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Isolation of neural stem cells from the postnatal cerebellum

Nature Neuroscience volume 8, pages 723–729 (2005)Cite this article

Abstract

The cerebellum is critical for motor coordination and cognitive function and is the target of transformation in medulloblastoma, the most common malignant brain tumor in children. Although the development of granule cells, the most abundant neurons in the cerebellum, has been studied in detail, the origins of other cerebellar neurons and glia remain poorly understood. Here we show that the murine postnatal cerebellum contains multipotent neural stem cells (NSCs). These cells can be prospectively isolated based on their expression of the NSC marker prominin-1 (CD133) and their lack of markers of neuronal and glial lineages (lin−). Purified prominin+lin− cells form self-renewing neurospheres and can differentiate into astrocytes, oligodendrocytes and neurons in vitro. Moreover, they can generate each of these lineages after transplantation into the cerebellum. Identification of cerebellar stem cells has important implications for the understanding of cerebellar development and the origins of medulloblastoma.

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Figure 1: Non–granule cell precursors can be purified from the postnatal cerebellum.
Figure 2: Prominin+ cells proliferate in response to bFGF.
Figure 3: Prominin+ cells are located in the cerebellar white matter.
Figure 4: Prominin+lin− cells generate neurospheres.
Figure 5: Prominin+lin− cells exhibit multipotency in vitro.
Figure 6: Prominin+lin− cells differentiate into neurons and glia after transplantation into the cerebellum.
Figure 7: Neurospheres derived from a single stem cell can generate neurons and glia in vivo.

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Acknowledgements

The authors thank M. Cook for flow cytometric analysis; E. Snyder and J.-P. Lee for assistance with intracranial injections; W. Salmon for help with microscopy; and A. Shetty, B. Barres and M. Rao for helpful discussions. R.W.R. is a Kimmel Foundation Scholar. This research was supported by a McDonnell Foundation 21st Century Award and by grant #1R01MH067916-01 from the US National Institute of Mental Health.

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Authors and Affiliations

  1. Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Audra Lee, Jessica D Kessler, Tracy-Ann Read, Constanze Kaiser & Robert J Wechsler-Reya

  2. Max-Planck Institute of Molecular Cell Biology and Genetics, Dresden, D-01307, Germany

    Denis Corbeil & Wieland B Huttner

  3. Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, 75390-9111, Texas, USA

    Jane E Johnson

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Correspondence to Robert J Wechsler-Reya.

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Supplementary information

Supplementary Fig. 1

Purification of cerebellar stem cells and assessment of neurosphere forming-efficiency. To enrich for stem cells, cells from the cerebellum of Math1-GFP mice (cerebellar suspension) were stained with antibodies specific for Prominin and for neuronal and glial lineage markers (O4, TAPA-1 and PSA-NCAM). Cells were then FACS-sorted into GFP+ (GCP) and GFP– (non-GCP) fractions, representing 90% and 10% of the starting population respectively. GFP– cells were further fractionated into Prominin– cells (7% of the cerebellar suspension), Prominin+Lineage+ cells (2.8%) and Prominin+Lineage– cells (0.2%). Each of these fractions was cultured at clonal density in bFGF and EGF for 10 days, and then the neurosphere forming efficiency (neurospheres/cells plated, shown in orange boxes) was determined. The starting cerebellar suspension formed one neurosphere for every 1000 cells plated. GCPs (Math1-GFP+ cells) could not form neurospheres when cultured at clonal density in the presence of bFGF + EGF or Sonic hedgehog; in contrast, Math1-GFP– cells showed a 10-fold enrichment in neurosphere-forming ability (1 neurosphere/100 cells) compared to the starting cell suspension. Prominin– cells survived poorly and did not form neurospheres at clonal density. Prominin+Lineage+ cells formed few free-floating neurospheres (<1 neurosphere/100 cells), but often gave rise to adherent colonies that underwent neuronal differentiation. Prominin+Lineage– cells formed one neurosphere for every 30 cells plated. (PDF 502 kb)

Supplementary Fig. 2

Self-renewal of cerebellar stem cells. (a) Primary and secondary neurospheres. FACS-sorted Prominin+Lin– cells were cultured at clonal density in media containing bFGF and EGF. After 10 days, primary (1°) neurospheres were photographed at 20X magnification. Primary neurospheres were then dissociated and replated under the conditions described above. Secondary (2°) neurospheres, photographed at 10 days, resembled primary neurospheres in morphology and frequency. Neurospheres could be propagated in this manner for up to 10 weeks in vitro. (b) Efficiency of neurosphere formation at clonal density and after repeated passage in culture. The efficiency of neurosphere formation was similar whether Prominin+Lineage– cells were cultured at a density of 1 cell per well or at a density of 1 cell per mm2 (clonal density). Moreover, neurosphere-forming efficiency was maintained in successive passages of neurospheres generated at clonal density. In all cases, approximately 1 neurosphere was generated for every 30 cells plated. (PDF 681 kb)

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Lee, A., Kessler, J., Read, TA. et al. Isolation of neural stem cells from the postnatal cerebellum. Nat Neurosci 8, 723–729 (2005). https://doi.org/10.1038/nn1473

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