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Cited by 202Extracellular vesicles in diseaseCellular transport and secretionMast cells and histamine

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Characterization of mRNA and microRNA in human mast cell-derived exosomes and their transfer to other mast cells and blood CD34 progenitor cells

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Ekström, Karin, Valadi, Hadi, Sjöstrand, Margareta, Malmhäll, Carina, Bossios, Apostolos, Eldh, Maria, Lötvall, Jan·2012·Journal of Extracellular Vesicles
DOI 10.3402/jev.v1i0.18389PMC PMC3760639
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Abstract

Background: Exosomes are nanosized vesicles of endocytic origin that are released into the extracellular environment by many different cells. It has been shown that exosomes from various cellular origins contain a substantial amount of RNA (mainly mRNA and microRNA). More importantly, exosomes are capable of delivering their RNA content to target cells, which is a novel way of cell‐to‐cell communication. The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34 + progenitor cells. Methods: The mRNA and microRNA content of exosomes from a human mast cell line, HMC‐1, was analysed by using microarray technology. Co‐culture experiments followed by flow cytometry analysis and confocal microscopy as well as radioactive labeling experiments were performed to examine the uptake of these exosomes and the shuttle of the RNA to other mast cells and CD34 + progenitor cells. Results: In this study, we show that human mast cells release RNA‐containing exosomes, with the capacity to shuttle RNA between cells. Interestingly, by using microRNA microarray analysis, 116 microRNAs could be identified in the exosomes and 134 microRNAs in the donor mast cells. Furthermore, DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. In addition, transfer experiments revealed that exosomes can shuttle RNA between human mast cells and to CD34 + hematopoietic progenitor cells. Conclusion: These findings suggest that exosomal shuttle RNA (esRNA) can play a role in the communication between cells, including mast cells and CD34 + progenitor cells, implying a role in cells maturation process.

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Figures

Fig. 1 Human mast cells produce exosomes that contain RNA. (a) Transmission electron microscopy picture of exosomes isolated from the supernatant of the human mast cell line HMC-1 by a method based on repeated centrifugation and filtration steps followed by high speed ultracentrifugation. Exosomes are visible as small 40–80 nm vesicles, scale bar 100 nm. (b-d) Flow cytometry detection and characterization of exosomes captured onto anti-CD63 coated beads. The exosome-bead complexes were immunostained against the tetraspanins (open curve) CD9 (b), CD63 (c) and CD81 (d) and compared with the appropriate isotype control (filled curve) (e, f) Bioanalyzer® results of total RNA isolated from exosomes (e) and their donor HMC-1 cells (f) shows the size distribution of RNA. The two dominant peaks in cellular RNA correspond to 18S and 28S rRNA, respectively. These ribosomal peaks are not dominant in the exosomal RNA (FU, fluorescence units).
Fig. 1 Human mast cells produce exosomes that contain RNA. (a) Transmission electron microscopy picture of exosomes isolated from the supernatant of the human mast cell line HMC-1 by a method based on repeated centrifugation and filtration steps followed by high speed ultracentrifugation. Exosomes are visible as small 40–80 nm vesicles, scale bar 100 nm. (b-d) Flow cytometry detection and characterization of exosomes captured onto anti-CD63 coated beads. The exosome-bead complexes were immunostained against the tetraspanins (open curve) CD9 (b), CD63 (c) and CD81 (d) and compared with the appropriate isotype control (filled curve) (e, f) Bioanalyzer® results of total RNA isolated from exosomes (e) and their donor HMC-1 cells (f) shows the size distribution of RNA. The two dominant peaks in cellular RNA correspond to 18S and 28S rRNA, respectively. These ribosomal peaks are not dominant in the exosomal RNA (FU, fluorescence units).CC BY · PubMed Central
Fig. 2 Characterization of exosomal mRNA. Affymetrix DNA microarray was applied to identify mRNA from exosomes and their donor HMC-1 cells (n=4). (a,b) Correlation of gene expression signals within exosomes and cells samples are shown. In total, 1,849 mRNAs could be found in exosomes and 12,346 in the cells. (c) There was no strong correlation between the gene expression signals in exosomes and cells. (d) Relative group functions of the exosomal mRNA according to Ingenuity Software Analysis.
Fig. 2 Characterization of exosomal mRNA. Affymetrix DNA microarray was applied to identify mRNA from exosomes and their donor HMC-1 cells (n=4). (a,b) Correlation of gene expression signals within exosomes and cells samples are shown. In total, 1,849 mRNAs could be found in exosomes and 12,346 in the cells. (c) There was no strong correlation between the gene expression signals in exosomes and cells. (d) Relative group functions of the exosomal mRNA according to Ingenuity Software Analysis.CC BY · PubMed Central
Fig. 3 Characterization of exosomal microRNA. miRCURY™ LNA array analysis was applied to detect microRNAs in exosomes (Hy3) and their donor HMC-1 cells (Hy5) (n=3). (a) In total, 116 microRNAs were detected in exosomes and 134 microRNAs in their donor HMC-1 cells. Twenty-seven microRNAs were only detected in exosomes, 45 were only detected in cells and 89 were found in both cells and exosomes. (b) Scatterplot showing the mean intensity signals for microRNAs in exosomes (Hy3) and cells (Hy5). A number of microRNAs were found to be differently expressed in exosomes compared to their donor cells. The annotations for microRNAs with mean signals>10,000 are indicated in the plot. miRPLus_are sequences not yet included in miRBase.
Fig. 3 Characterization of exosomal microRNA. miRCURY™ LNA array analysis was applied to detect microRNAs in exosomes (Hy3) and their donor HMC-1 cells (Hy5) (n=3). (a) In total, 116 microRNAs were detected in exosomes and 134 microRNAs in their donor HMC-1 cells. Twenty-seven microRNAs were only detected in exosomes, 45 were only detected in cells and 89 were found in both cells and exosomes. (b) Scatterplot showing the mean intensity signals for microRNAs in exosomes (Hy3) and cells (Hy5). A number of microRNAs were found to be differently expressed in exosomes compared to their donor cells. The annotations for microRNAs with mean signals>10,000 are indicated in the plot. miRPLus_are sequences not yet included in miRBase.CC BY · PubMed Central
Fig. 4 Network-based analysis of exosomal mRNA. IPA was applied to predict networks for the exosomal mRNA identified by Affymetrix DNA microarray. The top three predicted networks for exosomal mRNA were (a) “cellular development, hematological system development and function, hematopoiesis” (50 focus genes, score 34), (b) “protein synthesis, post-translational modification, protein folding” (34 focus genes, score 30) and (c) “cell death, RNA post-transcriptional modification, cellular assembly and organization” (47 focus genes, score 30). Continuous lines indicate direct interaction and dotted lines indirect interaction. Focus genes are labelled in grey and genes not included in the dataset are white. For more information please see the IPA web site (www.ingenuity.com/).
Fig. 4 Network-based analysis of exosomal mRNA. IPA was applied to predict networks for the exosomal mRNA identified by Affymetrix DNA microarray. The top three predicted networks for exosomal mRNA were (a) “cellular development, hematological system development and function, hematopoiesis” (50 focus genes, score 34), (b) “protein synthesis, post-translational modification, protein folding” (34 focus genes, score 30) and (c) “cell death, RNA post-transcriptional modification, cellular assembly and organization” (47 focus genes, score 30). Continuous lines indicate direct interaction and dotted lines indirect interaction. Focus genes are labelled in grey and genes not included in the dataset are white. For more information please see the IPA web site (www.ingenuity.com/).CC BY · PubMed Central
Fig. 5 Networks predicted to be regulated by exosomal microRNA. Predicted targets for the top 5 annotated miRNA in exosomes were searched using TargetScan to predict target mRNA and subsequently analysed by IPA software to determine predicted networks regulated by exosomal microRNA. The top 3 predicted networks regulated by exosomal microRNA were (a) “cellular development, cellular movement, hematological system development and function” (13 focus molecules, score 12), (b) “cellular function and maintenance, cellular development, neurological disease” (13 focus molecules, score 12) and (c) “cellular movement, hematological system development and function, immune cell trafficking” (12 focus molecules, score 11).
Fig. 5 Networks predicted to be regulated by exosomal microRNA. Predicted targets for the top 5 annotated miRNA in exosomes were searched using TargetScan to predict target mRNA and subsequently analysed by IPA software to determine predicted networks regulated by exosomal microRNA. The top 3 predicted networks regulated by exosomal microRNA were (a) “cellular development, cellular movement, hematological system development and function” (13 focus molecules, score 12), (b) “cellular function and maintenance, cellular development, neurological disease” (13 focus molecules, score 12) and (c) “cellular movement, hematological system development and function, immune cell trafficking” (12 focus molecules, score 11).CC BY · PubMed Central
Fig. 6 Exosomes can transfer RNA to HMC-1 cells and CD34 positive progenitor cells. PKH67-labelled (green) exosomes were added to HMC-1 or CD34 cells in culture. Cells were harvested, washed and analysed in the flow cytometer or by confocal microscopy at different time points. (a) Shows the percentage of HMC-1 cells positive for PKH67 after the different time points. (b) Confocal microscopy picture of green exosomes internalized by HMC-1 cells (red nuclei stained with 7-AAD). (c) CD34 positive cells take up PKH67-labelled exosomes after 20 hours compared to 0 hour. (d) Exosomes were radioactively labelled, by culturing HMC-1 donor cells in medium supplemented with 3H-uridine. Exosomes were isolated, washed and added to HMC-1 and CD34 cells in culture. Cells were harvested after 0 hour and 20 hours, washed and the radioactive signal quantified. The data show that exosomal RNA is shuttled to HMC-1 (n=2) and CD34 (n=1) cells. Values in (a) and (c) are mean ± SEM, n=4 and in C, *p<0.05.
Fig. 6 Exosomes can transfer RNA to HMC-1 cells and CD34 positive progenitor cells. PKH67-labelled (green) exosomes were added to HMC-1 or CD34 cells in culture. Cells were harvested, washed and analysed in the flow cytometer or by confocal microscopy at different time points. (a) Shows the percentage of HMC-1 cells positive for PKH67 after the different time points. (b) Confocal microscopy picture of green exosomes internalized by HMC-1 cells (red nuclei stained with 7-AAD). (c) CD34 positive cells take up PKH67-labelled exosomes after 20 hours compared to 0 hour. (d) Exosomes were radioactively labelled, by culturing HMC-1 donor cells in medium supplemented with 3H-uridine. Exosomes were isolated, washed and added to HMC-1 and CD34 cells in culture. Cells were harvested after 0 hour and 20 hours, washed and the radioactive signal quantified. The data show that exosomal RNA is shuttled to HMC-1 (n=2) and CD34 (n=1) cells. Values in (a) and (c) are mean ± SEM, n=4 and in C, *p<0.05.CC BY · PubMed Central

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  1. Karlsson M, 2001, “Tolerosomes” are produced by intestinal epithelial cells, 31, 2892

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Metadata

Authors
Ekström, K., Valadi, H., Sjöstrand, M., Malmhäll, C., Bossios, A., Eldh, M., & Lötvall, J.
Affiliations
University of Gothenburg (SE)
Corresponding author
Lötvall, Jan
ORCID
Venue
Journal of Extracellular Vesicles
Publisher
Wiley
Published
2012-01
Volume
1
Issue
1
ISSN
2001-3078 (ISSN-L)
DOI
10.3402/jev.v1i0.18389
PMC
PMC3760639
OpenAlex
W2132589340
Cited by
201
References
36
Citations by year
2026: 3 · 2025: 7 · 2024: 10 · 2023: 17 · 2022: 15 · 2021: 15 · 2020: 23 · 2019: 15 · 2018: 19 · 2017: 14 · 2016: 17 · 2015: 20 · 2014: 14 · 2013: 8 · 2012: 4
FWCI
5.91
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Extracellular Vesicles (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Vetenskapsrådet (K2008‐57X‐20676‐01‐3)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Immunology and Microbiology › Immunology › Mast cells and histamine
Keywords
exosomes, mast cells, microRNA, mRNA, cell-to-cell communication, CD34+ hematopoietic progenitor cells, HMC-1 cell line, extracellular vesicles, exosome uptake

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