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

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Sound strategies for hearing restoration. Science 344, 1241062 (2014).

  2. 2.

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

  3. 3.

    & New treatment options for hearing loss. Nat. Rev. Drug Discov. 14, 346–365 (2015).

  4. 4.

    , , & Age-related cochlear synaptopathy: an early-onset contributor to auditory functional decline. J. Neurosci. 33, 13686–13694 (2013).

  5. 5.

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

  6. 6.

    , , , & Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013).

  7. 7.

    , , , & Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells. Nat. Commun. 7, 11508 (2016).

  8. 8.

    , & Differential BMP signaling controls formation and differentiation of multipotent preplacodal ectoderm progenitors from human embryonic stem cells. Dev. Biol. 379, 208–220 (2013).

  9. 9.

    , , , & Identification of early requirements for preplacodal ectoderm and sensory organ development. PLoS Genet. 6, e1001133 (2010).

  10. 10.

    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).

  11. 11.

    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).

  12. 12.

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

  13. 13.

    , , , & Single-cell analysis delineates a trajectory toward the human early otic lineage. Proc. Natl. Acad. Sci. USA 113, 8508–8513 (2016).

  14. 14.

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

  15. 15.

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

  16. 16.

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

  17. 17.

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

  18. 18.

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

  19. 19.

    , & From shared lineage to distinct functions: the development of the inner ear and epibranchial placodes. Development 137, 1777–1785 (2010).

  20. 20.

    & Shaping sound in space: the regulation of inner ear patterning. Development 139, 245–257 (2012).

  21. 21.

    , , , & Wnt signals mediate a fate decision between otic placode and epidermis. Development 133, 865–875 (2006).

  22. 22.

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

  23. 23.

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

  24. 24.

    , , , & Identification and characterization of mouse otic sensory lineage genes. Front. Cell. Neurosci. 9, 79 (2015).

  25. 25.

    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).

  26. 26.

    , , , & Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear. Nat. Commun. 6, 8557 (2015).

  27. 27.

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

  28. 28.

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

  29. 29.

    , , & HCN channels expressed in the inner ear are necessary for normal balance function. J. Neurosci. 31, 16814–16825 (2011).

  30. 30.

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

  31. 31.

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

  32. 32.

    , , & Distinctive neurophysiological properties of embryonic trigeminal and geniculate neurons in culture. J. Neurophysiol. 88, 2058–2074 (2002).

  33. 33.

    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).

  34. 34.

    & Modeling mouse and human development using organoid cultures. Development 142, 3113–3125 (2015).

  35. 35.

    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).

  36. 36.

    , , & Production of hepatocyte-like cells from human pluripotent stem cells. Nat. Protoc. 8, 430–437 (2013).

  37. 37.

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

  38. 38.

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

  39. 39.

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

  40. 40.

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

  41. 41.

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

Download references

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.

Author information

Author notes

    • Xiao-Ping Liu

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

Affiliations

  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

Authors

  1. Search for Karl R Koehler in:

  2. Search for Jing Nie in:

  3. Search for Emma Longworth-Mills in:

  4. Search for Xiao-Ping Liu in:

  5. Search for Jiyoon Lee in:

  6. Search for Jeffrey R Holt in:

  7. Search for Eri Hashino in:

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.

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

PDF files

  1. 1.

    Supplementary Text and Figures

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

Videos

  1. 1.

    Supplementary Video 1

    Otic vesicles and epidermal core on day 35

  2. 2.

    Supplementary Video 2

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

  3. 3.

    Supplementary Video 3

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

  4. 4.

    Supplementary Video 4

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

  5. 5.

    Supplementary Video 5

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

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt.3840

Further reading