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Prospective isolation of adult neural stem cells from the mouse subependymal zone

Nature Protocols volume 6, pages 1981–1989 (2011)Cite this article

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Abstract

Neural stem cells (NSCs) have the remarkable capacity to self-renew and the lifelong ability to generate neurons in the adult mammalian brain. However, the molecular and cellular mechanisms contributing to these behaviors are still not understood. Now that prospective isolation of the NSCs has become feasible, these mechanisms can be studied. Here we describe a protocol for the efficient isolation of adult NSCs, by the application of a dual-labeling strategy on the basis of their glial identity and ciliated nature. The cells are isolated from the lateral ventricular subependymal zone (SEZ) of adult hGFAP-eGFP (human glial fibrillary acidic protein–enhanced green fluorescent protein) transgenic mice by fluorescence-activated cell sorting. Staining against prominin1 (CD133) allows the isolation of the NSCs (hGFAP-eGFP+/prominin1+), which can be further subdivided by labeling with the fluorescent epidermal growth factor. This protocol, which can be completed in 7 h, allows the assessment of quantitative changes in SEZ NSCs and the examination of their molecular and functional characteristics.

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Figure 1: Major cell types of the adult SEZ.
Figure 2
Figure 3: Dissection procedure of the lateral ventricular wall.
Figure 4: FACS plots for gate setting for the different marker analysis.
Figure 5: Isolation of NSCs and other SEZ cells.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 870, Go640/7.1, 8.1, 9.1, SFB 596), the Bundesministerium für Bildung und Forschung, the Helmholtz association (HELMA) and the European Union (EUTRACC, EduGlia).

Author information

Author notes

  1. Pratibha Tripathi

    Present address: Present address: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.,

  2. Judith Fischer and Ruth Beckervordersandforth: These authors contributed equally to this work.

Authors and Affiliations

  1. Helmholtz Center Munich, German Research Center for Environmental Health, Institute for Stem Cell Research, Neuherberg, Germany

    Judith Fischer, Ruth Beckervordersandforth, Pratibha Tripathi, Andrea Steiner-Mezzadri, Jovica Ninkovic & Magdalena Götz

  2. Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, Munich, Germany

    Jovica Ninkovic & Magdalena Götz

Authors
  1. Judith Fischer
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  4. Andrea Steiner-Mezzadri
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  5. Jovica Ninkovic
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  6. Magdalena Götz
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Contributions

J.F. and R.B. contributed to experimental design, execution and preparation of the manuscript. P.T. pioneered the establishment of the procedure. A.S.-M. provided support and technical assistance. J.N. provided conceptual advice, helped to design the experiments and assisted with the manuscript. M.G. developed, supervised and financed the project, and was involved in many discussions of the experimental approaches as well as in writing the manuscript.

Corresponding author

Correspondence to Magdalena Götz.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

(A-C) Dot plots depicting SEZ cells according to size and granularity (FCS-A vs. SSC-A; P1). (A′-C′) Dot plots illustrating cells positive for the marker of interest (for gate setting see Fig. 4). Colours depict cells expressing hGFAP-eGFP (A′; green), prominin1 (B′; red), EGFR (C′; blue) and indicate their positions according to FCS-A and SSC-A as a backprojection to P1 (A-C). Note that almost all hGFAP-GFP+, EGFR+ and most of the prominin1+ cells are located within the lower arm of the SEZ cell distribution, justifying the P1 gate in Fig 4. (PDF 147 kb)

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Fischer, J., Beckervordersandforth, R., Tripathi, P. et al. Prospective isolation of adult neural stem cells from the mouse subependymal zone. Nat Protoc 6, 1981–1989 (2011). https://doi.org/10.1038/nprot.2011.412

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