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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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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Y.S. and M.E. wrote the manuscript, and M.E. performed experiments and data analysis.
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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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DOI: https://doi.org/10.1038/nprot.2011.429
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