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Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture

Nature volume 500, pages 217–221 (2013)Cite this article

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Abstract

The inner ear contains sensory epithelia that detect head movements, gravity and sound. It is unclear how to develop these sensory epithelia from pluripotent stem cells, a process that will be critical for modelling inner ear disorders or developing cell-based therapies for profound hearing loss and balance disorders1,2. So far, attempts to derive inner ear mechanosensitive hair cells and sensory neurons have resulted in inefficient or incomplete phenotypic conversion of stem cells into inner-ear-like cells3,4,5,6,7. A key insight lacking from these previous studies is the importance of the non-neural and preplacodal ectoderm, two critical precursors during inner ear development8,9,10,11. Here we report the stepwise differentiation of inner ear sensory epithelia from mouse embryonic stem cells (ESCs) in three-dimensional culture12,13. We show that by recapitulating in vivo development with precise temporal control of signalling pathways, ESC aggregates transform sequentially into non-neural, preplacodal and otic-placode-like epithelia. Notably, in a self-organized process that mimics normal development, vesicles containing prosensory cells emerge from the presumptive otic placodes and give rise to hair cells bearing stereocilia bundles and a kinocilium. Moreover, these stem-cell-derived hair cells exhibit functional properties of native mechanosensitive hair cells and form specialized synapses with sensory neurons that have also arisen from ESCs in the culture. Finally, we demonstrate how these vesicles are structurally and biochemically comparable to developing vestibular end organs. Our data thus establish a new in vitro model of inner ear differentiation that can be used to gain deeper insight into inner ear development and disorder.

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Figure 1: Non-neural and preplacodal ectoderm induction in three-dimensional culture.
Figure 2: Otic induction from the preplacodal epithelium in vitro.
Figure 3: Stem-cell-derived otic vesicles generate functional inner ear hair cells.
Figure 4: Stem-cell-derived sensory epithelia are comparable to immature vestibular end organs.

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Acknowledgements

The authors would like to thank G. Oxford for contributing unpublished data and discussion; E. Beans, G. Kamocka, K. Dunn and C. Miller for technical assistance; P. Dolle, R. Romand, J. Williams, J. Meyer, X. Zhang and T. Cummins for comments and discussion; E. Tobin, R. Meadows, J. Hamilton, S. Majumdar and G. Wagner for editorial assistance. This work was supported by National Institutes of Health (NIH) grants RC1DC010706, R21DC012617 and R01GM086544. 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). A.I.M. was supported by NIH R01MH52619 (awarded to A. Shekhar).

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Authors and Affiliations

  1. Medical Neuroscience Graduate Program, Indiana University School of Medicine, Indianapolis, 46202, Indiana, USA

    Karl R. Koehler & Eri Hashino

  2. Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, 46202, Indiana, USA

    Karl R. Koehler, Andrew M. Mikosz, Andrei I. Molosh, Dharmeshkumar Patel & Eri Hashino

  3. Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, 46202, Indiana, USA

    Karl R. Koehler, Andrew M. Mikosz, Dharmeshkumar Patel & Eri Hashino

  4. Department of Psychiatry, Indiana University School of Medicine, Indianapolis, 46202, Indiana, USA

    Andrei I. Molosh

Authors
  1. Karl R. Koehler
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Contributions

K.R.K. conceived and designed the study, performed experiments, analysed data, created the figures and wrote the manuscript. A.M.M. and D.P. performed experiments and analysed data. A.I.M. generated electrophysiological data. E.H. helped to design the study, provided financial support, monitored the experiments and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Karl R. Koehler or Eri Hashino.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Figures 1-16 and additional references. (PDF 20951 kb)

3D reconstruction of a vesicle lined with Myo7a+ Sox2+ hair cells and Sox2+ supporting cells

A series of images and an animated 3D reconstruction of the vesicle seen in Figure 3. (MOV 12719 kb)

Characteristics of sensory epithelia displayed in vesicles containing hair cells

A series of images highlighting (1) the F-actin+ banding pattern on the luminal surface of each hair cell vesicle and (2) the F-actin+ Myo7a+ stereocilia-like protrusions emanating from the apical surface of hair cells into the lumen. Additionally, a 3D reconstruction of the sensory epithelium rotates in order to provide multiple views of the protruding stereocilia at day 20. A second 3D reconstruction of an epithelium at day 24 is stained for F-actin and acetylated-α- Tubulin to visualize more mature stereocilia bundles and kinocilium, respectively. (MOV 28835 kb)

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Koehler, K., Mikosz, A., Molosh, A. et al. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013). https://doi.org/10.1038/nature12298

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