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

Journal name:
Nature Protocols
Year published:
Published online


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.

At a glance


  1. Major cell types of the adult SEZ.
    Figure 1: Major cell types of the adult SEZ.

    (a,b) Schematic drawing of a sagittal section through an adult mouse brain. Red delineates the field of SEZ shown in the schematic drawing in b. (b) Simplified scheme depicting the cellular composition of adult SEZ. (c,d) Individual cells in the neurogenic lineage (c) or neurogenic niche (d). A small apical cilium (prominin1+; red) of a hGFAP-eGFP+ (green) adult neural stem cell contacts the ventricle, whereas basal end-feet contact the blood vessels31, 32. The expression of EGF receptor (blue line) has been suggested to mark activated neural stem cells (aNSCs), whereas quiescent neural stem cells (qNSCs) should not express EGFR17. Multiciliated ependymal cells are directly in contact with the liquid-filled ventricle and express prominin1. Niche astrocytes are located more basally in the SEZ and express hGFAP-eGFP. Cortex, cerebral cortex; LV, lateral ventricle; NB, neuroblast; OB, olfactory bulb; OPC, oligodendrocyte progenitor; RMS, rostral migratory stream; TAP, transient amplifying progenitor cell.

  2. Flow diagram depicting the major steps of the isolation protocol and possible further applications.
    Figure 2: Flow diagram depicting the major steps of the isolation protocol and possible further applications.
  3. Dissection procedure of the lateral ventricular wall.
    Figure 3: Dissection procedure of the lateral ventricular wall.

    (a,b) Photographs and schemes depicting the position of the transverse (at the level of the optic chiasm) and longitudinal (along the midline) cut (a) to isolate the anterior part of the forebrain hemispheres (b). (c) Medial view of both hemispheres with arrows pointing to rostral and caudal ends of the hippocampus. (d) Image and scheme depicting the removal of the hippocampus in order to uncover the underlying lateral wall of the lateral ventricle. (eh) Images and schemes demonstrating the dissection of the SEZ from the surrounding white matter (f) and striatum (h). (i) Image of the isolated SEZ. Scale bar, 5 mm. For a similar dissection protocol, see also refs. 24,33.

  4. FACS plots for gate setting for the different marker analysis.
    Figure 4: FACS plots for gate setting for the different marker analysis.

    (a,b) Assessment of the dead cells in the sample determined by propidium iodide (PI) labeling. (a) Dot plot depicting definition of PI gate according to sample without PI. (b) Dot plot of representative sample containing <5% of dying cells. If the percentage of dead cells in the SEZ sample exceeds 5%, the sample should not be further analyzed. (cg) Dot plots depicting the gate settings for different markers used to isolate the distinct SEZ cell types by FACS. (c) Select relevant and living cells by FCS-A and SSC-A (P1). (d) Cellular aggregates are excluded based on FSC-A and FSC-W gate (P2). (e,f) Gates for hGFAP-eGFP sorting (e) and EGF-Alexa Fluor 647–streptavidin complex (EGF-Alexa Fluor 647; f) were based on wild-type mice, which are negative for GFP and not incubated with a fluorescent ligand. (g) The gate for prominin1-PE (PE-conjugated CD133) was based on isotype-matched antibody control conjugated to PE. (hj) Dot plots depicting cells positive for hGFAP-eGFP (h), EGFR (i) and prominin1 (j). FACS data are reported as suggested by Alexander et al.34. APC, Allophycocyanin.

  5. Isolation of NSCs and other SEZ cells.
    Figure 5: Isolation of NSCs and other SEZ cells.

    (ac) Gate setting for hGFAP-eGFP (a), prominin1 (b) and EGF (c) (see also Fig. 4e–g). (d,e) Purification of distinct cell types from the SEZ. (d) Dot plot depicting the isolation of NSCs (blue box), ependymal cells (red box) and niche astrocytes/NSC progeny (green box). Note that because of its stability, GFP could be detected in the NSC progeny9. (e) Dot plot illustrating the separation of NSCs into the EGFR+ fraction (activated NSCs) and EGFR fraction (quiescent NSCs).


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Author information

  1. These authors contributed equally to this work.

    • Judith Fischer &
    • Ruth Beckervordersandforth


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

    • Pratibha Tripathi


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.

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The authors declare no competing financial interests.

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Supplementary information

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  1. Supplementary Fig. 1 (3M)

    (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.

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