Protocol | Published:

Generation of cortical neurons from mouse embryonic stem cells

Nature Protocols volume 4, pages 14541463 (2009) | Download Citation

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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References

  1. 1.

    & Induction and dorsoventral patterning of the telencephalon. Neuron 28, 641–651 (2000).

  2. 2.

    & Neocortical Development (Raven Press, New York, 1991).

  3. 3.

    et al. Beyond laminar fate: toward a molecular classification of cortical projection/pyramidal neurons. Dev. Neurosci. 25, 139–151 (2003).

  4. 4.

    , , & Neuronal subtype specification in the cerebral cortex. Nat. Rev. Neurosci. 8, 427–437 (2007).

  5. 5.

    , , , & The determination of projection neuron identity in the developing cerebral cortex. Curr. Opin. Neurobiol. 18, 28–35 (2008).

  6. 6.

    , & The incredible elastic brain: how neural stem cells expand our minds. Neuron 60, 420–429 (2008).

  7. 7.

    , & Neural stem and progenitor cells in cortical development. Novartis. Found. Symp. 288, 59–73 (2007).

  8. 8.

    & Cortical development: the art of generating cell diversity. Development 132, 3327–3332 (2005); discussion 73–78, 96–98.

  9. 9.

    & Molecular mechanisms underlying cell fate specification in the developing telencephalon. Curr. Opin. Neurobiol. 12, 26–34 (2002).

  10. 10.

    et al. An intrinsic mechanism of corticogenesis from embryonic stem cells. Nature 455, 351–357 (2008).

  11. 11.

    , & Specific effects of nerve growth factor on the differentiation pattern of mouse embryonic stem cells in vitro . Biomed. Biochim. Acta 47, 965–973 (1988).

  12. 12.

    , , , & Embryonic stem cells express neuronal properties in vitro . Dev. Biol. 168, 342–357 (1995).

  13. 13.

    , , , & Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro . Mech. Dev. 59, 89–102 (1996).

  14. 14.

    et al. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell Sci. 108 (Part 10): 3181–3188 (1995).

  15. 15.

    et al. Neural conversion of ES cells by an inductive activity on human amniotic membrane matrix. Proc. Natl. Acad. Sci. USA 103, 9554–9559 (2006).

  16. 16.

    et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31–40 (2000).

  17. 17.

    , , & Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397 (2002).

  18. 18.

    et al. Directed differentiation of embryonic stem cells into dorsal interneurons. FASEB J. 19, 252–254 (2005).

  19. 19.

    & Differentiation of ES cells into cerebellar neurons. Proc. Natl. Acad. Sci. USA 104, 2997–3002 (2007).

  20. 20.

    et al. Generation of cerebellar neuron precursors from embryonic stem cells. Dev. Biol. 290, 287–296 (2006).

  21. 21.

    , , , & Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat. Biotechnol. 18, 675–679 (2000).

  22. 22.

    , , & Neural differentiation of mouse embryonic stem cells in chemically defined medium. Brain Res. Bull. 68, 62–75 (2005).

  23. 23.

    et al. Directed differentiation of telencephalic precursors from embryonic stem cells. Nat. Neurosci. 8, 288–296 (2005).

  24. 24.

    et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3, 519–532 (2008).

  25. 25.

    et al. Differentiation of mouse embryonic stem cells into a defined neuronal lineage. Nat. Neurosci. 7, 1003–1009 (2004).

  26. 26.

    & Neural differentiation from embryonic stem cells: which way? Stem Cells Dev. 13, 372–381 (2004).

  27. 27.

    , , , & Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21, 183–186 (2003).

  28. 28.

    et al. The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. Nat. Neurosci. 9, 743–751 (2006).

  29. 29.

    & Cell-cycle control and cortical development. Nat. Rev. Neurosci. 8, 438–450 (2007).

  30. 30.

    & A stem cell niche for intermediate progenitor cells of the embryonic cortex. Cereb. Cortex 19 (Suppl 1): i70–i77 (2009).

  31. 31.

    , , & Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells. Nat. Protoc. 2, 1034–1043 (2007).

  32. 32.

    et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3, 519–532 (2008).

  33. 33.

    & Proposal of a model of mammalian neural induction. Dev. Biol. 308, 247–256 (2007).

  34. 34.

    , , & Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003).

  35. 35.

    , , & Inhibition of hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev. 16, 2743–2748 (2002).

  36. 36.

    et al. Directed differentiation of telencephalic precursors from embryonic stem cells. Nat. Neurosci. 8, 288–296 (2005).

  37. 37.

    , , & Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576 (1993).

  38. 38.

    , , & Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev. Biol. 275, 124–142 (2004).

  39. 39.

    et al. The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. Nat. Neurosci. 9, 743–751 (2006).

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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.

Author information

Author notes

    • Nicolas Gaspard
    •  & Tristan Bouschet

    These authors contributed equally to this work.

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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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.

Corresponding author

Correspondence to Pierre Vanderhaeghen.

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DOI

https://doi.org/10.1038/nprot.2009.157

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