Restoration of auditory evoked responses by human ES-cell-derived otic progenitors

Article metrics

Abstract

Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells1, is responsible for a substantial proportion of patients with hearing impairment2. Although the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss, as poor innervation limits the prospective performance of an implant3. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair-cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory-evoked response thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: FGF3 and FGF10 generate otic progenitors.
Figure 2: Otic epithelial progenitors and otic neuro progenitors.
Figure 3: Transplantation of otic progenitors restores a population of spiral ganglion neurons.
Figure 4: Transplanted cells provide a recovery of ABR thresholds.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray datasets have been deposited at the NCBI Gene Expression Omnibus and they can be retrieved with accession number GSE36754.

References

  1. 1

    Vlastarakos, P. V., Nikolopoulos, T. P., Tavoulari, E., Papacharalambous, G. & Korres, S. Auditory neuropathy: endocochlear lesion or temporal processing impairment? Implications for diagnosis and management. Int. J. Pediatr. Otorhinolaryngol. 72, 1135–1150 (2008)

  2. 2

    Uus, K. & Bamford, J. Effectiveness of population-based newborn hearing screening in England: ages of interventions and profile of cases. Pediatrics 117, e887–e893 (2006)

  3. 3

    Bradley, J., Beale, T., Graham, J. & Bell, M. Variable long-term outcomes from cochlear implantation in children with hypoplastic auditory nerves. Cochlear Implants Int. 9, 34–60 (2008)

  4. 4

    Li, H., Liu, H. & Heller, S. Pluripotent stem cells from the adult mouse inner ear. Nature Med. 9, 1293–1299 (2003)

  5. 5

    Li, H., Roblin, G., Liu, H. & Heller, S. Generation of hair cells by stepwise differentiation of embryonic stem cells. Proc. Natl Acad. Sci. USA 100, 13495–13500 (2003)

  6. 6

    Oshima, K. et al. Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. Cell 141, 704–716 (2010)

  7. 7

    Jeon, S. J., Oshima, K., Heller, S. & Edge, A. S. Bone marrow mesenchymal stem cells are progenitors in vitro for inner ear hair cells. Mol. Cell. Neurosci. 34, 59–68 (2007)

  8. 8

    Kondo, T., Johnson, S. A., Yoder, M. C., Romand, R. & Hashino, E. Sonic hedgehog and retinoic acid synergistically promote sensory fate specification from bone marrow-derived pluripotent stem cells. Proc. Natl Acad. Sci. USA 102, 4789–4794 (2005)

  9. 9

    Coleman, B., Fallon, J. B., Pettingill, L. N., de Silva, M. G. & Shepherd, R. K. Auditory hair cell explant co-cultures promote the differentiation of stem cells into bipolar neurons. Exp. Cell Res. 313, 232–243 (2007)

  10. 10

    Reyes, J. H. et al. Glutamatergic neuronal differentiation of mouse embryonic stem cells after transient expression of neurogenin 1 and treatment with BDNF and GDNF: in vitro and in vivo studies. J. Neurosci. 28, 12622–12631 (2008)

  11. 11

    Corrales, C. E. et al. Engraftment and differentiation of embryonic stem cell-derived neural progenitor cells in the cochlear nerve trunk: growth of processes into the organ of Corti. J. Neurobiol. 66, 1489–1500 (2006)

  12. 12

    Hildebrand, M. S. et al. Survival of partially differentiated mouse embryonic stem cells in the scala media of the guinea pig cochlea. J. Assoc. Res. Otolaryngol. 6, 341–354 (2005)

  13. 13

    Sekiya, T. et al. Transplantation of conditionally immortal auditory neuroblasts to the auditory nerve. Eur. J. Neurosci. 25, 2307–2318 (2007)

  14. 14

    Lang, H. et al. Transplantation of mouse embryonic stem cells into the cochlea of an auditory-neuropathy animal model: effects of timing after injury. J. Assoc. Res. Otolaryngol. 9, 225–240 (2008)

  15. 15

    Hu, Z., Ulfendahl, M. & Olivius, N. P. Central migration of neuronal tissue and embryonic stem cells following transplantation along the adult auditory nerve. Brain Res. 1026, 68–73 (2004)

  16. 16

    Rask-Andersen, H. et al. Regeneration of human auditory nerve. In vitro/in video demonstration of neural progenitor cells in adult human and guinea pig spiral ganglion. Hear. Res. 203, 180–191 (2005)

  17. 17

    Shi, F., Corrales, C. E., Liberman, M. C. & Edge, A. S. BMP4 induction of sensory neurons from human embryonic stem cells and reinnervation of sensory epithelium. Eur. J. Neurosci. 26, 3016–3023 (2007)

  18. 18

    Lee, G. et al. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nature Biotechnol. 25, 1468–1475 (2007)

  19. 19

    Chen, W. et al. Human fetal auditory stem cells can be expanded in vitro and differentiate into functional auditory neurons and hair cell-like cells. Stem Cells 27, 1196–1204 (2009)

  20. 20

    Martin, K. & Groves, A. K. Competence of cranial ectoderm to respond to Fgf signaling suggests a two-step model of otic placode induction. Development 133, 877–887 (2006)

  21. 21

    Freter, S., Muta, Y., Mak, S. S., Rinkwitz, S. & Ladher, R. K. Progressive restriction of otic fate: the role of FGF and Wnt in resolving inner ear potential. Development 135, 3415–3424 (2008)

  22. 22

    Alvarez, Y. et al. Requirements for FGF3 and FGF10 during inner ear formation. Development 130, 6329–6338 (2003)

  23. 23

    Wright, T. J. & Mansour, S. L. Fgf3 and Fgf10 are required for mouse otic placode induction. Development 130, 3379–3390 (2003)

  24. 24

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005)

  25. 25

    Schmiedt, R. A., Okamura, H. O., Lang, H. & Schulte, B. A. Ouabain application to the round window of the gerbil cochlea: a model of auditory neuropathy and apoptosis. J. Assoc. Res. Otolaryngol. 3, 223–233 (2002)

  26. 26

    Lang, H., Schulte, B. A. & Schmiedt, R. A. Ouabain induces apoptotic cell death in type I spiral ganglion neurons, but not type II neurons. J. Assoc. Res. Otolaryngol. 6, 63–74 (2005)

  27. 27

    McLean, W. J., Smith, K. A., Glowatzki, E. & Pyott, S. J. Distribution of the Na,K-ATPase alpha subunit in the rat spiral ganglion and organ of corti. J. Assoc. Res. Otolaryngol. 10, 37–49 (2009)

  28. 28

    Burkard, R., Boettcher, F., Voigt, H. & Mills, J. Comments on “Stimulus dependencies of the gerbil brain-stem auditory-evoked response (BAER). I: Effects of click level, rate and polarity” [J. Acoust. Soc. Am. 85, 2514–2525 (1989)]. J. Acoust. Soc. Am. 94, 2441–2442 (1993)

  29. 29

    Boettcher, F. A., Mills, J. H. & Norton, B. L. Age-related changes in auditory evoked potentials of gerbils. I. Response amplitudes. Hear. Res. 71, 137–145 (1993)

Download references

Acknowledgements

This work was supported primarily by grants from Action on Hearing Loss (RNID) to M.N.R. Other support included Deafness Research UK (M.N.R and W.M.), the Wellcome Trust (088719, W.M.), Medical Research Council (P.W.A, H.D.M. and M.N.R) and ESTOOLS (P.W.A). S.L.J. was supported by a Wellcome Trust VIP award and the RNID. W.M. and S.L.J. are Royal Society university research fellows. Confocal images were taken at the Light Microscopy Facility of the Department of Biomedical Sciences, University of Sheffield. We are grateful for the advice of M. Mulheran and I. Russell on the tests of auditory function, provided at the earlier stages of this project, and to the assistance of P. Gokhale on the use of the InCell Analyzer. M.N.R. acknowledges the support and encouragement of his late parents, Noemí Luján-Ceballos and Juan Carlos Rivolta.

Author information

W.C., N.J., L.A., S.J.E and J.K.T. collected and/or assembled, analysed and interpreted data. S.L.J., S.K. and W.M. collected and/or assembled, analysed and intepreted electrophysiology data. M.M. carried out biocomputational analysis of gene-array data. P.W.A. and H.D.M. provided study material and administrative support. M.N.R. was responsible for conceiving and designing the study, for obtaining financial support, collecting and/or assembling, analysing and interpreting data, for manuscript writing, and for final approval of the manuscript.

Correspondence to Marcelo N. Rivolta.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1 and 2 and Supplementary Figures 1-15. (PDF 1936 kb)

Supplementary Data

This zipped file contains Supplementary Tables 3-10. Supplementary Table 3 contains the genes differentially upregulated in the FGF condition when compared to undifferentiated hESCs, using a threshold of 1.5fold-change. Supplementary Table 4 contains the genes differentially upregulated in the FGF condition when compared to cells in DFNB, using a threshold of 1.5fold-change. Supplementary Table 5 contains the genes differentially downregulated in the FGF condition when compared to undifferentiated hESCs, using a threshold of 1.5fold-change. Supplementary Table 6 contains the genes differentially downregulated in the FGF condition when compared to cells in DFNB, using a threshold of 1.5fold-change. Supplementary Table 7 the top Gene Ontology BP5 terms enriched in the genes upregulated by a 1.5 fold-change in FGF when compared to undifferentiated hESCs. Supplementary Table 8 the top Gene Ontology BP5 terms enriched in the genes upregulated by a 1.5 fold-change in FGF when compared to cells in DFNB. Supplementary Table 9 contains the top Gene Ontology BP5 terms enriched in the genes downregulated by a 1.5 fold-change in FGF when compared to undifferentiated hESCs. Supplementary Table 10 contains the top Gene Ontology BP5 terms enriched in the genes downregulated by a 1.5 fold-change in FGF when compared to cells in DFNB. (ZIP 913 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chen, W., Jongkamonwiwat, N., Abbas, L. et al. Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature 490, 278–282 (2012) doi:10.1038/nature11415

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.