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Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks

Nature Protocols volume 7, pages 1836–1846 (2012)Cite this article

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Abstract

Efficient derivation of human cerebral neocortical neural stem cells (NSCs) and functional neurons from pluripotent stem cells (PSCs) facilitates functional studies of human cerebral cortex development, disease modeling and drug discovery. Here we provide a detailed protocol for directing the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to all classes of cortical projection neurons. We demonstrate an 80-d, three-stage process that recapitulates cortical development, in which human PSCs (hPSCs) first differentiate to cortical stem and progenitor cells that then generate cortical projection neurons in a stereotypical temporal order before maturing to actively fire action potentials, undergo synaptogenesis and form neural circuits in vitro. Methods to characterize cortical neuron identity and synapse formation are described.

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Figure 1: Morphological changes in PSC cultures during neural induction.
Figure 2: Confocal microscopy images of human PSC-derived neocortical cortical stem/progenitor cells.
Figure 4: Example of single-neuron electrophysiological properties at different stages of terminal differentiation.
Figure 5: Physical synapses visualized by super-resolution microscopy.
Figure 3: Confocal microscopy images of immunofluorescence staining for different classes of excitatory neurons and astrocytes derived from human PSCs.

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References

  1. Lindvall, O. & Kokaia, Z. Stem cells in human neurodegenerative disorders—time for clinical translation? J. Clin. Invest. 120, 29–40 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    CAS  PubMed  Google Scholar 

  4. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Park, I.H. et al. Disease-specific induced pluripotent stem cells. Cell 134, 877–886 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ebert, A.D. et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457, 277–280 (2009).

    Article  CAS  PubMed  Google Scholar 

  7. Lee, G. et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402–406 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Yagi, T. et al. Modeling familial Alzheimer's disease with induced pluripotent stem cells. Hum. Mol. Genet. 20, 4530–4539 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Shi, Y. et al. A human stem cell model of early Alzheimer's disease pathology in Down's syndrome. Sci. Transl. Med. 4, 124ra29 (2012).

    PubMed  PubMed Central  Google Scholar 

  10. Israel, M.A. et al. Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells. Nature 482, 216–220 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Devine, M.J. et al. Parkinson's disease induced pluripotent stem cells with triplication of the α-synuclein locus. Nat. Commun. 2, 440 (2011).

    Article  PubMed  Google Scholar 

  12. Shi, Y., Kirwan, P., Smith, J., Robinson, H.P. & Livesey, F.J. Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat. Neurosci. 15, 477–486 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Rakic, P. Evolution of the neocortex: a perspective from developmental biology. Nat. Rev. Neurosci. 10, 724–735 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hill, R.S. & Walsh, C.A. Molecular insights into human brain evolution. Nature 437, 64–67 (2005).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Eiraku, M. 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).

    Article  CAS  PubMed  Google Scholar 

  18. Au, E. & Fishell, G. Cortex shatters the glass ceiling. Cell Stem Cell 3, 472–474 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  21. Siegenthaler, J.A. et al. Retinoic acid from the meninges regulates cortical neuron generation. Cell 139, 597–609 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kim, D.S. et al. Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity. Stem Cell Rev. 6, 270–281 (2010).

    Article  CAS  Google Scholar 

  23. Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271–278 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Marchetto, M.C. et al. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143, 527–539 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Brennand, K.J. et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature 473, 221–225 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank all members of the Livesey group for feedback on the practical details of the protocol reported here. Y.S. was supported by a Biotechnology and Biological Sciences Research Council Dorothy Hodgkin Studentship. P.K. was supported by the University of Cambridge/Wellcome Trust PhD Programme in Developmental Biology. This research benefits from core support to the Gurdon Institute from the Wellcome Trust and Cancer Research UK and grants to F.J.L. from the Wellcome Trust and Alzheimer's Research UK.

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  1. Gurdon Institute, University of Cambridge, Cambridge, UK

    Yichen Shi, Peter Kirwan & Frederick J Livesey

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  1. Yichen Shi
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All authors contributed to the writing, editing and testing of this protocol.

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Correspondence to Frederick J Livesey.

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Shi, Y., Kirwan, P. & Livesey, F. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc 7, 1836–1846 (2012). https://doi.org/10.1038/nprot.2012.116

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