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Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes

Crescitelli, Rossella, Lässer, Cecilia, Szabó, Tamas G., Kittel, Agnes, Eldh, Maria, Dianzani, Irma, Buzás, Edit I., Lötvall, Jan·2013·Journal of Extracellular Vesicles
DOI 10.3402/jev.v2i0.20677PMC PMC3823106
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Abstract

Introduction: In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross‐contamination. Method: EVs released from three different kinds of cell lines: HMC‐1, TF‐1 and BV‐2 were isolated using two centrifugation‐based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer ® . Results: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF‐1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF‐1‐derived vesicles, as these cells do not express CD9. Conclusions: Our results demonstrate that centrifugation‐based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63‐coated beads for flow cytometry analysis.

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Fig. 1 Flow chart over two different differential centrifugation-based protocols. Apoptotic bodies (ABs) and microvesicles (MVs) were isolated separately using protocol 1 (A). ABs and MVs were isolated together (ABs+MVs) followed by exosome (EXO) isolation, using protocol 2 (B).
Fig. 1 Flow chart over two different differential centrifugation-based protocols. Apoptotic bodies (ABs) and microvesicles (MVs) were isolated separately using protocol 1 (A). ABs and MVs were isolated together (ABs+MVs) followed by exosome (EXO) isolation, using protocol 2 (B).CC BY · PubMed Central
Fig. 2 RNA profiles from different subpopulations of extracellular vesicles (EVs). RNA was extracted from vesicles released by three different cell lines; HMC-1 (human mast cell line), TF-1 (human erythroleukemia cells) and BV-2 (mouse microglia cells). The electropherograms show the size distribution in nucleotides (nt) and fluorescence intensity (FU) of total RNA in apoptotic bodies (ABs), microvesicles (MVs), ABs and MVs together (ABs+MVs) and exosomes (EXOs). The short peak at 25 nt is an internal standard. (A) In ABs the most dominant peaks are the 18S and 28S ribosomal RNA. (B) The 18S and 28S rRNA are not evident in MVs from HMC-1 and BV-2, but only obvious in MVs from TF-1, however at low concentrations. (C) 18S and 28S peaks are evident in the pellet composed by ABs and MVs together (ABs+MVs). (D) In EXOs small RNA is dominating, with no or very small rRNA peaks detected. (E–G) The overlapping profiles from ABs (in red) and MVs (in blue) and both collected together (ABs+MVs − in green), suggesting that the contribution of 18S and 28S rRNA is by ABs. The electropherograms are representative of n=4.
Fig. 2 RNA profiles from different subpopulations of extracellular vesicles (EVs). RNA was extracted from vesicles released by three different cell lines; HMC-1 (human mast cell line), TF-1 (human erythroleukemia cells) and BV-2 (mouse microglia cells). The electropherograms show the size distribution in nucleotides (nt) and fluorescence intensity (FU) of total RNA in apoptotic bodies (ABs), microvesicles (MVs), ABs and MVs together (ABs+MVs) and exosomes (EXOs). The short peak at 25 nt is an internal standard. (A) In ABs the most dominant peaks are the 18S and 28S ribosomal RNA. (B) The 18S and 28S rRNA are not evident in MVs from HMC-1 and BV-2, but only obvious in MVs from TF-1, however at low concentrations. (C) 18S and 28S peaks are evident in the pellet composed by ABs and MVs together (ABs+MVs). (D) In EXOs small RNA is dominating, with no or very small rRNA peaks detected. (E–G) The overlapping profiles from ABs (in red) and MVs (in blue) and both collected together (ABs+MVs − in green), suggesting that the contribution of 18S and 28S rRNA is by ABs. The electropherograms are representative of n=4.CC BY · PubMed Central
Fig. 3 RNA profiles from different subpopulations of extracellular vesicles (EVs) after TRAIL-induced apoptosis. The electropherograms show the RNA size distribution in nucleotides (nt) and fluorescence intensity (FU) in apoptotic bodies (ABs), microvesicles (MVs), ABs and MVs together (ABs+MVs) and exosomes (EXOs) in TF-1 cells with and without TRAIL treatment. The short peak at 25 nt is an internal standard. (A–C) RNA profiles from ABs released by TF-1 cells after 4, 24, 48 hours of TRAIL treatment (in blue) and without TRAIL (in red). After 4 hours (A), 24 hours (B) and 48 hours (C) of TRAIL treatment, in ABs, the peaks of 18S and 28S rRNAs are more prominent comparing with ABs released in the absence of TRAIL. (D–F) RNA profiles from MVs, ABs+MVs and EXOs released by TF-1 cells after 48 hours of TRAIL treatment (in blue) and without TRAIL (in red). (D) The low 18S and 28S rRNA peaks in MVs without TRAIL (in red) become much more prominent after TRAIL treatment (in blue). (E) The highest rRNA peaks are seen in the pellet composed by ABs and MVs together (ABs+MVs). (F) After 48 hours of TRAIL-induced apoptosis, increased amount of small RNAs is observed in exosomes (EXOs). The electropherograms are representative of n=2.
Fig. 3 RNA profiles from different subpopulations of extracellular vesicles (EVs) after TRAIL-induced apoptosis. The electropherograms show the RNA size distribution in nucleotides (nt) and fluorescence intensity (FU) in apoptotic bodies (ABs), microvesicles (MVs), ABs and MVs together (ABs+MVs) and exosomes (EXOs) in TF-1 cells with and without TRAIL treatment. The short peak at 25 nt is an internal standard. (A–C) RNA profiles from ABs released by TF-1 cells after 4, 24, 48 hours of TRAIL treatment (in blue) and without TRAIL (in red). After 4 hours (A), 24 hours (B) and 48 hours (C) of TRAIL treatment, in ABs, the peaks of 18S and 28S rRNAs are more prominent comparing with ABs released in the absence of TRAIL. (D–F) RNA profiles from MVs, ABs+MVs and EXOs released by TF-1 cells after 48 hours of TRAIL treatment (in blue) and without TRAIL (in red). (D) The low 18S and 28S rRNA peaks in MVs without TRAIL (in red) become much more prominent after TRAIL treatment (in blue). (E) The highest rRNA peaks are seen in the pellet composed by ABs and MVs together (ABs+MVs). (F) After 48 hours of TRAIL-induced apoptosis, increased amount of small RNAs is observed in exosomes (EXOs). The electropherograms are representative of n=2.CC BY · PubMed Central
Fig. 4 Flow chart over the original and modified protocol 2. (A) In the modification of protocol 2, a 2,000×g step was added to isolate apoptotic bodies (ABs) and microvesicles (MVs) separately, prior to EXOs isolation (here called protocol 2b). (B) The RNA profiles from the different subpopulation of extracellular vesicles (EVs) collected using protocol 2a and 2b. RNA was extracted from vesicles releases from two different cell lines; HMC-1 and TF-1. Shown here are the overlapping profiles from ABs (ABs – in red), MVs (MVs – in blue) and both of them collected together (ABs+MVs − in green), indicating that the contribution of 18S and 28S rRNA is primarily by ABs. The electopherograms show the size distribution in nucleotides (nt) and fluorescence intensity (FU) of total RNA. The peak at 25 nt is an internal standard. The electropherograms are representative of n=3.
Fig. 4 Flow chart over the original and modified protocol 2. (A) In the modification of protocol 2, a 2,000×g step was added to isolate apoptotic bodies (ABs) and microvesicles (MVs) separately, prior to EXOs isolation (here called protocol 2b). (B) The RNA profiles from the different subpopulation of extracellular vesicles (EVs) collected using protocol 2a and 2b. RNA was extracted from vesicles releases from two different cell lines; HMC-1 and TF-1. Shown here are the overlapping profiles from ABs (ABs – in red), MVs (MVs – in blue) and both of them collected together (ABs+MVs − in green), indicating that the contribution of 18S and 28S rRNA is primarily by ABs. The electopherograms show the size distribution in nucleotides (nt) and fluorescence intensity (FU) of total RNA. The peak at 25 nt is an internal standard. The electropherograms are representative of n=3.CC BY · PubMed Central
Fig. 5 Analysis of ABs, MVs and EXOs by TEM. Micrographs of vesicles released from three different cell lines; HMC-1 (human mast cell line), TF-1 (human erythroleukemia cells), and BV-2 (mouse microglia cells) are shown. (A1–3) Dense structures show the chromatin substance in the generally round shaped apoptotic bodies (ABs) with a size of 800–5,000 nm. (B1–3) Microvesicles (MVs) are diverse in their shape and density, with a size range between 200 and 800 nm. (C1–3) In the pellet obtained by centrifugation at 16,500×g presents the mixture of ABs and MVs. (D1–3) The exosome (EXO) fraction from HMC-1 (D1), TF-1 (D2) and BV-2 (D3) cells were found to have a diameter of approximately 40–100 nm.
Fig. 5 Analysis of ABs, MVs and EXOs by TEM. Micrographs of vesicles released from three different cell lines; HMC-1 (human mast cell line), TF-1 (human erythroleukemia cells), and BV-2 (mouse microglia cells) are shown. (A1–3) Dense structures show the chromatin substance in the generally round shaped apoptotic bodies (ABs) with a size of 800–5,000 nm. (B1–3) Microvesicles (MVs) are diverse in their shape and density, with a size range between 200 and 800 nm. (C1–3) In the pellet obtained by centrifugation at 16,500×g presents the mixture of ABs and MVs. (D1–3) The exosome (EXO) fraction from HMC-1 (D1), TF-1 (D2) and BV-2 (D3) cells were found to have a diameter of approximately 40–100 nm.CC BY · PubMed Central
Fig. 6 Detection and characterization of extracellular vesicles (EVs) by flow cytometry. The CD9, CD63 and CD81 expression on HMC-1 and TF-1 cells (A) and their expression on different vesicles, using anti-CD63-coated beads, are shown. (B) Cells and vesicles were immunostained against the tetraspanin (open curve) CD9 (in black), CD63 (in blue) and CD81 (in red) and compared with their appropriate isotype control (filled curve). The graphs are representative of n=3.
Fig. 6 Detection and characterization of extracellular vesicles (EVs) by flow cytometry. The CD9, CD63 and CD81 expression on HMC-1 and TF-1 cells (A) and their expression on different vesicles, using anti-CD63-coated beads, are shown. (B) Cells and vesicles were immunostained against the tetraspanin (open curve) CD9 (in black), CD63 (in blue) and CD81 (in red) and compared with their appropriate isotype control (filled curve). The graphs are representative of n=3.CC BY · PubMed Central

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Research works printed but not linked: 29

  1. 10.1038/nri2567 · DOI
  2. 10.1016/j.imlet.2006.09.005 · DOI
  3. 10.1016/j.jprot.2010.06.006 · DOI
  4. 10.1016/j.tcb.2008.11.003 · DOI · Paper page
  5. 10.1034/j.1600-0854.2002.30502.x · DOI
  6. 10.1093/jb/mvj128 · DOI
  7. 10.1016/j.bcmd.2005.12.001 · DOI
  8. 10.1073/pnas.0603838103 · DOI · Paper page
  9. 10.4172/jpb.10000e10 · DOI
  10. 10.1007/s00018-011-0689-3 · DOI
  11. 10.1021/pr200842h · DOI
  12. Jong OG, 2012, Cellular stress conditions are reflected in the protein and RNA content of endothelial cell‐derived exosomes, J Extracell Vesicles, 1
  13. 10.3402/jev.v1i0.18374 · DOI
  14. 10.3402/jev.v2i0.20389 · DOI
  15. 10.1124/pr.112.005983 · DOI
  16. 10.1007/s00281-010-0233-9 · DOI
  17. 10.1111/j.1538-7836.2010.04074.x · DOI
  18. 10.1016/j.jprot.2011.05.023 · DOI
  19. 10.1007/978-1-62703-453-1_9 · DOI
  20. 10.1016/j.canlet.2011.06.016 · DOI
  21. 10.1038/sj.onc.1204282 · DOI
  22. Berent‐Maoz B, 2013, Human mast cells undergo TRAIL‐induced apoptosis, J Immunol, 176, 2272, 10.4049/jimmunol.176.4.2272 · DOI
  23. 10.1074/jbc.273.32.20121 · DOI · Paper page
  24. 10.3402/jev.v1i0.18397 · DOI
  25. Tatischeff I, 2012, Fast characterisation of cell‐derived extracellular vesicles by nanoparticles tracking analysis, cryo‐electron microscopy, and Raman tweezers microspectroscopy, J Extracell Vesicles, 1, 10.3402/jev.v1i0.19179 · DOI
  26. 10.1158/0008-5472.CAN-05-4579 · DOI
  27. 10.1038/cdd.2008.104 · DOI
  28. 10.1158/0008-5472.CAN-07-6538 · DOI · Paper page
  29. 10.1111/j.1538-7836.2009.03679.x · DOI

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Metadata

Authors
Crescitelli, R., Lässer, C., Szabó, T. G., Kittel, A., Eldh, M., Dianzani, I., Buzás, E. I., & Lötvall, J.
Affiliations
  • Department of Internal Medicine and Clinical Nutrition Krefting Research Centre University of Gothenburg Gothenburg Sweden
  • Department of Genetics Cell and Immunobiology Semmelweis University Budapest Hungary
  • Institute of Experimental Medicine Hungarian Academy of Sciences Budapest Hungary
  • Department of Health Sciences University of Eastern Piedmont Novara Italy
Venue
Journal of Extracellular Vesicles
Publisher
Wiley
Published
2013-01
Volume
2
Issue
1
ISSN
2001-3078
DOI
10.3402/jev.v2i0.20677
PMC
PMC3823106
Cited by
876
References
5
Language
en
Licence
creativecommons.org/licenses/by-nc/3.0/ (vor, 254 d)
Funders
Vetenskapsrådet (K2011‐56X‐20676‐04‐6)

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