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Generation of cortical neurons from mouse embryonic stem cells

Nature Protocols volume 4, pages 1454–1463 (2009)Cite this article

Abstract

Embryonic stem cells (ESCs) constitute a tool of great potential in neurobiology, enabling the directed differentiation of specific neural cell types. We have shown recently that neurons of the cerebral cortex can be generated from mouse ESCs cultured in a chemically defined medium that contains no morphogen, but in the presence of the sonic hedgehog inhibitor cyclopamine. Corticogenesis from ESCs recapitulates the most important steps of cortical development, leading to the generation of multipotent cortical progenitors that sequentially produce cortical pyramidal neurons displaying distinct layer-specific identities. The protocol provides a most reductionist cellular model to tackle the complex mechanisms of cortical development and function, thereby opening new perspectives for the modeling of cortical diseases and the design of novel neurological treatments, while offering an alternative to animal use. In this protocol, we describe a method by which millions of cortical neurons can be generated in 2–3 weeks, starting from a single frozen vial of ESCs.

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Figure 1: Timetable of corticogenesis from ESCs.
Figure 2: ESCs and ESC-derived neural progenitors and neurons in the DDM–cyclopamine protocol.

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Acknowledgements

We are grateful to other members of the lab and IRIBHM for their help and advice. This work was funded by the Belgian FNRS/FRSM, the Belgian Queen Elizabeth Medical Foundation, the Simone et Pierre Clerdent Foundation, the Action de Recherches Concertées (ARC) Programs, the Interuniversity Attraction Poles Program (IUAP), Belgian State, Federal Office, the Walloon Region Excellence Program CIBLES (to P.V.) and the EU Marie Curie Fellowship Program (to T.B. and P.V.). P.V. is a Senior Research Associate of the FNRS and N.G. and T.B. were funded as Research Fellows of the FNRS. T.B. is a Fellow of the EU Marie Curie Program.

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Author notes

  1. Nicolas Gaspard and Tristan Bouschet: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Interdisciplinary Research on Human and Molecular Biology (IRIBHM), Université Libre de Bruxelles (ULB), Brussels, Belgium

    Nicolas Gaspard, Tristan Bouschet, Adèle Herpoel, Gilles Naeije, Jelle van den Ameele & Pierre Vanderhaeghen

  2. Department of Neurology, Université Libre de Bruxelles (ULB), Brussels, Belgium

    Nicolas Gaspard

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  1. Nicolas Gaspard
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Contributions

N.G., T.B., A.H., G.N. and J.v.d.A. performed all experiments. All authors contributed to the design and analysis of experiment. N.G., T.B. and P.V. wrote the paper.

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Correspondence to Pierre Vanderhaeghen.

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Gaspard, N., Bouschet, T., Herpoel, A. et al. Generation of cortical neurons from mouse embryonic stem cells. Nat Protoc 4, 1454–1463 (2009). https://doi.org/10.1038/nprot.2009.157

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