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Isolation of Extracellular Vesicles from Subventricular Zone Neural Stem Cells

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Stem Cell Niche

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2002))

Abstract

The neonatal subventricular zone (SVZ) is a neurogenic niche that contains neural stem cells (NSCs). NSCs release particles called extracellular vesicles (EVs) that contain biological material. EVs are transferred to cells, including immune cells in the brain called microglia. A standard approach to identify EV functions is to isolate and transplant EVs. Here, a detailed protocol is provided that will allow one to culture neonatal SVZ NSCs and to isolate, label, and transplant EVs. The protocol will permit careful and thorough examination of EVs in a wide range of physiological and pathophysiological conditions.

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References

  1. Cocucci E, Meldolesi J (2015) Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends Cell Biol 25:364–372

    Article  CAS  Google Scholar 

  2. Théry C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579

    Article  Google Scholar 

  3. Zhang H, Freitas D, Kim HS, Fabijanic K, Li Z, Chen H, Mark MT, Molina H, Martin AB, Bojmar L, Fang J, Rampersaud S, Hoshino A, Matei I, Kenific CM, Nakajima M, Mutvei AP, Sansone P, Buehring W, Wang H, Jimenez JP, Cohen-Gould L, Paknejad N, Brendel M, Manova-Todorova K, Magalhães A, Ferreira JA, Osório H, Silva AM, Massey A, Cubillos-Ruiz JR, Galletti G, Giannakakou P, Cuervo AM, Blenis J, Schwartz R, Brady MS, Peinado H, Bromberg J, Matsui H, Reis CA, Lyden D (2018) Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat Cell Biol 20:332–343

    Article  CAS  Google Scholar 

  4. Ramachandran S, Palanisamy V (2012) Horizontal transfer of RNAs: exosomes as mediators of intercellular communication. Wiley Interdiscip Rev RNA 3:286–293

    Article  CAS  Google Scholar 

  5. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659

    Article  CAS  Google Scholar 

  6. Eckard SC, Rice GI, Fabre A, Badens C, Gray EE, Hartley JL, Crow YJ, Stetson DB (2014) The SKIV2L RNA exosome limits activation of the RIG-I-like receptors. Nat Immunol 15:839–845

    Article  CAS  Google Scholar 

  7. Iraci N, Gaude E, Leonardi T, Costa ASH, Cossetti C, Peruzzotti-Jametti L, Bernstock JD, Saini HK, Gelati M, Vescovi AL, Bastos C, Faria N, Occhipinti LG, Enright AJ, Frezza C, Pluchino S (2017) Extracellular vesicles are independent metabolic units with asparaginase activity. Nat Chem Biol 13:951–955

    Article  CAS  Google Scholar 

  8. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13:423–433

    Article  CAS  Google Scholar 

  9. Lobb RJ, Becker M, Wen SW, Wong CSF, Wiegmans AP, Leimgruber A, Möller A (2015) Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell vesicles 4:27031

    Article  Google Scholar 

  10. Momen-Heravi F, Saha B, Kodys K, Catalano D, Satishchandran A, Szabo G (2015) Increased number of circulating exosomes and their microRNA cargos are potential novel biomarkers in alcoholic hepatitis. J Transl Med 13:261

    Article  Google Scholar 

  11. Livshits MA, Khomyakova E, Evtushenko EG, Lazarev VN, Kulemin NA, Semina SE, Generozov EV, Govorun VM, Govorun VM (2015) Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol. Sci Rep 5:17319

    Article  Google Scholar 

  12. Bobrie A, Colombo M, Krumeich S, Raposo G, Théry C (2012) Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation. J Extracell Vesicles 1:18397

    Article  CAS  Google Scholar 

  13. Taylor DD, Shah S (2015) Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods 87:3–10

    Article  CAS  Google Scholar 

  14. 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 30:3.22.1–3.22.29

    Article  Google Scholar 

  15. Linares R, Tan S, Gounou C, Arraud N, Brisson AR (2015) High-speed centrifugation induces aggregation of extracellular vesicles. J. Extracell. Vesicles. 4:29509

    Article  Google Scholar 

  16. Lim DA, Alvarez-Buylla A (2016) The adult ventricular-subventricular zone (V-SVZ) and olfactory bulb (OB) neurogenesis. Cold Spring Harb Perspect Biol 8:a018820

    Article  Google Scholar 

  17. Obernier K, Cebrian-Silla A, Thomson M, Parraguez JI, Anderson R, Guinto C, Rodas Rodriguez J, Garcia-Verdugo J-M, Alvarez-Buylla A (2018) Adult neurogenesis is sustained by symmetric self-renewal and differentiation. Cell Stem Cell 22:221–234.e8

    Article  CAS  Google Scholar 

  18. Bjornsson CS, Apostolopoulou M, Tian Y, Temple S (2015) It takes a village: constructing the neurogenic niche. Dev Cell 32:435–446

    Article  CAS  Google Scholar 

  19. Luskin MB (1993) Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11:173–189

    Article  CAS  Google Scholar 

  20. Lois C, Garcia-Verdugo J-M, Alvarez-Buylla A (1996) Chain migration of neuronal precursors. Science 271:978–981

    Article  CAS  Google Scholar 

  21. Petreanu L, Alvarez-Buylla A (2002) Maturation and death of adult-born olfactory bulb granule neurons: role of olfaction. J Neurosci 22:6106–6113

    Article  CAS  Google Scholar 

  22. Imayoshi I, Sakamoto M, Ohtsuka T, Takao K, Miyakawa T, Yamaguchi M, Mori K, Ikeda T, Itohara S, Kageyama R (2008) Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain. Nat Neurosci 11:1153–1161

    Article  CAS  Google Scholar 

  23. Bouab M, Paliouras GN, Aumont A, Forest-Bérard K, Fernandes KJL (2011) Aging of the subventricular zone neural stem cell niche: evidence for quiescence-associated changes between early and mid-adulthood. Neuroscience 173:135–149

    Article  CAS  Google Scholar 

  24. Daynac M, Morizur L, Chicheportiche A, Mouthon M-A, Boussin FD (2016) Age-related neurogenesis decline in the subventricular zone is associated with specific cell cycle regulation changes in activated neural stem cells. Sci Rep 6:21505

    Article  CAS  Google Scholar 

  25. Maslov AY, Barone TA, Plunkett RJ, Pruitt SC (2004) Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice. J Neurosci 24:1726–1733

    Article  CAS  Google Scholar 

  26. Tramontin AD, García-Verdugo JM, Lim DA, Alvarez-Buylla A (2003) Postnatal development of radial glia and the ventricular zone (VZ): a continuum of the neural stem cell compartment. Cereb Cortex 13:580–587

    Article  Google Scholar 

  27. Mirzadeh Z, Merkle FT, Soriano-Navarro M, Garcia-Verdugo JM, Alvarez-Buylla A (2008) Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell 3:265–278

    Article  CAS  Google Scholar 

  28. Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, Samokhvalov IM, Merad M (2010) Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330:841–845

    Article  CAS  Google Scholar 

  29. Cunningham CL, Martínez-Cerdeño V, Noctor SC (2013) Microglia regulate the number of neural precursor cells in the developing cerebral cortex. J Neurosci 33:4216–4233

    Article  CAS  Google Scholar 

  30. Swinnen N, Smolders S, Avila A, Notelaers K, Paesen R, Ameloot M, Brône B, Legendre P, Rigo J-M (2013) Complex invasion pattern of the cerebral cortex bymicroglial cells during development of the mouse embryo. Glia 61:150–163

    Article  Google Scholar 

  31. Battista D, Ferrari CC, Gage FH, Pitossi FJ (2006) Neurogenic niche modulation by activated microglia: transforming growth factor β increases neurogenesis in the adult dentate gyrus. Eur J Neurosci 23:83–93

    Article  Google Scholar 

  32. Zhu P, Hata R, Cao F, Gu F, Hanakawa Y, Hashimoto K, Sakanaka M (2008) Ramified microglial cells promote astrogliogenesis and maintenance of neural stem cells through activation of Stat3 function. FASEB J 22:3866–3877

    Article  CAS  Google Scholar 

  33. Antony JM, Paquin A, Nutt SL, Kaplan DR, Miller FD (2011) Endogenous microglia regulate development of embryonic cortical precursor cells. J Neurosci Res 89:286–298

    Article  CAS  Google Scholar 

  34. Snyder EY, Yoon C, Flax JD, Macklis JD (1997) Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex. Proc Natl Acad Sci U S A 94:11663–11668

    Article  CAS  Google Scholar 

  35. Morton MC, Neckles VN, Seluzicki CM, Holmberg JC, Feliciano DM (2018) Neonatal subventricular zone neural stem cells release extracellular vesicles that act as a microglial morphogen. Cell Rep 23:78–89

    Article  CAS  Google Scholar 

  36. Cossetti C, Iraci N, Mercer TR, Leonardi T, Alpi E, Drago D, Alfaro-Cervello C, Saini HK, Davis MP, Schaeffer J, Vega B, Stefanini M, Zhao C, Muller W, Garcia-Verdugo JM, Mathivanan S, Bachi A, Enright AJ, Mattick JS, Pluchino S (2014) Extracellular vesicles from neural stem cells transfer IFN-γ via Ifngr1 to activate Stat1 signaling in target cells. Mol Cell 56:193–204

    Article  CAS  Google Scholar 

  37. Asai H, Ikezu S, Tsunoda S, Medalla M, Luebke J, Haydar T, Wolozin B, Butovsky O, Kügler S, Ikezu T (2015) Depletion of microglia and inhibition of exosome synthesis halt tau propagation. Nat Neurosci 18:1584–1593

    Article  CAS  Google Scholar 

  38. Ray J, Raymon HK, Gage FH (1995) Generation and culturing of precursor cells and neuroblasts from embryonic and adult central nervous system. Methods Enzymol 254:20–37

    Article  CAS  Google Scholar 

  39. Palmer TD, Ray J, Gage FH (1995) FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol Cell Neurosci 6:474–486

    Article  CAS  Google Scholar 

  40. Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707–1710

    Article  CAS  Google Scholar 

  41. Reynolds BA, Weiss S (1996) Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 175:1–13

    Article  CAS  Google Scholar 

  42. Walker TL, Kempermann G (2014) One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice. J Vis Exp. https://doi.org/10.3791/51225

  43. Azari H, Rahman M, Sharififar S, Reynolds BA (2010) Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay. J Vis Exp. https://doi.org/10.3791/2393

  44. Bez A, Corsini E, Curti D, Biggiogera M, Colombo A, Nicosia RF, Pagano SF, Parati EA (2003) Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization. Brain Res 993:18–29

    Article  CAS  Google Scholar 

  45. Suslov ON, Kukekov VG, Ignatova TN, Steindler DA (2002) Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres. Proc Natl Acad Sci U S A 99:14506–14511

    Article  CAS  Google Scholar 

  46. Parmar M, Sjöberg A, Björklund A, Kokaia Z (2003) Phenotypic and molecular identity of cells in the adult subventricular zone. in vivo and after expansion in vitro. Mol Cell Neurosci 24:741–752

    Article  CAS  Google Scholar 

  47. Babu H, Claasen J-H, Kannan S, Rünker AE, Palmer T, Kempermann G (2011) A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus. Front Neurosci 5:89

    Article  Google Scholar 

  48. Aligholi H, Rezayat SM, Azari H, Ejtemaei Mehr S, Akbari M, Modarres Mousavi SM, Attari F, Alipour F, Hassanzadeh G, Gorji A (2016) Preparing neural stem/progenitor cells in PuraMatrix hydrogel for transplantation after brain injury in rats: a comparative methodological study. Brain Res 1642:197–208

    Article  CAS  Google Scholar 

  49. Thonhoff JR, Lou DI, Jordan PM, Zhao X, Wu P (2008) Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro. Brain Res 1187:42–51

    Article  CAS  Google Scholar 

  50. Moradi F, Bahktiari M, Joghataei MT, Nobakht M, Soleimani M, Hasanzadeh G, Fallah A, Zarbakhsh S, Hejazian LB, Shirmohammadi M, Maleki F (2012) BD PuraMatrix peptide hydrogel as a culture system for human fetal Schwann cells in spinal cord regeneration. J Neurosci Res 90:2335–2348

    Article  CAS  Google Scholar 

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Acknowledgments

David M. Feliciano is supported by grants from the Whitehall Foundation and National Institutes of Health 1R15NS096562.

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Authors and Affiliations

  1. Department of Biological Sciences, Clemson University, Clemson, SC, USA

    Mary C. Morton, Victoria N. Neckles & David M. Feliciano

Authors
  1. Mary C. Morton
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  2. Victoria N. Neckles
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  3. David M. Feliciano
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Corresponding author

Correspondence to David M. Feliciano .

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Editors and Affiliations

  1. Ottawa Hospital Research Institute, Ottawa, ON, Canada

    Kursad Turksen

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Morton, M.C., Neckles, V.N., Feliciano, D.M. (2018). Isolation of Extracellular Vesicles from Subventricular Zone Neural Stem Cells. In: Turksen, K. (eds) Stem Cell Niche. Methods in Molecular Biology, vol 2002. Humana, New York, NY. https://doi.org/10.1007/7651_2018_183

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  • DOI: https://doi.org/10.1007/7651_2018_183

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9507-3

  • Online ISBN: 978-1-4939-9508-0

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