Isolation of small extracellular vesicles from regenerating muscle tissue using tangential flow filtration and size exclusion chromatography.
Myoregeneration
Skeletal muscle
Tangential flow filtration
Tissue derived extracellular vesicles
Journal
Skeletal muscle
ISSN: 2044-5040
Titre abrégé: Skelet Muscle
Pays: England
ID NLM: 101561193
Informations de publication
Date de publication:
11 Oct 2024
11 Oct 2024
Historique:
received:
14
02
2024
accepted:
30
09
2024
medline:
12
10
2024
pubmed:
12
10
2024
entrez:
11
10
2024
Statut:
epublish
Résumé
We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.
Identifiants
pubmed: 39394606
doi: 10.1186/s13395-024-00355-1
pii: 10.1186/s13395-024-00355-1
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
22Subventions
Organisme : US National Institutes for Health
ID : R01AR044031
Organisme : Canadian Institutes for Health Research
ID : FDN-148387
Informations de copyright
© 2024. The Author(s).
Références
Kalluri R, LeBleu VS. The biology,function, and biomedical applications of exosomes. Science (1979) 2020;367. https://doi.org/10.1126/science.aau6977
Van Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19:213–28. https://doi.org/10.1038/nrm.2017.125 .
doi: 10.1038/nrm.2017.125
pubmed: 29339798
Abbott A. FedEx for your cells: this biological delivery service could treat disease. Nature. 2023;621:462–4. https://doi.org/10.1038/d41586-023-02906-w .
doi: 10.1038/d41586-023-02906-w
pubmed: 37726444
Herrmann IK, Wood MJA, Fuhrmann G. Extracellular vesicles as a next-generation drug delivery platform. Nat Nanotechnol. 2021;16:748–59. https://doi.org/10.1038/s41565-021-00931-2 .
doi: 10.1038/s41565-021-00931-2
pubmed: 34211166
Zhao Z, Wijerathne H, Godwin AK, Soper SA. Isolation and analysis methods of extracellular vesicles (EVs). Extracell Vesicles Circ Nucl Acids 2021. https://doi.org/10.20517/evcna.2021.07
Zhang Q, Jeppesen DK, Higginbotham JN, Franklin JL, Coffey RJ. Comprehensive isolation of extracellular vesicles and nanoparticles. Nat Protoc. 2023. https://doi.org/10.1038/s41596-023-00811-0 .
doi: 10.1038/s41596-023-00811-0
pubmed: 38036926
pmcid: 11406548
Zhang H, Freitas D, Kim HS, Fabijanic K, Li Z, Chen H, et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat Cell Biol. 2018;20:332–43. https://doi.org/10.1038/s41556-018-0040-4 .
doi: 10.1038/s41556-018-0040-4
pubmed: 29459780
pmcid: 5931706
Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7. https://doi.org/10.1080/20013078.2018.1535750 .
Van Deun J, Mestdagh P, Agostinis P, Akay Ö, Anand S, Anckaert J, et al. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nat Methods. 2017;14:228–32. https://doi.org/10.1038/nmeth.4185 .
doi: 10.1038/nmeth.4185
pubmed: 28245209
Li S, Man Q, Gao X, Lin H, Wang J, Su F, et al. Tissue-derived extracellular vesicles in cancers and non‐cancer diseases: Present and future. J Extracell Vesicles. 2021;10. https://doi.org/10.1002/jev2.12175 .
Qin B, Hu X, Su Z, Zeng X, Ma H, Xiong K. Tissue-derived extracellular vesicles: Research progress from isolation to application. Pathol Res Pract. 2021;226:153604. https://doi.org/10.1016/j.prp.2021.153604 .
doi: 10.1016/j.prp.2021.153604
pubmed: 34500372
van Niel G, Carter DRF, Clayton A, Lambert DW, Raposo G, Vader P. Challenges and directions in studying cell–cell communication by extracellular vesicles. Nat Rev Mol Cell Biol. 2022;23:369–82. https://doi.org/10.1038/s41580-022-00460-3 .
doi: 10.1038/s41580-022-00460-3
pubmed: 35260831
Akbar A, Malekian F, Baghban N, Kodam SP, Ullah M. Methodologies to isolate and purify clinical Grade Extracellular vesicles for medical applications. Cells. 2022;11:186. https://doi.org/10.3390/cells11020186 .
doi: 10.3390/cells11020186
pubmed: 35053301
pmcid: 8774122
Ji S, Ma P, Cao X, Wang J, Yu X, Luo X, et al. Myoblast-derived exosomes promote the repair and regeneration of injured skeletal muscle in mice. FEBS Open Bio. 2022;12:2213–26. https://doi.org/10.1002/2211-5463.13504 .
doi: 10.1002/2211-5463.13504
pubmed: 36325691
pmcid: 9714366
Rome S, Forterre A, Mizgier ML, Bouzakri K. Skeletal muscle-released Extracellular vesicles: state of the art. Front Physiol. 2019;10. https://doi.org/10.3389/fphys.2019.00929 .
Yedigaryan L, Sampaolesi M. Extracellular vesicles and Duchenne muscular dystrophy pathology: modulators of disease progression. Front Physiol. 2023;14. https://doi.org/10.3389/fphys.2023.1130063 .
Watanabe S, Sudo Y, Makino T, Kimura S, Tomita K, Noguchi M, et al. Skeletal muscle releases extracellular vesicles with distinct protein and microRNA signatures that function in the muscle microenvironment. PNAS Nexus. 2022;1. https://doi.org/10.1093/pnasnexus/pgac173 .
Wang K, Frey N, Garcia A, Man K, Yang Y, Gualerzi A, et al. Nanotopographical cues Tune the therapeutic potential of Extracellular vesicles for the treatment of aged skeletal muscle injuries. ACS Nano. 2023;17:19640–51. https://doi.org/10.1021/acsnano.3c02269 .
doi: 10.1021/acsnano.3c02269
pubmed: 37797946
pmcid: 10603813
Ran N, Gao X, Dong X, Li J, Lin C, Geng M et al. Effects of exosome-mediated delivery of myostatin propeptide on functional recovery of mdx mice. Biomaterials 2020;236. https://doi.org/10.1016/j.biomaterials.2020.119826
Nakamura Y, Miyaki S, Ishitobi H, Matsuyama S, Nakasa T, Kamei N, et al. Mesenchymal-stem-cell-derived exosomes accelerate skeletal muscle regeneration. FEBS Lett. 2015;589:1257–65. https://doi.org/10.1016/j.febslet.2015.03.031 .
doi: 10.1016/j.febslet.2015.03.031
pubmed: 25862500
Aminzadeh MA, Rogers RG, Fournier M, Tobin RE, Guan X, Childers MK et al. Exosome-mediated benefits of cell therapy in mouse and human models of Duchenne muscular dystrophy. Stem Cell Reports 2018;10. https://doi.org/10.1016/j.stemcr.2018.01.023
Clevers H, Loh KM, Nusse R. An integral program for tissue renewal and regeneration: wnt signaling and stem cell control. Science. 1979;2014346. https://doi.org/10.1126/science.1248012 .
von Maltzahn J, Bentzinger CF, Rudnicki MA. Wnt7a-Fzd7 signalling directly activates the Akt/mTOR anabolic growth pathway in skeletal muscle. Nat Cell Biol. 2012;14:186–91. https://doi.org/10.1038/ncb2404 .
doi: 10.1038/ncb2404
Le Grand F, Jones AE, Seale V, Scimè A, Rudnicki MA. Wnt7a activates the Planar Cell Polarity Pathway to drive the symmetric expansion of Satellite Stem cells. Cell Stem Cell. 2009;4:535–47. https://doi.org/10.1016/j.stem.2009.03.013 .
doi: 10.1016/j.stem.2009.03.013
pubmed: 19497282
pmcid: 2743383
Bentzinger CF, von Maltzahn J, Dumont NA, Stark DA, Wang YX, Nhan K, et al. Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength. J Cell Biol. 2014;205:97–111. https://doi.org/10.1083/jcb.201310035 .
doi: 10.1083/jcb.201310035
pubmed: 24711502
pmcid: 3987134
von Maltzahn J, Renaud J-MM, Parise G, Rudnicki MA. Wnt7a treatment ameliorates muscular dystrophy. Proc Natl Acad Sci U S A. 2012;109:20614–9. https://doi.org/10.1073/pnas.1215765109 .
doi: 10.1073/pnas.1215765109
Bentzinger CF, Wang YX, von Maltzahn J, Soleimani VD, Yin H, Rudnicki MA. Fibronectin regulates Wnt7a signaling and Satellite Cell expansion. Cell Stem Cell. 2013;12:75–87. https://doi.org/10.1016/j.stem.2012.09.015 .
doi: 10.1016/j.stem.2012.09.015
pubmed: 23290138
pmcid: 3539137
Janda CY, Waghray D, Levin AM, Thomas C, Garcia KC. Structural basis of Wnt recognition by frizzled. Science (1979) 2012;336:59–64. https://doi.org/10.1126/science.1222879
Mehta S, Hingole S, Chaudhary V. The emerging mechanisms of wnt secretion and signaling in Development. Front Cell Dev Biol. 2021;9. https://doi.org/10.3389/fcell.2021.714746 .
Gross JC, Chaudhary V, Bartscherer K, Boutros M. Active wnt proteins are secreted on exosomes. Nat Cell Biol. 2012;14:1036–45. https://doi.org/10.1038/ncb2574 .
doi: 10.1038/ncb2574
pubmed: 22983114
Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR, Chiu E, et al. Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell. 2012;151:1542–56. https://doi.org/10.1016/j.cell.2012.11.024 .
doi: 10.1016/j.cell.2012.11.024
pubmed: 23260141
Menck K, Klemm F, Gross JC, Pukrop T, Wenzel D, Binder C. Induction and transport of wnt 5a during macrophage-induced malignant invasion is mediated by two types of extracellular vesicles. Oncotarget. 2013;4:2057–66. https://doi.org/10.18632/oncotarget.1336 .
doi: 10.18632/oncotarget.1336
pubmed: 24185202
pmcid: 3875769
Gurriaran-Rodriguez U, Datzkiw D, Radusky LG, Esper M, Xiao F, Ming H, et al. Wnt binding to Coatomer proteins directs secretion on exosomes independently of palmitoylation. BioRxiv. 2023. https://doi.org/10.1101/2023.05.30.542914 .
doi: 10.1101/2023.05.30.542914
pubmed: 37398399
pmcid: 10312507
Gurriaran-Rodriguez U, Kodippili K, Datzkiw D, Javandoost E, Xiao F, Teresa Rejas M et al. Wnt7a is required for regeneration of Dystrophic Skeletal Muscle n.d. https://doi.org/10.1101/2024.01.24.577041
Le Gall L, Ouandaogo ZG, Anakor E, Connolly O, Butler Browne G, Laine J, et al. Optimized method for extraction of exosomes from human primary muscle cells. Skelet Muscle. 2020;10:20. https://doi.org/10.1186/s13395-020-00238-1 .
doi: 10.1186/s13395-020-00238-1
pubmed: 32641118
pmcid: 7341622
Le Bihan MC, Bigot A, Jensen SS, Dennis JL, Rogowska-Wrzesinska A, Lainé J, et al. In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts. J Proteom. 2012;77:344–56. https://doi.org/10.1016/j.jprot.2012.09.008 .
doi: 10.1016/j.jprot.2012.09.008
Crescitelli R, Lä C, Lö J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat Protoc n d. https://doi.org/10.1038/s41596-020-00466-1
Visan KS, Lobb RJ, Ham S, Lima LG, Palma C, Edna CPZ, et al. Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles. J Extracell Vesicles. 2022;11. https://doi.org/10.1002/jev2.12266 .
Busatto S, Vilanilam G, Ticer T, Lin W-L, Dickson D, Shapiro S, et al. Tangential Flow Filtration for highly efficient concentration of Extracellular vesicles from large volumes of Fluid. Cells. 2018;7:273. https://doi.org/10.3390/cells7120273 .
doi: 10.3390/cells7120273
pubmed: 30558352
pmcid: 6315734
Guo J, Wu C, Lin X, Zhou J, Zhang J, Zheng W, et al. Establishment of a simplified dichotomic size-exclusion chromatography for isolating extracellular vesicles toward clinical applications. J Extracell Vesicles. 2021;10. https://doi.org/10.1002/jev2.12145 .
Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of Exosomes from Cell Culture supernatants and Biological fluids. Curr Protoc Cell Biol. 2006;30. https://doi.org/10.1002/0471143030.cb0322s30 .
Konoshenko MYu, Lekchnov EA, Vlassov AV, Laktionov PP. Isolation of Extracellular vesicles: General methodologies and latest trends. Biomed Res Int. 2018;2018:1–27. https://doi.org/10.1155/2018/8545347 .
doi: 10.1155/2018/8545347
Zhi Z, Sun Q, Tang W. Research advances and challenges in tissue-derived extracellular vesicles. Front Mol Biosci. 2022;9. https://doi.org/10.3389/fmolb.2022.1036746 .
Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of Exosomes from Cell Culture supernatants. Curr Protoc Cell Biol. 2006;3:3221–32229.
Musumeci T, Leonardi A, Bonaccorso A, Pignatello R, Puglisi G. Tangential Flow Filtration Technique: An Overview on Nanomedicine Applications 2018:48–60. https://doi.org/10.2174/2211738506666180306160921
Gurriarán-Rodríguez U, Santos-Zas I, González-Sánchez J, Beiroa D, Moresi V, Mosteiro CS, et al. Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling. Mol Ther. 2015;23. https://doi.org/10.1038/mt.2015.40 .