Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells

Abstract

The derivation of human inner ear tissue from pluripotent stem cells would enable in vitro screening of drug candidates for the treatment of hearing and balance dysfunction and may provide a source of cells for cell-based therapies of the inner ear. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and WNT signaling to generate multiple otic-vesicle-like structures from a single stem-cell aggregate. Over 2 months, the vesicles develop into inner ear organoids with sensory epithelia that are innervated by sensory neurons. Additionally, using CRISPR–Cas9, we generate an ATOH1-2A-eGFP cell line to detect hair cell induction and demonstrate that derived hair cells exhibit electrophysiological properties similar to those of native sensory hair cells. Our culture system should facilitate the study of human inner ear development and research on therapies for diseases of the inner ear.

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Figure 1: Step-wise induction of otic-placode-like epithelia.
Figure 2: WNT signaling activation initiates self-organization and maturation of inner ear organoids containing vestibular-like hair cells.
Figure 3: Development of an ATOH1 fluorescent reporter hESC line for tracking hair cell induction.
Figure 4: hESC-derived hair cells have similar electrophysiological properties as native hair cells and form synapse-like contacts with sensory neurons.

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Acknowledgements

This work was supported by National Institute of Health (NIH) grants R01 DC013294 (E.H. and J.R.H.) and R03 DC015624 (K.R.K.), Action on Hearing Loss International Project Grant (E.H.), and an Indiana Clinical and Translational Sciences Institute Core Grant (NIH UL1 TR001108; K.R.K.). This work was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR020128-01 from the National Center for Research Resources, NIH. The authors would like to thank A. Mikosz, P.-C. Tang, R. Nelson, S. Winfree, and M. Kamocka for their comments and technical assistance, and J. Bartles (Northwestern University) for the espin antibody.

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Authors

Contributions

K.R.K. conceived, designed, and led the study, performed experiments, analyzed data, and drafted the manuscript with input from all authors. J.N. generated the ATOH1-2A-eGFP cell line, performed experiments, and wrote the manuscript. E.L.-M. performed experiments, data analysis and wrote the manuscript. X.-P.L. performed electrophysiology experiments and wrote the manuscript. J.L. performed experiments and data analysis. J.R.H. designed and analyzed electrophysiology experiments and wrote the manuscript. E.H. designed and oversaw the study and wrote the manuscript.

Corresponding authors

Correspondence to Karl R Koehler or Eri Hashino.

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Competing interests

K.R.K. and E.H., with the Indiana University Research and Technology Corporation, have applied for a patent on the cell culture method described in this manuscript.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16, Supplementary Tables 1–5 and Supplementary Data (PDF 22944 kb)

Supplementary Video 1

Otic vesicles and epidermal core on day 35 (MOV 27157 kb)

Supplementary Video 2

Multi-chambered inner ear organoid viewed through the surface of a day 48 aggregate using DIC imaging. (MOV 21044 kb)

Supplementary Video 3

Inner ear organoids with ATOH1-2A-eGFP+ hair cells (day 100 live cell imaging). (MOV 23867 kb)

Supplementary Video 4

Multi-chambered inner ear organoid with ATOH-2A-eGFP+ hair cells in flat-mount preparation (day 100) (MOV 28752 kb)

Supplementary Video 5

Inner ear organoid with ESPN+ eGFP+ hair cells with innervation by NEFH+ sensory-like neurons. (MOV 25533 kb)

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Koehler, K., Nie, J., Longworth-Mills, E. et al. Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells. Nat Biotechnol 35, 583–589 (2017). https://doi.org/10.1038/nbt.3840

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