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Stepwise differentiation of pluripotent stem cells into retinal cells

Nature Protocols volume 4, pages 811–824 (2009)Cite this article

Abstract

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. They can maintain an undifferentiated state indefinitely and can differentiate into derivatives of all three germ layers, namely ectoderm, endoderm and mesoderm. Although much progress has been made in the propagation and differentiation of ES cells, induction of photoreceptors has generally required coculture with or transplantation into developing retinal tissue. Here, we describe a protocol for generating retinal cells from ES cells by stepwise treatment with defined factors. This method preferentially induces photoreceptor and retinal pigment epithelium (RPE) cells from mouse and human ES cells. In our protocol, differentiation of RPE and photoreceptors from mouse ES cells requires 28 d and the differentiation of human ES cells into mature RPE and photoreceptors requires 120 and 150 d, respectively. This differentiation system and the resulting pluripotent stem cell-derived retinal cells will facilitate the development of transplantation therapies for retinal diseases, drug testing and in vitro disease modeling. It will also improve our understanding of the development of the central nervous system, especially the eye.

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Figure 1: Localization of neural stem/progenitor cells in adult eye tissue.
Figure 2: Multistep commitment in the development of retinal cells.
Figure 3: Schematic diagram of mouse and human ES cell differentiation into retinal cells.
Figure 4: Undifferentiated mouse ES cells, formed aggregates and FACS analysis.
Figure 5: Morphology of differentiated pelleted mouse ES cells after sorting.
Figure 6: Immunocytochemical analyses of the differentiated cells from mouse ES cells.
Figure 7: Morphology of undifferentiated human ES cells and detachment of ES cell colonies.
Figure 8: Morphology of floating aggregates formed from human ES cells.
Figure 9: Morphology of human ES cell aggregates in adherent culture.
Figure 10: Morphology of RPE cells differentiated from human ES cells.
Figure 11: RPE derived from human ES cells.
Figure 12: Morphology of putative photoreceptors differentiated from human ES cells.
Figure 13: Photoreceptors derived from human ES cells.

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Acknowledgements

We thank H. Suemori and N. Nakatsuji (Kyoto University) for providing the human ES cell line; K. Watanabe, M. Ueno and N. Osakada for valuable comments; and members of the Takahashi laboratory, the Sasai laboratory and the Akaike laboratory for helpful discussions and technical assistance. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, and the Leading Project (M.T. and Y.S.). This study was also supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Mochida Memorial Foundation for Medical and Pharmaceutical Research (F.O.).

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Authors and Affiliations

  1. Laboratory for Retinal Regeneration, Center for Developmental Biology, RIKEN, Kobe, Japan

    Fumitaka Osakada, Hanako Ikeda & Masayo Takahashi

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

    Fumitaka Osakada, Hanako Ikeda & Yoshiki Sasai

  3. Shiga Medical Center for Adults, Shiga, Japan

    Hanako Ikeda

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  1. Fumitaka Osakada
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Correspondence to Fumitaka Osakada.

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Osakada, F., Ikeda, H., Sasai, Y. et al. Stepwise differentiation of pluripotent stem cells into retinal cells. Nat Protoc 4, 811–824 (2009). https://doi.org/10.1038/nprot.2009.51

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