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Directed differentiation of telencephalic precursors from embryonic stem cells

Nature Neuroscience volume 8, pages 288–296 (2005)Cite this article

Abstract

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells (∼90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation (∼35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6+ cells yield up to 75% of Bf1+ cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1+ and/or Islet1/2+ cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.

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Figure 1: Efficient neural conversion of mouse ES cells in the SFEB culture with Dkk1 and LeftyA.
Figure 2: Selective biases do not have a major role in neural induction in SFEB.
Figure 3: Differentiation of Bf1+ cells in SFEB-treated ES cells and further enhancement by Dkk1 and LeftyA.
Figure 4: Differential control of dorsal and ventral telencephalic specification by soluble patterning signals.
Figure 5: Expression of ventral telencephalic markers by Wnt and Shh signals.

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Acknowledgements

We are grateful to members of the Sasai lab and to T. Era, S. Kuratani, R. Ladher, I. Matsuo, F. Matsuzaki, H. Niwa, T. Watabe, R. Shigemoto and S. Kaneko for invaluable comments and advice on this work; to S. Nishikawa, S.-i. Nishikawa and M. Takeichi for labeled E-cadherin antibody; to A. Smith for Sox1-GFP ES cells and to N. Sasai for advice on antibody production. K.W. is thankful to S. Iwamizu-Watanabe for discussion and constant encouragement. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (Y.S., Y.W.), the Kobe Cluster Project (Y.S.) and the Leading Project (Y.S.).

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Authors and Affiliations

  1. Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe, 650-0047, Japan

    Kiichi Watanabe, Daisuke Kamiya, Ayaka Nishiyama, Tomoko Katayama, Kenji Mizuseki & Yoshiki Sasai

  2. Department of Medical Embryology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan

    Kiichi Watanabe, Daisuke Kamiya & Yoshiki Sasai

  3. Department of Physiology, Graduate School of Medicine, Osaka City University, Osaka, 545-8585, Japan

    Satoshi Nozaki & Yasuyoshi Watanabe

  4. Department of Molecular and System Neurobiology, Graduate School of Medicine, University of Tokyo, Tokyo, 113-0033, Japan

    Hiroshi Kawasaki

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Supplementary information

Supplementary Fig. 1

Immunostaining analysis of SFEB-treated ES cell aggregates. (PDF 1190 kb)

Supplementary Fig. 2

Analysis of the effects of Wnt, Nodal and BMP antagonists. (PDF 650 kb)

Supplementary Fig. 3

Expression of primitive endodermal and mesodermal markers in SFEB. (PDF 1019 kb)

Supplementary Fig. 4

BrdU uptake and TUNEL analyses in SFEB/BMP-treated ES cells. (PDF 207 kb)

Supplementary Fig. 5

Expression of rostral-caudal markers in differentiating ES cells. (PDF 998 kb)

Supplementary Fig. 6

RT-PCR analysis and the role of E-cad function in SDIA-treated ES cells. (PDF 1086 kb)

Supplementary Fig. 7

Systematic induction of pallial and subpallial telencephalic tissues. (PDF 486 kb)

Supplementary Fig. 8

Expression of basal telencephalic markers in SFEB/Shh-induced cells and the embryonal brain. (PDF 1246 kb)

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Watanabe, K., Kamiya, D., Nishiyama, A. et al. Directed differentiation of telencephalic precursors from embryonic stem cells. Nat Neurosci 8, 288–296 (2005). https://doi.org/10.1038/nn1402

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