Letter | Published:

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

Nature Biotechnology volume 35, pages 583589 (2017) | Download Citation


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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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.

Author information

Author notes

    • Xiao-Ping Liu

    Present address: Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA.


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

    • Karl R Koehler
    • , Jing Nie
    • , Emma Longworth-Mills
    • , Jiyoon Lee
    •  & Eri Hashino
  2. Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

    • Emma Longworth-Mills
  3. Departments of Otolaryngology and Neurology, F.M. Kirby Neurobiology Center Boston Children's Hospital, and Harvard Medical School, Boston, Massachusetts, USA.

    • Xiao-Ping Liu
    •  & Jeffrey R Holt
  4. Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA.

    • Eri Hashino


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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.

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.

Corresponding authors

Correspondence to Karl R Koehler or Eri Hashino.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–16, Supplementary Tables 1–5 and Supplementary Data


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    Supplementary Video 1

    Otic vesicles and epidermal core on day 35

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    Supplementary Video 2

    Multi-chambered inner ear organoid viewed through the surface of a day 48 aggregate using DIC imaging.

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    Supplementary Video 3

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

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    Supplementary Video 4

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

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    Supplementary Video 5

    Inner ear organoid with ESPN+ eGFP+ hair cells with innervation by NEFH+ sensory-like neurons.

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