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

Nature Protocols volume 6, pages 214228 (2011) | Download Citation

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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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.

Author information

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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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Benedikt Berninger.

Supplementary information

Videos

  1. 1.

    Supplementary Video 1

    Direct visualisation of the astroglia-to-neuron metamorphosis.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).ReferencesRieger, 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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DOI

https://doi.org/10.1038/nprot.2010.188

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