is known that EVs can mediate an array of biological messages to recipient cells- including surface-to-surface antigen presentation and receptors activation- and can deliver RNA (e.g. mRNA, miRNA) cargo to recipient cells[1][7]. However, the presence and function of DNA as a cargo on the outside of EVs is less explored. EVs have previously been associated with cell-free DNA that carries retrotransposon elements and oncogenes[3][4], but overall EV-associated DNA has been extensively characterized. A recent report[5] emphasizes the presence of dsDNA inside of the EVs, whereas we find that a majority of DNA from the human mast cell line is associated with outer perimeter of EVs, since it is sensitive to DNase treatment without lysing the EVs. Our study also indicates, for the first time, that DNA covering floated EVs can lead to an increase in the net negative charge of the vesicles. This was confirmed by reduction of net negative charge from EVs by DNase treatment. We were also able to monitor the increase in particle numbers after DNase treatment, indirectly suggesting that EV-DNA may lead to aggregation of EVs. These results are in line with previous observation[8][9] made in various electron micrographs, showing clustering of EVs, which could have occurred because of DNA on the surface of these vesicles. The aggregation of EVs may be secondary to nonspecific aggregation of EVs during ultracentrifugation, but our study suggests that EV-related DNA can contribute to this observation. As we observed that the extracellular DNA floated in the density gradient, we argue strongly that it is associated with the floating vesicles with relatively low density. Also, it is has been shown that exogenous plasmids DNA if associated with EVs are taken up more efficiently in recipient cells[10] than free DNA, again arguing that association to EVs could be involved in DNA uptake. Similarly, we also observed a time-dependent increase in cytoplasmic DNA foci in EV treated recipient human mesenchymal stem cells. Overall, this study highlights the need to define the EV-associated DNA in delivering biological function in cells that take up these EVs.

    Conclusionslink

    We conclude that cell-free DNA can be associated with the outside of EVs, which can cause aggregation of these EVs, possibly influencing the effects of EVs in recipient cells.

    Limitationslink

    This work is based on cell-line-produced EVs, which may not reflect a biological function in vivo.

    Alternative Explanationslink

    It is not known whether the EVs leave the producing cells with DNA on their surface, or whether that occurs extracellularly. DNA from the cells or supernatant of the cell culture could have been sticking to the released EVs at an early stage of the different isolation steps, which in that case putatively could be considered to be a contamination to the EVs.

    Conjectureslink

    In the future, the detailed nature of the EV-associated DNA needs to be determined, in relation to sequences and intracellular origin. Also, the role of the EV-associated DNA in recipient cells may be studied.

    Methodslink

    EVs were isolated from conditional medium from HMC1.2 cells cultured in medium supplemented with exosomes-depleted FBS[11]. Cells used had a viability exceeding 99%. We used differential centrifugations to remove cells (300 x g, 10 min), apoptotic bodies and microvesicles together (16,000 x g, 20 min), with a final pelleting of remaining EVs at 120,000 x g for 3 h.

    [12]The EVs were further separated from any other pelleted material using a density gradient (at the 20–24% iodixanol concentration). EVs were treated with DNase against double stranded DNA (DNase-I; Turbo-DNase) and RNase, without lysing the EVs. Later, DNA was isolated from the EVs using Qiamp DNA isolation kits, and visualized by nucleic acid stain after agarose gel electrophoresis. Zeta potential on EVs and total particle numbers were measured per unit of protein using a nanoparticle tracking analysis system (ZetaViewer®, Particle Metrix, Germany). Uptake of DNase treated EVs was visualized by DAPI in recipient human mesenchymal stem cells, using fluorescent microscopy.

    Funding Statementlink

    This work was funded by the Swedish Cancer Foundation, the Swedish Research Council, Assar Gabrielssons Fond, the Swedish Heart and Lung Foundation as well as VBG-group Centre for allergy and asthma research.

    Conflict of interestlink

    The authors do declare conflicts of interest:

    Jan Lötvall is an employee of Codiak BioSciences, a company developing exosomes as a platform for therapy in disease, and has several patents in the area of using exosomes as therapeutics and diagnostic tools.
    Ethics Statementlink

    Ethical approval: Not applicable.

    No animal was used in the experiments. Therefore, no ethical approval is required.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

    Referenceslink
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      Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells
      Nature Cell Biology, 9/2007, pages 654-659 DOI: 10.1038/ncb1596chrome_reader_mode
    2. Kahlert Christoph, Melo Sonia A., Protopopov Alexei,more_horiz, Kalluri Raghu
      Identification of Double-stranded Genomic DNA Spanning All Chromosomes with Mutated KRAS and p53 DNA in the Serum Exosomes of Patients with Pancreatic Cancer
      Journal of Biological Chemistry, 289/2014, pages 3869-3875 DOI: 10.1074/jbc.c113.532267chrome_reader_mode
    3. Tae Hoon Lee, Shilpa Chennakrishnaiah, Eric Audemard,more_horiz, Janusz Rak
      Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells
      Biochemical and Biophysical Research Communications, 451/2014, pages 295-301 DOI: 10.1016/j.bbrc.2014.07.109chrome_reader_mode
    4. Leonora Balaj, Ryan Lessard, Lixin Dai,more_horiz, Johan Skog
      Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences
      Nature Communications, 2/2011, page 180 DOI: 10.1038/ncomms1180chrome_reader_mode
    5. Basant Kumar Thakur, Haiying Zhang, Annette Becker,more_horiz, David Lyden
      Double-stranded DNA in exosomes: a novel biomarker in cancer detection
      Cell Research, 24/2014, pages 766-769 DOI: 10.1038/cr.2014.44chrome_reader_mode
    6. Michele Guescini, Susanna Genedani, Vilberto Stocchi, Luigi Francesco Agnati
      Astrocytes and Glioblastoma cells release exosomes carrying mtDNA
      Journal of Neural Transmission, 117/2009, pages 1-4 DOI: 10.1007/s00702-009-0288-8chrome_reader_mode
    7. Joseph Wolfers, Anne Lozier, Graça Raposo,more_horiz, Laurence Zitvogel
      Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming
      Nature Medicine, 7/2001, pages 297-303 DOI: 10.1038/85438chrome_reader_mode
    8. Kevin E. Petersen, Eliana Manangon, Joshua L. Hood,more_horiz, Bruce K. Gale
      A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM
      Analytical and Bioanalytical Chemistry, 406/2014, pages 7855-7866 DOI: 10.1007/s00216-014-8040-0chrome_reader_mode
    9. Harry F.G. Heijnen, Anja E. Schiel, Rob Fijnheer,more_horiz, And Jan J. Sixma
      Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules.
      Blood, 94(11)/1999, pages 3791-9 chrome_reader_mode
    10. Tek N. Lamichhane, Rahul S. Raiker, Steven M. Jay
      Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery
      Molecular Pharmaceutics, 12/2015, pages 3650-3657 DOI: 10.1021/acs.molpharmaceut.5b00364chrome_reader_mode
    11. Ganesh Vilas Shelke, Cecilia Lässer, Yong Song Gho, Jan Lötvall
      Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum
      Journal of Extracellular Vesicles, 3/2014 DOI: 10.3402/jev.v3.24783chrome_reader_mode
    12. Aleksander Cvjetkovic, Jan Lötvall, Cecilia Lässer
      The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles
      Journal of Extracellular Vesicles, 3/2014 DOI: 10.3402/jev.v3.23111chrome_reader_mode
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