Abstract
The sensory epithelia of the inner ear contain mechanosensitive hair cells that detect sound and head acceleration. This protocol details a 3D differentiation method to generate inner ear organoids containing sensory epithelia with hair cells. Human pluripotent stem cells are aggregated in low-binding 96-well plates and treated in chemically defined media with extracellular matrix to promote epithelialization. Small molecules and recombinant proteins are applied in a stepwise manner to recapitulate the morphogenic cues (BMP, TGF-β, FGF, and WNT) present during inner ear development in vivo. These treatments induce the sequential formation of nonneural ectoderm, otic-epibranchial progenitor domain, and otic placodes. The derived otic placodes then undergo self-guided morphogenesis to form otic vesicles, which eventually give rise to sensory epithelia containing hair cells and supporting cells, as well as neurons with synaptic formations to hair cells. This human stem cell–derived inner ear organoid system provides an ideal platform to study human inner ear development and disease in vitro.
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Acknowledgments
We would like to thank Jing Nie for the protocol optimization. This work was supported by a National Institutes of Health grant R01 DC015788 and a Department of Defense U.S. Army Medical Research and Materiel Command Congressionally Directed Medical Research Program grant W81XWH-18-1-0062 (to E.H.).
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Ueda, Y., Moore, S.T., Hashino, E. (2021). Directed Differentiation of Human Pluripotent Stem Cells into Inner Ear Organoids. In: Turksen, K. (eds) Embryonic Stem Cell Protocols . Methods in Molecular Biology, vol 2520. Humana, New York, NY. https://doi.org/10.1007/7651_2021_448
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DOI: https://doi.org/10.1007/7651_2021_448
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Online ISBN: 978-1-0716-2437-1
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