Abstract
Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, released from mesenchymal stem cells under different conditions and carry information capable of reestablishing homeostasis. EVs derived from MSCs transfer molecules (such as DNA, proteins/peptides, mRNA, microRNA, and lipids) and/or organelles with reparative and anti-inflammatory properties to recipient cells. EVs present many advantages over MSCs, such as homing ability to target tissue, preventing undesired accumulation in other organs, and absence of any innate toxicity or association with long-term maldifferentiated engrafted cells, tumor generation, or immune rejection after stem cell injection. The paracrine anti-inflammatory effects promoted by MSC-derived EVs have attracted significant interest in the regenerative medicine field, including for potential use in different diseases. This chapter addresses methods used to induce the release of extracellular vesicles from mesenchymal stem cells, the methods of extracting these vesicles, and processing and conditioning techniques used after extraction. We aim to enlighten the reader on how to choose the appropriate protocol and methods to obtain extracellular vesicles, characterize them, and use them in preclinical studies.
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References
Amiri F, Halabian R, Salimian M et al (2014) Induction of multipotency in umbilical cord-derived mesenchymal stem cells cultivated under suspension conditions. Cell Stress Chaperones 19:657–666. https://doi.org/10.1007/s12192-014-0491-x
Bæk R, Søndergaard EKL, Varming K, Jørgensen MM (2016) The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray. J Immunol Methods 438:11–20. https://doi.org/10.1016/j.jim.2016.08.007
Barile L, Lionetti V, Cervio E et al (2014) Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction. Cardiovasc Res 103:530–541. https://doi.org/10.1093/cvr/cvu167
Bellingham SA, Coleman BM, Hill AF (2012) Small RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells. Nucleic Acids Res 40:10937–10949. https://doi.org/10.1093/nar/gks832
Bian S, Zhang L, Duan L et al (2014) Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model. J Mol Med (Berl) 92:387–397. https://doi.org/10.1007/s00109-013-1110-5
Bieback K, Hecker A, Kocaömer A et al (2009) Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 27:2331–2341. https://doi.org/10.1002/stem.139
Böing AN, Van Der Pol E, Grootemaat AE et al (2014) Single-step isolation of extracellular vesicles from plasma by size-exclusion chromatography. J Extracell Vesicles 3:118. https://doi.org/10.3402/jev.v3.23430
Bosch S, de Beaurepaire L, Allard M et al (2016) Trehalose prevents aggregation of exosomes and cryodamage. Sci Rep 6:36162. https://doi.org/10.1038/srep36162
Brownlee Z, Lynn KD, Thorpe PE, Schroit AJ (2014) A novel “salting-out” procedure for the isolation of tumor-derived exosomes. J Immunol Methods 407:120–126. https://doi.org/10.1016/j.jim.2014.04.003
Bruno S, Grange C, Collino F et al (2012) Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury. PLoS One 7:e33115. https://doi.org/10.1371/journal.pone.0033115
Chase LG, Lakshmipathy U, Solchaga LA et al (2010) A novel serum-free medium for the expansion of human mesenchymal stem cells. Stem Cell Res Ther 1:8. https://doi.org/10.1186/scrt8
Choi D-S, Lee J-M, Park GW et al (2007) Proteomic analysis of microvesicles derived from human colorectal cancer cells. J Proteome Res 6:4646–4655. https://doi.org/10.1021/pr070192y
Choi D-S, Park JO, Jang SC et al (2011) Proteomic analysis of microvesicles derived from human colorectal cancer ascites. Proteomics 11:2745–2751. https://doi.org/10.1002/pmic.201100022
Clayton A, Court J, Navabi H et al (2001) Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods 247:163–174. https://doi.org/10.1016/S0022-1759(00)00321-5
Collino F, Pomatto M, Bruno S et al (2017) Exosome and microvesicle-enriched fractions isolated from mesenchymal stem cells by gradient separation showed different molecular signatures and functions on renal tubular epithelial cells. Stem Cell Rev 13:226–243. https://doi.org/10.1007/s12015-016-9713-1
Conforti A, Scarsella M, Starc N et al (2014) Microvescicles derived from mesenchymal stromal cells are not as effective as their cellular counterpart in the ability to modulate immune responses in vitro. Stem Cells Dev 23:2591–2599. https://doi.org/10.1089/scd.2014.0091
Cruz FF, Borg ZD, Goodwin M et al (2015) Systemic administration of human bone marrow-derived mesenchymal stromal cell extracellular vesicles ameliorates aspergillus hyphal extract-induced allergic airway inflammation in immunocompetent mice. Stem Cells Transl Med 4:1302. https://doi.org/10.5966/sctm.2014-0280
Cvjetkovic A, Lötvall J, Lässer C (2014) The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J Extracell Vesicles 3:3–4. https://doi.org/10.3402/jev.v3.23111
de Castro LL, Xisto DG, Kitoko JZ et al (2017) Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma. Stem Cell Res Ther 8:151. https://doi.org/10.1186/s13287-017-0600-8
Eitan E, Zhang S, Witwer KW, Mattson MP (2015) Extracellular vesicle-depleted fetal bovine and human sera have reduced capacity to support cell growth. J Extracell Vesicles 4:1–10. https://doi.org/10.3402/jev.v4.26373
Franquesa M, Hoogduijn MJ, Ripoll E et al (2014) Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells. Front Immunol 5. https://doi.org/10.3389/fimmu.2014.00525
Fu WL, Jia ZQ, Wang WP et al (2011) Proliferation and apoptosis property of mesenchymal stem cells derived from peripheral blood under the culture conditions of hypoxia and serum deprivation. Chin Med J 124:3959–3967. https://doi.org/10.3760/cma.j.issn.0366-6999.2011.23.022
Gallart-Palau X, Serra A, Wong ASW et al (2015) Extracellular vesicles are rapidly purified from human plasma by PRotein organic solvent PRecipitation (PROSPR). Sci Rep 5:14664. https://doi.org/10.1038/srep14664
György B, Módos K, Pállinger É et al (2011) Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters. Blood 117:e39. https://doi.org/10.1182/blood-2010-09-307595
Haga H, Yan IK, Takahashi K et al (2017) Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from lethal hepatic failure in mice. Stem Cells Transl Med 6:1262–1272. https://doi.org/10.1002/sctm.16-0226
Ismail N, Wang Y, Dakhlallah D et al (2013) Macrophage microvesicles induce macrophage differentiation and miR-223 transfer. Blood 121:984–995. https://doi.org/10.1182/blood-2011-08-374793
Jeppesen DK, Hvam ML, Primdahl-Bengtson B et al (2014) Comparative analysis of discrete exosome fractions obtained by differential centrifugation. J Extracell Vesicles 3. https://doi.org/10.3402/jev.v3.25011
Kim G, Yoo CE, Kim M et al (2012) Noble polymeric surface conjugated with zwitterionic moieties and antibodies for the isolation of exosomes from human serum. Bioconjug Chem 23:2114–2120. https://doi.org/10.1021/bc300339b
Kim D-K, Nishida H, An SY et al (2015) Chromatographically isolated CD63+CD81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after TBI. Proc Natl Acad Sci U S A 113:170. https://doi.org/10.1073/pnas.1522297113
Kordelas L, Rebmann V, Ludwig A et al (2014) MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leukemia 28:970–973. https://doi.org/10.1038/leu.2014.41
Lacroix R, Judicone C, Poncelet P et al (2012) Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol. J Thromb Haemost 10:437–446. https://doi.org/10.1111/j.1538-7836.2011.04610.x
Lamparski HG, Metha-Damani A, Yao JY et al (2002) Production and characterization of clinical grade exosomes derived from dendritic cells. J Immunol Methods 270:211–226. https://doi.org/10.1016/S0022-1759(02)00330-7
Livshts MA, Khomyakova E, Evtushenko EG et al (2015) Isolation of exosomes by differential centrifugation: theoretical analysis of a commonly used protocol. Sci Rep 5:17319. https://doi.org/10.1038/srep17319
Lőrincz ÁM, Timár CI, Marosvári KA et al (2014) Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes. J Extracell Vesicles 3:25465. https://doi.org/10.3402/jev.v3.25465\r25465
Marędziak M, Marycz K, Lewandowski D et al (2015) Static magnetic field enhances synthesis and secretion of membrane-derived microvesicles (MVs) rich in VEGF and BMP-2 in equine adipose-derived stromal cells (EqASCs)-a new approach in veterinary regenerative medicine. In Vitro Cell Dev Biol Anim 51:230–240. https://doi.org/10.1007/s11626-014-9828-0
Mathivanan S, Lim JWE, Tauro BJ et al (2010) Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature. Mol Cell Proteomics 9:197–208. https://doi.org/10.1074/mcp.M900152-MCP200
Momen-Heravi F, Balaj L, Alian S et al (2012) Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles. Front Physiol 3. https://doi.org/10.3389/fphys.2012.00162
Monsel A, Zhu YG, Gennai S et al (2015) Therapeutic effects of human mesenchymal stem cell-derived microvesicles in severe pneumonia in mice. Am J Respir Crit Care Med 192:324. https://doi.org/10.1164/rccm.201410-1765OC
Nordin JZ, Lee Y, Vader P et al (2015) Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomed Nanotechnol Biol Med 11:879–883. https://doi.org/10.1016/j.nano.2015.01.003
Oskowitz A, McFerrin H, Gutschow M et al (2011) Serum-deprived human multipotent mesenchymal stromal cells (MSCs) are highly angiogenic. Stem Cell Res 6:215–225. https://doi.org/10.1016/j.scr.2011.01.004
Potier E, Ferreira E, Meunier A et al (2007) Prolonged hypoxia concomitant with serum deprivation induces massive human mesenchymal stem cell death. Tissue Eng 13:1325–1331. https://doi.org/10.1089/ten.2006.0325
Progen OptiPrep datasheet (2017). https://www.progen.com/media/downloads/datasheets/1114542.pdf. Accessed 8 Jan 2017
Quah BJ, O’Neill HC (2007) Mycoplasma contaminants present in exosome preparations induce polyclonal B cell responses. J Leukoc Biol 82:1070–1082. jlb.0507277 [pii]. https://doi.org/10.1189/jlb.0507277
Rad F, Pourfathollah AA, Yari F et al (2016) Microvesicles preparation from mesenchymal stem cells. Med J Islam Repub Iran 30:398
Ragni E, Banfi F, Barilani M et al (2017) Extracellular vesicle-shuttled mRNA in mesenchymal stem cell communication. Stem Cells 35:1093–1105. https://doi.org/10.1002/stem.2557
Rana S, Yue S, Stadel D, Zöller M (2012) Toward tailored exosomes: the exosomal tetraspanin web contributes to target cell selection. Int J Biochem Cell Biol 44:1574–1584. https://doi.org/10.1016/j.biocel.2012.06.018
Rani S, Ryan AE, Griffin MD, Ritter T (2015) Mesenchymal stem cell-derived extracellular vesicles: toward cell-free therapeutic applications. Mol Ther 23:812–823. https://doi.org/10.1038/mt.2015.44
Rickwood D, Ford T, Steensgaard J (1994) Centrifugation: essential data. Wiley, Chichester, UK
Sáenz-Cuesta M, Arbelaiz A, Oregi A et al (2015) Methods for extracellular vesicles isolation in a hospital setting. Front Immunol 6. https://doi.org/10.3389/fimmu.2015.00050
Shelke GV, Lässer C, Gho YS, Lötvall J (2014) Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J Extracell Vesicles 3. https://doi.org/10.3402/jev.v3.24783
Singh PP, Li L, Schorey JS (2015) Exosomal RNA from mycobacterium tuberculosis-infected cells is functional in recipient macrophages. Traffic 16:555–571. https://doi.org/10.1111/tra.12278
Tauro BJ, Greening DW, Mathias RA et al (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56:293–304. https://doi.org/10.1016/j.ymeth.2012.01.002
Tetta C, Ghigo E, Silengo L et al (2013) Extracellular vesicles as an emerging mechanism of cell-to-cell communication. Endocrine 44:11–19
Théry C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3:Unit 3.22. https://doi.org/10.1002/0471143030.cb0322s30, 30, 3.22.1
Van Deun J, Mestdagh P, Sormunen R et al (2014) The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles 3. https://doi.org/10.3402/jev.v3.24858
Wang F, Zhou H, Du Z et al (2015) Cytoprotective effect of melatonin against hypoxia/serum deprivation-induced cell death of bone marrow mesenchymal stem cells in vitro. Eur J Pharmacol 748:157–165. https://doi.org/10.1016/j.ejphar.2014.09.033
Wen S, Dooner M, Cheng Y et al (2016) Mesenchymal stromal cell-derived extracellular vesicles rescue radiation damage to murine marrow hematopoietic cells. Leukemia 30:2221–2231. https://doi.org/10.1038/leu.2016.107
Willms E, Johansson HJ, Mäger I et al (2016) Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci Rep 6:22519. https://doi.org/10.1038/srep22519
Witwer KW, Buzás EI, Bemis LT et al (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2:1–25. https://doi.org/10.3402/jev.v2i0.20360
Wubbolts R, Leckie RS, Veenhuizen PTM et al (2003) Proteomic and biochemical analyses of human B cell-derived exosomes: potential implications for their function and multivesicular body formation. J Biol Chem 278:10963–10972. https://doi.org/10.1074/jbc.M207550200
Yang C, Chalasani G, Ng YH, Robbins PD (2012) Exosomes released from mycoplasma infected tumor cells activate inhibitory B cells. PLoS One 7:e36138. https://doi.org/10.1371/journal.pone.0036138
Yoo PB, Woock JP, Grill WM (2008) Somatic innervation of the feline lower urinary tract. Brain Res 1246:80–87. https://doi.org/10.1016/j.brainres.2008.09.053
Yuan A, Farber EL, Rapoport AL et al (2009) Transfer of microRNAs by embryonic stem cell microvesicles. PLoS One 4:e4722. https://doi.org/10.1371/journal.pone.0004722
Zhang H-C, Liu X-B, Huang S et al (2012) Microvesicles derived from human umbilical cord mesenchymal stem cells stimulated by hypoxia promote angiogenesis both in vitro and in vivo. Stem Cells Dev 21:3289–3297. https://doi.org/10.1089/scd.2012.0095
Zhu W, Chen J, Cong X et al (2006) Hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells. Stem Cells 24:416–425. https://doi.org/10.1634/stemcells.2005-0121
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Cruz, F.F., de Castro, L.L., Rocco, P.R.M. (2018). Preparation of Extracellular Vesicles from Mesenchymal Stem Cells. In: Pham, P. (eds) Stem Cell Drugs - A New Generation of Biopharmaceuticals. Stem Cells in Clinical Applications. Springer, Cham. https://doi.org/10.1007/978-3-319-99328-7_3
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