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Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex

Nature Protocols volume 6, pages 214–228 (2011)Cite this article

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Abstract

Instructing glial cells to generate neurons may prove to be a strategy to replace neurons that have degenerated. Here, we describe a robust protocol for the efficient in vitro conversion of postnatal astroglia from the mouse cerebral cortex into functional, synapse-forming neurons. This protocol involves two steps: (i) expansion of astroglial cells (7 d) and (ii) astroglia-to-neuron conversion induced by persistent and strong retroviral expression of Neurog2 (encoding neurogenin-2) or Mash1 (also referred to as achaete-scute complex homolog 1 or Ascl1) and/or distal-less homeobox 2 (Dlx2) for generation of glutamatergic or GABAergic neurons, respectively (7–21 d for different degrees of maturity). Our protocol of astroglia-to-neuron conversion by a single neurogenic transcription factor provides a stringent experimental system to study the specification of a selective neuronal subtype, thus offering an alternative to the use of embryonic or neural stem cells. Moreover, it can be a useful model for studies of lineage conversion from non-neuronal cells, with potential for brain regenerative medicine.

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Figure 1
Figure 2: Schematic structure of the recombinant retroviral vector of pCAG-Neurog2-IRES-DsRed.
Figure 3: Primary culture of adherent astroglial cells from the mouse postnatal cerebral cortex.
Figure 4: Postnatal cortical astroglia differentiate into βIII tubulin-positive neurons on forced expression of neurogenic fate determinants.
Figure 5: Postnatal cortical astroglia give rise to distinct types of neurons following forced expression of Neurog2 or Dlx2.

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Acknowledgements

This work was supported by grants from the Deutsche Forschungsgemeinschaft to B.B. and M.G. (BE 4182/1-3 and GO 640/9-1) and to T.S.-E. (SCHR 1142/1-1), and by the Bavarian State Ministry of Sciences, Research and the Arts (ForNeuroCell) to B.B. and M.G. We thank S. Bauer for virus production.

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Authors and Affiliations

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

    Christophe Heinrich, Sergio Gascón, Giacomo Masserdotti, Alexandra Lepier, Rodrigo Sanchez, Tatiana Simon-Ebert, Magdalena Götz & Benedikt Berninger

  2. Institute for Stem Cell Research, National Research Center for Environment and Health, Neuherberg, Germany

    Sergio Gascón, Timm Schroeder, Magdalena Götz & Benedikt Berninger

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  1. Christophe Heinrich
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Contributions

C.H. contributed to protocol design, generation and characterization of the astroglia-derived neurons and preparation of the manuscript; S.G. to the design of the transfection procedure and single-cell tracking by time-lapse video microscopy; G.M. to the characterization of the astroglia-derived neurons; A.L. and R.S. to viral vector design; T.S.-E. to astroglia culture and immunocytochemistry; and T.S. to single-cell tracking by time-lapse video microscopy. M.G. pioneered this protocol for astroglia-to-neuron conversion and contributed to the current protocol design and the manuscript; B.B. contributed to protocol design, electrophysiological characterization of the astroglia-derived neurons 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 Video 1

Direct visualisation of the astroglia-to-neuron metamorphosis. (AVI 8843 kb)

Temporal sequence of astroglia-to-neuron conversion by Neurog2 of a fate-mapped astroglia derived from GLAST::CreERT2/Z/EG mice following recombination in vivo between P2-P7. The movie starts with a GFP-fluorescence and bright field image at the beginning of the time-lapse experiment. The GFP-positive cell is marked with a red arrow throughout the sequence. The cell is subsequently followed using bright field images. At the point when DsRed fluorescence is detectable, a GFP fluorescence image is shown to demonstrate the co-expression of DsRed and GFP in the very same cell. Subsequently, DsRed fluorescence images (grey scale) monitor the transduced cell and its gradual metamorphosis into a neuron. At the end of the imaging sequence a GFP fluorescence image is shown to confirm that the neuron is indeed GFP reporter-positive. The time is indicated in each image. For details on the single-cell tracking method see Rieger et al. (2009).

References

Rieger, M. A., Hoppe, P. S., Smejkal, B. M., Eitelhuber, A. C. & Schroeder, T. Hematopoietic cytokines can instruct lineage choice. Science 325, 217–218 (2009).

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Heinrich, C., Gascón, S., Masserdotti, G. et al. Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex. Nat Protoc 6, 214–228 (2011). https://doi.org/10.1038/nprot.2010.188

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