Abstract
The neurosphere assay is the most widely used in vitro tool to determine the proliferative and differentiation potential of adult neural precursor cells in rodents. Although originally developed for, and predominantly applied to, the growth of embryonic and adult subventricular zone-derived stem cells, hippocampal neurospheres are now routinely cultured by many laboratories. As hippocampal neurospheres are fewer in number, on average smaller in size, and more slowly growing than their ventricular counterparts, the methodology traditionally used to isolate and culture neurospheres from the subventricular zone is not optimal for hippocampal neurosphere growth. Here, we provide a detailed description of an optimized protocol for the microdissection, dissociation, and neurosphere generation from adult hippocampal dentate gyrus tissue. We also outline the protocols required to perform downstream passaging, differentiation, and immunohistological determination of the multipotentiality of hippocampal neurospheres.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Altman J (1962) Are new neurons formed in the brains of adult mammals? Science 135:1128–1129
Altman J (1962) Autoradiographic study of degenerative and regenerative proliferation of neuroglia cells with tritiated thymidine. Exp Neurol 5:302–318
Altman J, Das GD (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124(3):319–335
Boldrini M, Fulmore CA, Tartt AN, Simeon LR, Pavlova I, Poposka V, Rosoklija GB, Stankov A, Arango V, Dwork AJ, Hen R, Mann JJ (2018) Human hippocampal neurogenesis persists throughout aging. Cell Stem Cell 22(4):589–599.e585. https://doi.org/10.1016/j.stem.2018.03.015
Moreno-Jimenez EP, Flor-Garcia M, Terreros-Roncal J, Rabano A, Cafini F, Pallas-Bazarra N, Avila J, Llorens-Martin M (2019) Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer's disease. Nat Med 25(4):554–560. https://doi.org/10.1038/s41591-019-0375-9
Spalding KL, Bhardwaj RD, Buchholz BA, Druid H, Frisen J (2005) Retrospective birth dating of cells in humans. Cell 122(1):133–143
Tobin MK, Musaraca K, Disouky A, Shetti A, Bheri A, Honer WG, Kim N, Dawe RJ, Bennett DA, Arfanakis K, Lazarov O (2019) Human hippocampal neurogenesis persists in aged adults and Alzheimer's disease patients. Cell Stem Cell 24(6):974–982.e973. https://doi.org/10.1016/j.stem.2019.05.003
Reynolds BA, Tetzlaff W, Weiss S (1992) A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 12(11):4565–4574
Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255(5052):1707–1710
Babu H, Claasen JH, Kannan S, Runker 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. https://doi.org/10.3389/fnins.2011.00089
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
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):1–13
Jessberger S, Clemenson GD Jr, Gage FH (2007) Spontaneous fusion and nonclonal growth of adult neural stem cells. Stem Cells 25(4):871–874. https://doi.org/10.1634/stemcells.2006-0620. 2006-0620 [pii]
Pastrana E, Silva-Vargas V, Doetsch F (2011) Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell 8(5):486–498. https://doi.org/10.1016/j.stem.2011.04.007. S1934-5909(11)00172-X [pii]
Bernas SN, Leiter O, Walker TL, Kempermann G (2017) Isolation, culture and differentiation of adult hippocampal precursor cells. Bio-Protocol 7(21):e2603. https://doi.org/10.21769/BioProtoc.2603
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 (84). https://doi.org/10.3791/51225
Hagihara H, Toyama K, Yamasaki N, Miyakawa T (2009) Dissection of hippocampal dentate gyrus from adult mouse. J Vis Exp (33). https://doi.org/10.3791/1543. 1543 [pii]
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rust, R., Walker, T.L. (2022). Isolation and Culture of Adult Hippocampal Precursor Cells as Free-Floating Neurospheres. In: Deleyrolle, L.P. (eds) Neural Progenitor Cells. Methods in Molecular Biology, vol 2389. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1783-0_3
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1783-0_3
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1782-3
Online ISBN: 978-1-0716-1783-0
eBook Packages: Springer Protocols