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A ROCK inhibitor permits survival of dissociated human embryonic stem cells

Nature Biotechnology volume 25, pages 681–686 (2007)Cite this article

Abstract

Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor1,2, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from ∼1% to ∼27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture3 and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1+ cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.

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Figure 1: The ROCK inhibitor Y-27632 markedly increases the cloning efficiency of hES cells (KhES-1) without affecting their pluripotency.
Figure 2: Y-27632 directly enhances the cloning efficiency of hES cells (KhES-1).
Figure 3: Y-27632 prevents apoptosis and promotes survival of dissociated hES cells (KhES-1) in suspension culture.

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Acknowledgements

We are grateful to S. Narumiya and H. Bito for discussions and invaluable advice about ROCK inhibitors, to M. Hirose for kind advice and help in cell cycle analysis, to H. Niwa, H. Enomoto and N. Love for discussion and technical advice. Y.S. is thankful to Kenzo Sasai and Tetsuro Haraguchi, who passed away while this project was underway, for continuous encouragement. This work was supported by grants-in-aid from Ministry of Education, Culture, Sports, Science and Technology, the Kobe Cluster Project and the Leading Project (Y.S., S.N., J.B.T.). The hES cells (KhES-1, 2 and 3) were a gift from N. Nakatsuji and H. Suemori (Kyoto University) and the drug-selectable plasmid pCAGGS-Venus-Hygro was a gift from H. Niwa.

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Author notes

  1. Kiichi Watanabe

    Present address: Present address: Division of Biology 216-76, California Institute of Technology, Pasadena, California 91125, USA.,

Authors and Affiliations

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

    Kiichi Watanabe, Morio Ueno, Daisuke Kamiya, Ayaka Nishiyama, Michiru Matsumura, Takafumi Wataya, Keiko Muguruma & Yoshiki Sasai

  2. Stem Cell Biology Group, Center for Developmental Biology, RIKEN, Kobe, 650-0047, Japan

    Satomi Nishikawa & Shin-ichi Nishikawa

  3. Department of Ophthalmology, National Center for Geriatrics and Gerontology, Obu, 474-8511, Japan

    Morio Ueno

  4. Department of Neurosurgery and Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, 606-8507, Japan

    Takafumi Wataya & Jun B Takahashi

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Contributions

K.W. and Y.S. designed the project; Y.S. wrote the report; all authors performed experiments.

Corresponding author

Correspondence to Yoshiki Sasai.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Analysis of hES cells cultured in the presence of Y-27632 at low density (PDF 1174 kb)

Supplementary Fig. 2

Neural differentiation of hES cells (KhES-1) in suspension culture involving dissociation/reaggregation in the presence of Y-27632 (PDF 421 kb)

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Watanabe, K., Ueno, M., Kamiya, D. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25, 681–686 (2007). https://doi.org/10.1038/nbt1310

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