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Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues

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Abstract

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9–10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

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Figure 1: Schematic diagram of SFEBq methods for retinal and cortical tissue differentiation from mouse ESCs.
Figure 2: SFEBq culture of mouse ESCs.
Figure 3: Self-organizing optic cup formation in 3D ESC culture.
Figure 4: Generation of stratified retina from the isolated retinal epithelium.
Figure 5: Induction of polarized cortical tissues from mouse ESCs.

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Acknowledgements

We are grateful to M. Tanaka and M. Kawada for critical reading. The Sox1∷GFP ES cell line was a kind gift from A. Smith (University of Cambridge). This work was supported by grants-in-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) (Y.S., M.E.), the Knowledge Cluster Initiative at Kobe and the Leading Project for Realization of Regenerative Medicine (Y.S.).

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Authors and Affiliations

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

    Mototsugu Eiraku & Yoshiki Sasai

  2. Unit for Four-Dimensional Tissue Analysis, RIKEN Center for Developmental Biology, Kobe, Japan

    Mototsugu Eiraku & Yoshiki Sasai

Authors
  1. Mototsugu Eiraku
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  2. Yoshiki Sasai
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Contributions

Y.S. and M.E. wrote the manuscript, and M.E. performed experiments and data analysis.

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Correspondence to Mototsugu Eiraku or Yoshiki Sasai.

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The authors declare no competing financial interests.

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Eiraku, M., Sasai, Y. Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues. Nat Protoc 7, 69–79 (2012). https://doi.org/10.1038/nprot.2011.429

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