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Ontogeny-recapitulating generation and tissue integration of ES cell–derived Purkinje cells

Nature Neuroscience volume 13, pages 1171–1180 (2010)Cite this article

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Abstract

Purkinje cells are the sole output neurons of the cerebellar cortex and their dysfunction causes severe ataxia. We found that Purkinje cells could be robustly generated from mouse embryonic stem (ES) cells by recapitulating the self-inductive signaling microenvironments of the isthmic organizer. The cell-surface marker Neph3 enabled us to carry out timed prospective selection of Purkinje cell progenitors, which generated morphologically characteristic neurons with highly arborized dendrites that expressed mature Purkinje cell–specific markers such as the glutamate receptor subunit GluRδ2. Similar to mature Purkinje cells, these neurons also showed characteristic spontaneous and repeated action potentials and their postsynaptic excitatory potentials were generated exclusively through nonNMDA glutamate receptors. Fetal transplantation of precursors isolated by fluorescence-activated cell sorting showed orthotopic integration of the grafted neurons into the Purkinje cell layer with their axons extending to the deep cerebellar nuclei and dendrites receiving climbing and parallel fibers. This selective preparation of bona fide Purkinje cells should aid future investigation of this important neuron.

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Figure 1: Fgf2 and insulin cooperatively induce generation of midbrain-hindbrain identity in ES cells in SFEBq culture.
Figure 2: Sequential treatment of Fgf2 and a Shh inhibitor in the presence of insulin induces cerebellar plate precursors in SFEBq culture.
Figure 3: Generation of Purkinje cells in SFEBq/FIC culture in Transwell culture.
Figure 4: ES cell–derived neuroepithelial rosettes express cerebellar progenitor markers in SFEBq culture.
Figure 5: Neph3+ progenitors on day 13 in SFEBq/FIC culture efficiently generate Purkinje cells with morphological and functional characteristics.
Figure 6: Orthotopic integration of ES cell–derived Purkinje cells into the cerebellum.

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Acknowledgements

We thank H. Enomoto and M. Eiraku for comments, T. Wataya for help in tissue preparation and members of the Sasai laboratory for discussion and advice. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (Japan), the Kobe Cluster Project, the Leading Project (Y.S.) and the National Center of Neurology and Psychiatry (K.M.).

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Authors and Affiliations

  1. Organogenesis and Neurogenesis Group, RIKEN Center for Developmental Biology, Kobe, Japan

    Keiko Muguruma, Ayaka Nishiyama & Yoshiki Sasai

  2. Group for Neuronal Differentiation, KAN Institute, Kobe, Japan

    Yuichi Ono & Eri Mizuhara

  3. Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan

    Hiroyuki Miyawaki & Tomoo Hirano

  4. Laboratory of Functional Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan

    Seiji Hori & Akira Kakizuka

  5. Obata Research Unit, RIKEN Brain Science Institute, Wako, Japan

    Kunihiko Obata

  6. Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi, Japan

    Yuchio Yanagawa

  7. Japan Science and Technology Agency, CREST, Tokyo, Japan

    Yuchio Yanagawa

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  1. Keiko Muguruma
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Contributions

K.M. performed the experiments that comprise the main body of this work. A.N. assisted with cell culture and histology. Y.O. and E.M. contributed to the FACS analysis. H.M. and T.H. performed electrophysiological analysis. S.H. and A.K. contributed to the ataxia model mouse analysis. K.O. and Y.Y. provided GAD-GFP transgenic mice. Y.S. wrote the manuscript and designed the experiments with K.M.

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Correspondence to Yoshiki Sasai.

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Muguruma, K., Nishiyama, A., Ono, Y. et al. Ontogeny-recapitulating generation and tissue integration of ES cell–derived Purkinje cells. Nat Neurosci 13, 1171–1180 (2010). https://doi.org/10.1038/nn.2638

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