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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Géléoc, G.S.G. & Holt, J.R. Sound strategies for hearing restoration. Science 344, 1241062 (2014).

    Article  Google Scholar 

  2. 2

    Rosenhall, U. Degenerative patterns in the aging human vestibular neuro-epithelia. Acta Otolaryngol. (Stockh.) 76, 208–220 (1973).

    CAS  Article  Google Scholar 

  3. 3

    Müller, U. & Barr-Gillespie, P.G. New treatment options for hearing loss. Nat. Rev. Drug Discov. 14, 346–365 (2015).

    Article  Google Scholar 

  4. 4

    Sergeyenko, Y., Lall, K., Liberman, M.C. & Kujawa, S.G. Age-related cochlear synaptopathy: an early-onset contributor to auditory functional decline. J. Neurosci. 33, 13686–13694 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Koehler, K.R. & Hashino, E. 3D mouse embryonic stem cell culture for generating inner ear organoids. Nat. Protoc. 9, 1229–1244 (2014).

    CAS  Article  Google Scholar 

  6. 6

    Koehler, K.R., Mikosz, A.M., Molosh, A.I., Patel, D. & Hashino, E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013).

    CAS  Article  Google Scholar 

  7. 7

    Liu, X.-P., Koehler, K.R., Mikosz, A.M., Hashino, E. & Holt, J.R. Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells. Nat. Commun. 7, 11508 (2016).

    CAS  Article  Google Scholar 

  8. 8

    Leung, A.W., Kent Morest, D. & Li, J.Y.H. Differential BMP signaling controls formation and differentiation of multipotent preplacodal ectoderm progenitors from human embryonic stem cells. Dev. Biol. 379, 208–220 (2013).

    CAS  Article  Google Scholar 

  9. 9

    Kwon, H.-J., Bhat, N., Sweet, E.M., Cornell, R.A. & Riley, B.B. Identification of early requirements for preplacodal ectoderm and sensory organ development. PLoS Genet. 6, e1001133 (2010).

    Article  Google Scholar 

  10. 10

    Chen, J.-R. et al. Effects of genetic correction on the differentiation of hair cell-like cells from iPSCs with MYO15A mutation. Cell Death Differ. 23, 1347–1357 (2016).

    CAS  Article  Google Scholar 

  11. 11

    Tang, Z.-H. et al. Genetic correction of induced pluripotent stem cells from a deaf patient with MYO7A mutation results in morphologic and functional recovery of the derived hair cell-like cells. Stem Cells Transl. Med. 5, 561–571 (2016).

    CAS  Article  Google Scholar 

  12. 12

    Ohnishi, H. et al. Limited hair cell induction from human induced pluripotent stem cells using a simple stepwise method. Neurosci. Lett. 599, 49–54 (2015).

    CAS  Article  Google Scholar 

  13. 13

    Ealy, M., Ellwanger, D.C., Kosaric, N., Stapper, A.P. & Heller, S. Single-cell analysis delineates a trajectory toward the human early otic lineage. Proc. Natl. Acad. Sci. USA 113, 8508–8513 (2016).

    CAS  Article  Google Scholar 

  14. 14

    Ronaghi, M. et al. Inner ear hair cell-like cells from human embryonic stem cells. Stem Cells Dev. 23, 1275–1284 (2014).

    CAS  Article  Google Scholar 

  15. 15

    Chen, W. et al. Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature 490, 278–282 (2012).

    CAS  Article  Google Scholar 

  16. 16

    Lim, R. & Brichta, A.M. Anatomical and physiological development of the human inner ear. Hear. Res. 338, 9–21 (2016).

    CAS  Article  Google Scholar 

  17. 17

    Roberts, R.M. et al. Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be? Reproduction 147, D1–D12 (2014).

    CAS  Article  Google Scholar 

  18. 18

    Chambers, S.M. et al. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat. Biotechnol. 30, 715–720 (2012).

    CAS  Article  Google Scholar 

  19. 19

    Ladher, R.K., O'Neill, P. & Begbie, J. From shared lineage to distinct functions: the development of the inner ear and epibranchial placodes. Development 137, 1777–1785 (2010).

    CAS  Article  Google Scholar 

  20. 20

    Groves, A.K. & Fekete, D.M. Shaping sound in space: the regulation of inner ear patterning. Development 139, 245–257 (2012).

    CAS  Article  Google Scholar 

  21. 21

    Ohyama, T., Mohamed, O.A., Taketo, M.M., Dufort, D. & Groves, A.K. Wnt signals mediate a fate decision between otic placode and epidermis. Development 133, 865–875 (2006).

    CAS  Article  Google Scholar 

  22. 22

    DeJonge, R.E. et al. Modulation of Wnt signaling enhances inner ear organoid development in 3D culture. PLoS One 11, e0162508 (2016).

    Article  Google Scholar 

  23. 23

    Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547–551 (2011).

    CAS  Article  Google Scholar 

  24. 24

    Hartman, B.H., Durruthy-Durruthy, R., Laske, R.D., Losorelli, S. & Heller, S. Identification and characterization of mouse otic sensory lineage genes. Front. Cell. Neurosci. 9, 79 (2015).

    Article  Google Scholar 

  25. 25

    Nelson, R.F. et al. Selective cochlear degeneration in mice lacking the F-box protein, Fbx2, a glycoprotein-specific ubiquitin ligase subunit. J. Neurosci. 27, 5163–5171 (2007).

    CAS  Article  Google Scholar 

  26. 26

    Burns, J.C., Kelly, M.C., Hoa, M., Morell, R.J. & Kelley, M.W. Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear. Nat. Commun. 6, 8557 (2015).

    CAS  Article  Google Scholar 

  27. 27

    Oghalai, J.S. et al. Ionic currents and electromotility in inner ear hair cells from humans. J. Neurophysiol. 79, 2235–2239 (1998).

    CAS  Article  Google Scholar 

  28. 28

    Géléoc, G.S.G., Risner, J.R. & Holt, J.R. Developmental acquisition of voltage-dependent conductances and sensory signaling in hair cells of the embryonic mouse inner ear. J. Neurosci. 24, 11148–11159 (2004).

    Article  Google Scholar 

  29. 29

    Horwitz, G.C., Risner-Janiczek, J.R., Jones, S.M. & Holt, J.R. HCN channels expressed in the inner ear are necessary for normal balance function. J. Neurosci. 31, 16814–16825 (2011).

    CAS  Article  Google Scholar 

  30. 30

    Levin, M.E. & Holt, J.R. The function and molecular identity of inward rectifier channels in vestibular hair cells of the mouse inner ear. J. Neurophysiol. 108, 175–186 (2012).

    CAS  Article  Google Scholar 

  31. 31

    Appler, J.M. & Goodrich, L.V. Connecting the ear to the brain: molecular mechanisms of auditory circuit assembly. Prog. Neurobiol. 93, 488–508 (2011).

    CAS  Article  Google Scholar 

  32. 32

    Grigaliunas, A., Bradley, R.M., MacCallum, D.K. & Mistretta, C.M. Distinctive neurophysiological properties of embryonic trigeminal and geniculate neurons in culture. J. Neurophysiol. 88, 2058–2074 (2002).

    Article  Google Scholar 

  33. 33

    Beers, J. et al. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat. Protoc. 7, 2029–2040 (2012).

    CAS  Article  Google Scholar 

  34. 34

    Huch, M. & Koo, B.-K. Modeling mouse and human development using organoid cultures. Development 142, 3113–3125 (2015).

    CAS  Article  Google Scholar 

  35. 35

    Nasu, M. et al. Robust formation and maintenance of continuous stratified cortical neuroepithelium by laminin-containing matrix in mouse ES cell culture. PLoS One 7, e53024 (2012).

    CAS  Article  Google Scholar 

  36. 36

    Hannan, N.R.F., Segeritz, C.-P., Touboul, T. & Vallier, L. Production of hepatocyte-like cells from human pluripotent stem cells. Nat. Protoc. 8, 430–437 (2013).

    CAS  Article  Google Scholar 

  37. 37

    Hama, H. et al. ScaleS: an optical clearing palette for biological imaging. Nat. Neurosci. 18, 1518–1529 (2015).

    CAS  Article  Google Scholar 

  38. 38

    Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  Article  Google Scholar 

  39. 39

    Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011).

    CAS  Article  Google Scholar 

  40. 40

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  Article  Google Scholar 

  41. 41

    Maruyama, T. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542 (2015).

    CAS  Article  Google Scholar 

Download references


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




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.

Ethics declarations

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)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing