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

Journal name:
Nature Protocols
Volume:
6,
Pages:
1981–1989
Year published:
DOI:
doi:10.1038/nprot.2011.412
Published online

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.

At a glance

Figures

  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).

References

  1. Kriegstein, A. & Alvarez-Buylla, A. The glial nature of embryonic and adult neural stem cells. Annu. Rev. Neurosci. 32, 149184 (2009).
  2. Ninkovic, J. & Gotz, M. Signaling in adult neurogenesis: from stem cell niche to neuronal networks. Curr. Opin. Neurobiol. 17, 338344 (2007).
  3. Curtis, M.A., Kam, M. & Faull, R.L. Neurogenesis in humans. Eur. J. Neurosci. 33, 11701174 (2011).
  4. Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z. & Lindvall, O. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat. Med. 8, 963970 (2002).
  5. Brill, M.S. et al. Adult generation of glutamatergic olfactory bulb interneurons. Nat. Neurosci. 12, 15241533 (2009).
  6. Carlen, M. et al. Forebrain ependymal cells are Notch-dependent and generate neuroblasts and astrocytes after stroke. Nat. Neurosci. 12, 259267 (2009).
  7. Goings, G.E., Sahni, V. & Szele, F.G. Migration patterns of subventricular zone cells in adult mice change after cerebral cortex injury. Brain Res. 996, 213226 (2004).
  8. Jablonska, B. et al. Chordin-induced lineage plasticity of adult SVZ neuroblasts after demyelination. Nat. Neurosci. 13, 541550 (2010).
  9. Beckervordersandforth, R. et al. In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells. Cell Stem Cell 7, 744758 (2010).
  10. Pinto, L. et al. Prospective isolation of functionally distinct radial glial subtypes—lineage and transcriptome analysis. Mol. Cell Neurosci. 38, 1542 (2008).
  11. Weigmann, A., Corbeil, D., Hellwig, A. & Huttner, W.B. Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells. Proc. Natl Acad. Sci. USA 94, 1242512430 (1997).
  12. Mirzadeh, Z., Merkle, F.T., Soriano-Navarro, M., Garcia-Verdugo, J.M. & Alvarez-Buylla, A. Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell 3, 265278 (2008).
  13. Nolte, C. et al. GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue. Glia 33, 7286 (2001).
  14. Capela, A. & Temple, S. LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal. Neuron 35, 865875 (2002).
  15. Corti, S. et al. Isolation and characterization of murine neural stem/progenitor cells based on Prominin-1 expression. Exp. Neurol. 205, 547562 (2007).
  16. Kawaguchi, A. et al. Nestin-EGFP transgenic mice: visualization of the self-renewal and multipotency of CNS stem cells. Mol. Cell Neurosci. 17, 259273 (2001).
  17. Pastrana, E., Cheng, L.C. & Doetsch, F. Simultaneous prospective purification of adult subventricular zone neural stem cells and their progeny. Proc. Natl Acad. Sci. USA 106, 63876392 (2009).
  18. Rietze, R.L. et al. Purification of a pluripotent neural stem cell from the adult mouse brain. Nature 412, 736739 (2001).
  19. Azari, H. et al. Purification of immature neuronal cells from neural stem cell progeny. PLoS One 6, e20941 (2011).
  20. Barraud, P., Thompson, L., Kirik, D., Bjorklund, A. & Parmar, M. Isolation and characterization of neural precursor cells from the Sox1-GFP reporter mouse. Eur. J. Neurosci. 22, 15551569 (2005).
  21. Hack, M.A., Sugimori, M., Lundberg, C., Nakafuku, M. & Gotz, M. Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6. Mol. Cell Neurosci. 25, 664678 (2004).
  22. Gabay, L., Lowell, S., Rubin, L.L. & Anderson, D.J. Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron 40, 485499 (2003).
  23. Costa, M.R. et al. Continuous live imaging of adult neural stem cell division and lineage progression in vitro. Development 138, 10571068 (2011).
  24. Ortega, F. et al. An adherent cell culture of the mouse subependymal zone to study the behavior of adult neural stem cells on a single cell level. Nat. Protoc. 6, 18471857 (2011).
  25. Ciccolini, F., Mandl, C., Holzl-Wenig, G., Kehlenbach, A. & Hellwig, A. Prospective isolation of late development multipotent precursors whose migration is promoted by EGFR. Dev. Biol. 284, 112125 (2005).
  26. Colak, D. et al. Adult neurogenesis requires Smad4-mediated bone morphogenic protein signaling in stem cells. J. Neurosci. 28, 434446 (2008).
  27. Zhuo, L. et al. Live astrocytes visualized by green fluorescent protein in transgenic mice. Dev. Biol. 187, 3642 (1997).
  28. Malatesta, P., Hartfuss, E. & Gotz, M. Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage. Development 127, 52535263 (2000).
  29. Malatesta, P. et al. Neuronal or glial progeny: regional differences in radial glia fate. Neuron 37, 751764 (2003).
  30. Requardt, R.P. et al. Quality control of astrocyte-directed Cre transgenic mice: the benefits of a direct link between loss of gene expression and reporter activation. Glia 57, 680692 (2009).
  31. Shen, Q. et al. Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell 3, 289300 (2008).
  32. Tavazoie, M. et al. A specialized vascular niche for adult neural stem cells. Cell Stem Cell 3, 279288 (2008).
  33. Mirzadeh, Z., Doetsch, F., Sawamoto, K., Wichterle, H. & Alvarez-Buylla, A. The subventricular zone en-face: wholemount staining and ependymal flow. J. Vis. Exp. 39 Published online, doi:10.3791/1938 (2010).
  34. Alexander, C.M. et al. Separating stem cells by flow cytometry: reducing variability for solid tissues. Cell Stem Cell 5, 579583 (2009).

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Author information

  1. These authors contributed equally to this work.

    • Judith Fischer &
    • Ruth Beckervordersandforth

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

    • Pratibha Tripathi

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.

Competing financial interests

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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