3D mouse embryonic stem cell culture for generating inner ear organoids

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This protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 96-well plates in medium containing extracellular matrix proteins to promote epithelialization. During the first 14 d, a series of precisely timed protein and small-molecule treatments sequentially induce epithelia that represent the mouse embryonic non-neural ectoderm, preplacodal ectoderm and otic vesicle epithelia. Ultimately, these tissues develop into cysts with a pseudostratified epithelium containing inner ear hair cells and supporting cells after 16–20 d. Concurrently, sensory-like neurons generate synapse-like structures with the derived hair cells. We have designated the stem cell–derived epithelia harboring hair cells, supporting cells and sensory-like neurons as inner ear organoids. This method provides a reproducible and scalable means to generate inner ear sensory tissue in vitro.

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Figure 1: Overview of inner ear induction protocol.
Figure 2: Development of the definitive ectoderm epithelium.
Figure 3: Morphology changes after BMP/SB-FGF/LDN treatment.
Figure 4: Aggregate organization on day 8 of differentiation.
Figure 5: Cellular rearrangement between days 8 and 12 of differentiation.
Figure 6: Hair cell induction and organoid types.
Figure 7: Inner ear organoid dissection for electrophysiological analysis.


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We thank A. Mikosz, D. Patel and S. Majumdar for their technical assistance and comments on the manuscript. This work was supported by US National Institutes of Health (NIH) grants RC1DC010706, R21DC012617, and R01DC013294 and an Indiana University School of Medicine Biomedical Research Grant (to E.H.). K.R.K. was supported by a Paul and Carole Stark Neurosciences Fellowship and an Indiana Clinical and Translational Science Institute Predoctoral Fellowship (NIH TL1RR025759).

Author information

K.R.K. designed the protocol, made figures and wrote the manuscript. E.H. edited the manuscript.

Correspondence to Karl R Koehler or Eri Hashino.

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Competing interests

K.R.K. and E.H. have disclosed these findings to the Indiana University Research and Technology Corporation, which has filed a patent to ensure broad use of the methods for studying inner-ear development and disease mechanisms. All protocols and methods remain freely available for academic and nonprofit research in perpetuity.

Integrated supplementary information

Supplementary Figure 1 Aggregate imaging chamber.

a, Typical imaging chamber constructed from two cover glasses and a silicone isolator. One aggregate (sample) is loaded in the chamber. Note the air bubble that forms during the loading process. b, Projection drawing of an aggregate imaging chamber. Note that this chamber design is an example and can be easily customized using different sized cover glasses or silicone isolators.

Supplementary information

Supplementary Figure 1

Aggregate imaging chamber. (PDF 1922 kb)

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Koehler, K., Hashino, E. 3D mouse embryonic stem cell culture for generating inner ear organoids. Nat Protoc 9, 1229–1244 (2014) doi:10.1038/nprot.2014.100

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