Protocol | Published:

Production of neurons, astrocytes and oligodendrocytes from mammalian CNS stem cells

Nature Protocols volume 3, pages 935940 (2008) | Download Citation



The isolation and expansion of precursor cells in a serum-free culture system allows for the systematic characterization of their properties and the intrinsic and extrinsic signals that regulate their function. The discovery of neural stem cells in the adult mouse brain was made possible by the creation of a novel culture system subsequently termed the neurosphere assay. Therein, the dissociated adult mouse periventricular area was plated in the presence of epidermal growth factor, but in the absence of adhesive substrates, which resulted in the generation of spheres of proliferating cells that detached from the plate bottom and remained suspended in the media. Since its inception, the neurosphere culture system has been widely used in the neural precursor cell field and has been extensively adapted for the isolation and expansion of corneal, cardiac, skin, prostate, mammary and brain tumor stem cells. The original neurosphere culture protocol, which takes approximately 10 d to complete, is described here in detail.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    & Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710 (1992).

  2. 2.

    Neurogenesis in the adult brain: death of a dogma. Nat. Rev. Neurosci. 1, 67–73 (2000).

  3. 3.

    & Neural stem cells in mammalian development. Curr. Opin. Cell Biol. 18, 704–709 (2006).

  4. 4.

    et al. Human corneal endothelial cell precursors isolated by sphere-forming assay. Invest. Ophthalmol. Vis. Sci. 46, 1626–1631 (2005).

  5. 5.

    et al. Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart. J. Cell Biol. 170, 1135–1146 (2005).

  6. 6.

    et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat. Cell Biol. 3, 778–784 (2001).

  7. 7.

    , , , & Isolation and functional characterization of murine prostate stem cells. Proc. Natl. Acad. Sci. USA 104, 181–186 (2007).

  8. 8.

    et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 17, 1253–1270 (2003).

  9. 9.

    et al. Identification of human brain tumour initiating cells. Nature 432, 396–401 (2004).

  10. 10.

    & Strengths and limitations of the neurosphere culture system. Mol. Neurobiol. 34, 153–161 (2006).

  11. 11.

    , & Using the neurosphere assay to quantify neural stem cells in vivo. Curr. Pharm. Biotechnol. 8, 141–145 (2007).

  12. 12.

    , , & Neurosphere assays: growth factors and hormone differences in tumor and nontumor studies. Stem Cells 24, 2851–2857 (2006).

  13. 13.

    , & Redox regulation of precursor cell function: insights and paradoxes. Antioxid. Redox Signal. 7, 1456–1467 (2005).

  14. 14.

    & Isolation of a novel platelet-derived growth factor-responsive precursor from the embryonic ventral forebrain. J. Neurosci. 24, 10888–10899 (2004).

  15. 15.

    et al. Bone morphogenetic proteins induce astroglial differentiation of oligodendroglial–astroglial progenitor cells. J. Neurosci. 17, 4112–4120 (1997).

  16. 16.

    , & A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J. Neurosci. 12, 4565–4574 (1992).

  17. 17.

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

  18. 18.

    & CNTF/LIF/gp130 receptor complex signaling maintains a VZ precursor differentiation gradient in the developing ventral forebrain. Development 132, 565–578 (2005).

  19. 19.

    et al. Pregnancy-stimulated neurogenesis in the adult female forebrain mediated by prolactin. Science 299, 117–120 (2003).

  20. 20.

    et al. Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nat. Protoc. 2, 1044–1051 (2007).

  21. 21.

    & Isolation and culture of adult neurons and neurospheres. Nat. Protoc. 2, 1490–1498 (2007).

Download references


We thank Dorothea Livingstone and Rozina Hassam for technical comments on the manuscript. This work was supported by the Canadian Institutes of Health Research. S.W. is an Alberta Heritage Foundation for Medical Research Scientist.

Author information


  1. Hotchkiss Brain Institute, Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, Calgary, Alberta Canada, T2N 4N1.

    • Andrew Chojnacki
    •  & Samuel Weiss


  1. Search for Andrew Chojnacki in:

  2. Search for Samuel Weiss in:

Corresponding author

Correspondence to Samuel Weiss.

About this article

Publication history



Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.