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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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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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