Advertisement

Astrocytes pp 485-498 | Cite as

Reprogramming of Postnatal Astroglia of the Mouse Neocortex into Functional, Synapse-Forming Neurons

  • Christophe Heinrich
  • Magdalena Götz
  • Benedikt BerningerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 814)

Abstract

Direct conversion of glia into neurons by cellular reprogramming represents a novel approach toward a cell-based therapy of neurodegenerative processes. Here we describe a protocol that allows for the direct and efficient in vitro reprogramming of mouse astroglia from the early postnatal neocortex by forced expression of single neurogenic fate determinants. By selective retrovirus-mediated expression of neurogenin-2 (Neurog2) on the one hand, or the mouse homologue of Distal-less Dlx2 or the mammalian homologue of achaete-schute-1 (Mash1) on the other, it is possible to drive postnatal astroglia in culture toward the genesis of fully functional, synapse-forming, glutamatergic, i.e., excitatory, and GABAergic, i.e., inhibitory, neurons, respectively.

Key words

Astrocyte Glutamatergic GABAergic Neurogenin2 Dlx2 Transcription factor 

Notes

Acknowledgments

This work was supported by grants from the Deutsche Forschungsgemeinschaft (BE 4182/1-3 and GO 640/9-1), the BMBF, EUTRACC, HELMA, and the Bavarian State Ministry of Sciences, Research and the Arts (ForNeuroCell). We wish to thank Tatiana Simon-Ebert for her excellent technical help in optimizing the protocol and Dr. Alex Lepier and Simone Bauer for virus production.

References

  1. 1.
    Nicholas, C. R., and Kriegstein, A. R. (2010) Regenerative medicine: Cell reprogramming gets direct, Nature 463, 1031–1032.PubMedCrossRefGoogle Scholar
  2. 2.
    Robel, S., Berninger, B., and Goetz, M. (2011) The stem cell potential of glia: lessons from reactive gliosis Nature Reviews Neuroscience In press.Google Scholar
  3. 3.
    Malatesta, P., Hartfuss, E., and Gotz, M. (2000) Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage, Development 127, 5253–5263.PubMedGoogle Scholar
  4. 4.
    Noctor, S. C., Flint, A. C., Weissman, T. A., Dammerman, R. S., and Kriegstein, A. R. (2001) Neurons derived from radial glial cells establish radial units in neocortex, Nature 409, 714–720.PubMedCrossRefGoogle Scholar
  5. 5.
    Miyata, T., Kawaguchi, A., Okano, H., and Ogawa, M. (2001) Asymmetric inheritance of radial glial fibers by cortical neurons, Neuron 31, 727–741.PubMedCrossRefGoogle Scholar
  6. 6.
    Beckervordersandforth, R., Tripathi, P., Ninkovic, J., Bayam, E., Lepier, A., Stempfhuber, B., Kirchhoff, F., Hirrlinger, J., Haslinger, A., Lie, D. C., Beckers, J., Yoder, B., Irmler, M., and Gotz, M. (2010) In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells, Cell Stem Cell 7, 744–758.PubMedCrossRefGoogle Scholar
  7. 7.
    Kriegstein, A., and Alvarez-Buylla, A. (2009) The glial nature of embryonic and adult neural stem cells, Annu Rev Neurosci 32, 149–184.PubMedCrossRefGoogle Scholar
  8. 8.
    Berninger, B., Costa, M. R., Koch, U., Schroeder, T., Sutor, B., Grothe, B., and Gotz, M. (2007) Functional properties of neurons derived from in vitro reprogrammed postnatal astroglia, J Neurosci 27, 8654–8664.PubMedCrossRefGoogle Scholar
  9. 9.
    Blum, R., Heinrich, C., Sanchez, R., Lepier, A., Gundelfinger, E. D., Berninger, B., and Gotz, M. (2010) Neuronal Network Formation from Reprogrammed Early Postnatal Rat Cortical Glial Cells, Cereb Cortex [Epub ahead of print].Google Scholar
  10. 10.
    Heinrich, C., Blum, R., Gascon, S., Masserdotti, G., Tripathi, P., Sanchez, R., Tiedt, S., Schroeder, T., Gotz, M., and Berninger, B. (2010) Directing astroglia from the cerebral cortex into subtype specific functional neurons, PLoS Biol 8, e1000373.PubMedCrossRefGoogle Scholar
  11. 11.
    Heins, N., Malatesta, P., Cecconi, F., Nakafuku, M., Tucker, K. L., Hack, M. A., Chapouton, P., Barde, Y. A., and Gotz, M. (2002) Glial cells generate neurons: the role of the transcription factor Pax6, Nat Neurosci 5, 308–315.PubMedCrossRefGoogle Scholar
  12. 12.
    Tashiro, A., Zhao, C., and Gage, F. H. (2006) Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo, Nat Protoc 1, 3049–3055.PubMedCrossRefGoogle Scholar
  13. 13.
    Ory, D. S., Neugeboren, B. A., and Mulligan, R. C. (1996) A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes, Proc Natl Acad Sci U S A 93, 11400–11406.PubMedCrossRefGoogle Scholar
  14. 14.
    Hong, E. J., McCord, A. E., and Greenberg, M. E. (2008) A biological function for the neuronal activity-dependent component of Bdnf transcription in the development of cortical inhibition, Neuron 60, 610–624.PubMedCrossRefGoogle Scholar
  15. 15.
    Berninger, B., Guillemot, F., and Gotz, M. (2007) Directing neurotransmitter identity of neurones derived from expanded adult neural stem cells, Eur J Neurosci 25, 2581–2590.PubMedCrossRefGoogle Scholar
  16. 16.
    Nolte, C., Matyash, M., Pivneva, T., Schipke, C. G., Ohlemeyer, C., Hanisch, U. K., Kirchhoff, F., and Kettenmann, H. (2001) GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue, Glia 33, 72–86.PubMedCrossRefGoogle Scholar
  17. 17.
    Mori, T., Tanaka, K., Buffo, A., Wurst, W., Kuhn, R., and Gotz, M. (2006) Inducible gene deletion in astroglia and radial glia--a valuable tool for functional and lineage analysis, Glia 54, 21–34.PubMedCrossRefGoogle Scholar
  18. 18.
    Buffo, A., Rite, I., Tripathi, P., Lepier, A., Colak, D., Horn, A. P., Mori, T., and Gotz, M. (2008) Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain, Proc Natl Acad Sci U S A 105, 3581–3586.PubMedCrossRefGoogle Scholar
  19. 19.
    Brewer, G. J., and Cotman, C. W. (1989) Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen, Brain Res 494, 65–74.PubMedCrossRefGoogle Scholar
  20. 20.
    Bottenstein, J. E., and Sato, G. H. (1979) Growth of a rat neuroblastoma cell line in serum-free supplemented medium, Proc Natl Acad Sci U S A 76, 514–517.PubMedCrossRefGoogle Scholar
  21. 21.
    Heinrich, C., Gascon, S., Masserdotti, G., Lepier, A., Sanchez, R., Simon-Ebert, T., Schroeder, T., Gotz, M., and Berninger, B. (2011) Generation of subtype specific neurons from postnatal astroglia of the mouse cerebral cortex, Nature Protocols In press 6, 214–28.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Christophe Heinrich
    • 1
  • Magdalena Götz
    • 1
  • Benedikt Berninger
    • 1
    Email author
  1. 1.Department of Physiological Genomics, Institute of PhysiologyLudwig-Maximilians University MunichMunichGermany

Personalised recommendations