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Using an adherent cell culture of the mouse subependymal zone to study the behavior of adult neural stem cells on a single-cell level

Nature Protocols volume 6, pages 1847–1859 (2011)Cite this article

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Abstract

A comprehensive understanding of the cell biology of adult neural stem cells (aNSCs) requires direct observation of aNSC division and lineage progression in the absence of niche-dependent signals. Here we describe a culture preparation of the adult mouse subependymal zone (SEZ), which allows for continuous single-cell tracking of aNSC behavior. The protocol involves the isolation (∼3 h) and culture of cells from the adult SEZ at low density in the absence of mitogenic growth factors in chemically defined medium and subsequent live imaging using time-lapse video microscopy (5–7 d); these steps are followed by postimaging immunocytochemistry to identify progeny (∼7 h). This protocol enables the observation of the progression from slow-dividing aNSCs of radial/astroglial identity up to the neuroblast stage, involving asymmetric and symmetric cell divisions of distinct fast-dividing precursors. This culture provides an experimental system for studying instructive or permissive effects of signal molecules on aNSC modes of cell division and lineage progression.

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Figure 1: Schematic diagram illustrating the major steps of the protocol.
Figure 2: Preparation of the adult SEZ.
Figure 3: Prototypical lineage trees observed by single-cell tracking of SEZ cultures.
Figure 4: Clonal analysis using retroviral-mediated transduction.
Figure 5: Examples of asymmetric lineage trees.

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Acknowledgements

This work was supported by grants from the Deutsche Forschungsgemeinschaft to B.B. and M.G. (BE 4182/2-2 and GO 640/9-2) and to T.S. (SCHR 1142/1-2), and from the Bundesministerium für Bildung und Forschung (NewNeurons) and the Bavarian State Ministry of Sciences, Research and the Arts (ForNeuroCell) to B.B. and M.G.

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Author notes

  1. Felipe Ortega and Marcos R Costa: These authors contributed equally to this work.

Authors and Affiliations

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

    Felipe Ortega, Tatiana Simon-Ebert, Magdalena Götz & Benedikt Berninger

  2. Brain Institute, Universidade Federal do Rio Grande do Norte, Natal, Brazil

    Marcos R Costa

  3. Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany

    Timm Schroeder, Magdalena Götz & Benedikt Berninger

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  1. Felipe Ortega
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Contributions

F.O. and M.R.C. contributed to protocol design, experimental analysis of the culture preparation by single-cell tracking, postimaging ICC and clonal analysis, and the preparation of the manuscript; T.S.-E. contributed to the culture preparation; T.S. contributed to single-cell tracking by time-lapse video microscopy; M.G. contributed to protocol design and preparation of the manuscript; B.B. developed the culture preparation and contributed to protocol design, experimental analysis and preparation of the manuscript.

Corresponding author

Correspondence to Benedikt Berninger.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Summary of lineage trees derived from a single live imaging experiment. Each lineage tree was reconstructed from phase contrast image sequences using TTT. Shown are all lineage trees derived from cells which underwent cell division. After imaging, the identity of each cell was determined by post-imaging immunocytochemistry for βIII tubulin (neuronal) and GFAP (astroglial). N, neuron; G, astroglia; ?, marker negative; X, cell death. (PDF 449 kb)

Supplementary Video 1

Tracking options in TTT software. The video shows the three different outcomes labelled (1) cell division, (2) apoptosis (including any kind of cell death) and (3) cell lost (due to formation of cell aggregates as shown in the video or exit from field of view by the tracked cells), as assigned by the investigator during single cell tracking with the support by the TTT software. (AVI 43101 kb)

Supplementary Video 2

Adult neural stem cell lineage tree. The video shows the lineage progression of the aNSC (black arrow) depicted in Fig. 5a,c. The left panel shows the sequence of phase contrast images and the right panel the corresponding development of the lineage trees. Note the generation of both neuronal and astroglial progeny, as indicated by the final image following post-imaging immunocytochemistry. (AVI 89373 kb)

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Ortega, F., Costa, M., Simon-Ebert, T. et al. Using 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, 1847–1859 (2011). https://doi.org/10.1038/nprot.2011.404

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