Abstract
The production of induced neuronal (iN) cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells by the forced expression of proneural transcription factors is rapid, efficient and reproducible. The ability to generate large numbers of human neurons in such a robust manner enables large-scale studies of human neural differentiation and neuropsychiatric diseases. Surprisingly, similar transcription factor–based approaches for converting mouse ESCs into iN cells have been challenging, primarily because of low cell survival. Here, we provide a detailed approach for the efficient and reproducible generation of functional iN cells from mouse ESC cultures by the genetically induced expression of neurogenin-2. The resulting iN cells display mature pre- and postsynaptic specializations and form synaptic networks. Our method provides the basis for studying neuronal development and enables the direct comparison of cellular phenotypes in mouse and human neurons generated in an equivalent way. The procedure requires 14 d and can be carried out by users with expertise in stem cell culture.
Key points
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Following lentiviral infection of mouse embryonic stem cells with FUW-M2rtTA and Tet-O-FUW-NGN2-T2A-PURO, neurons are induced with doxycycline and selected with puromycin. The selected cells are co-cultured with mouse primary glia, eliminating the clustering typical of mature mouse NGN2-induced neuronal cells.
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After 2 weeks of co-culturing, homogeneous cultures of induced neuronal cells exhibit functional neuronal properties, including the ability to form functional synapses that integrate into spontaneously active networks.
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Data availability
Source data for the figures in this study are available at https://doi.org/10.5281/zenodo.7625605.
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Acknowledgements
We thank C. E. Ang, B. Zhou and J. Janas for discussion on the design of, and useful comments on, the experiments and the rest of the Wernig lab for helpful discussion. The project described was supported by U19MH104172 from the National Institutes of Health (NIH).
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Y.L. and M.W. conceived the project. Y.L. and M.W. designed and planned the experiments. Y.L. executed the experiments. J.W. contributed to the electrophysiology assay. T.C.S. and M.W. provided critical advice. Y.L. and M.W. analyzed the data and wrote the manuscript.
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Nature Protocols thanks Brady Maher and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Zijian, Z. et al. Nat. Cell Biol. 23, 198–208 (2021): https://doi.org/10.1038/s41556-020-00625-2
ChangHui, P. et al. Proc. Natl. Acad. Sci. USA 118, e2025598118 (2021): https://doi.org/10.1073/pnas.2025598118
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Liu, Y., Wang, J., Südhof, T.C. et al. Efficient generation of functional neurons from mouse embryonic stem cells via neurogenin-2 expression. Nat Protoc 18, 2954–2974 (2023). https://doi.org/10.1038/s41596-023-00863-2
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DOI: https://doi.org/10.1038/s41596-023-00863-2
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