Inner Ear Organoids: Recapitulating Inner Ear Development in 3D Culture

  • Alhasan N. Elghouche
  • Rick F. Nelson
  • Eri HashinoEmail author


The inner ear contains sensory epithelia composed of mechanosensitive hair cells, supporting cells, and sensory neurons that work in concert to detect sound and positional information and transmit those signals to the brain. Within the backdrop of embryogenesis, inner ear development follows an intricate pathway of signaling cues and morphological changes, leading to its complex final three-dimensional (3D) structure. Application of various small molecules and recombinant proteins to mouse embryonic stem cells at specific time points in vitro has enabled recapitulation of developmental cues with subsequent formation of inner ear organoids. This has resulted in a model system of inner ear development that is easily derived, manipulated, and analyzed. These organoids contain functional mechanosensitive hair cells, supporting cells, and sensory neurons, which phenocopy functional components of the inner ear responsible for detection of positional information. The potential applications of this system include investigation of inner ear development, disease modeling, drug screening, and therapy development. This chapter highlights the process of in vivo inner ear development, the rationale and process behind inner ear organoid formation, and potential applications and limitations of this in vitro model system.


Inner ear Hearing Balance Embryonic development Stem cells 3D culture Organogenesis 



The authors would like to thank Atsushi Shimomura for the schematic drawing and Rachel DeJonge and Andrew Mikosz for some of the image data. This work was supported by a National Institutes of Health grant R01 DC013294 (to E.H.), an Indiana Clinical and Translational Research Institute predoctoral fellowship (to A.N.E.), and a Centralized Otolaryngology Research Effort (CORE) grant (to R.F.N.).


  1. Ahrens K, Schlosser G (2005) Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis. Dev Biol 288(1):40–59CrossRefPubMedGoogle Scholar
  2. Bailey AP, Streit A (2005) Sensory organs: making and breaking the pre-placodal region. In: Current topics in developmental biology, vol 72. Academic, San Diego/London, pp 167–204Google Scholar
  3. Baker C, Bronner-Fraser M (2001) Vertebrate cranial placodes I. Embryonic induction. Dev Biol 232:1–61CrossRefPubMedGoogle Scholar
  4. Bhat N, Kwon H, Riley B (2013) A gene network that coordinates preplacodal competence and neural crest specification in zebrafish. Dev Biol 373(1):107–117CrossRefPubMedGoogle Scholar
  5. Bouchard M, Andersson E, Novitch B, Muhr J (2004) Tissue-specific expression of cre recombinase from the Pax8 locus. Genesis 38(3):105–109CrossRefPubMedGoogle Scholar
  6. Breneman K, Brownell W, Rabbitt R (2009) Hair cell bundles: flexoelectric motors of the inner ear. PLoS One 4(4):e5201CrossRefPubMedPubMedCentralGoogle Scholar
  7. Brugmann S, Pandur P, Kenyon K, Pignoni F, Moody S (2004) Six1 promotes a placodal fate within the lateral neurogenic ectoderm by functioning as both a transcriptional activator and repressor. Development 131:5871–5881CrossRefPubMedGoogle Scholar
  8. Chambers S, Fasano C, Papapetrou E (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Nanotechnol 27:275–280Google Scholar
  9. Chen W, Jongkamonwiwat N, Abbas L et al (2012) Restoration of auditory evodked responses by human ES-cell-derived otic progenitors. Nature 490:278–284CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chi N, Epstein JA (2002) Getting your Pax straight: PAX proteins in development and disease. Trends Genet 18(1):41–47CrossRefPubMedGoogle Scholar
  11. Christophorou N, Bailey A, Hanson S, Streit A (2009) Activation of Six1 target genes is required for sensory placode formation. Dev Biol 336:327–336CrossRefPubMedGoogle Scholar
  12. Christophorou N, Mende M, Lleras-Forero L, Grocott T, Streit A (2010) Pax2 coordinates epithelial morphogenesis and cell fate in the inner ear. Dev Biol 345:180–190CrossRefPubMedPubMedCentralGoogle Scholar
  13. Dekkers J, Wiegerinck C, de Jonge H et al (2013) A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med 19(7):939–948CrossRefPubMedGoogle Scholar
  14. Ding Q, Regan S, Xia Y, Oostrom L, Cowan C, Musunuru K (2013) Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12(4):393–394CrossRefPubMedPubMedCentralGoogle Scholar
  15. Eiraku M, Watanabe K, Matsuo-Takasaki M et al (2008) Self-Organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3(5):519–532CrossRefPubMedGoogle Scholar
  16. Eiraku M, Takata N, Ishibashi H et al (2011) Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472:51–56CrossRefPubMedGoogle Scholar
  17. Esterberg R, Fritz A (2009) dlx3b/4b are required for the formation of the preplacodal region and otic placode through local modulation of BMP activity. Dev Biol 325(1):189–199CrossRefPubMedGoogle Scholar
  18. Forge A, Schacht J (2000) Aminoglycoside antibiotics. Audiol Neurootol 5(1):3–22CrossRefPubMedGoogle Scholar
  19. Freter S, Muta Y, Mak S, Rinkwitz S, Ladher R (2008) Progressive restriction of otic fate: the role of FGF and Wnt in resolving inner ear potential. Development 135:3415–3424CrossRefPubMedGoogle Scholar
  20. Géléoc G, Holt J (2014) Sound strategies for hearing restoration. Science 344(6184):596–605CrossRefGoogle Scholar
  21. Glavic A, Honoré SM, Feijóo CG, Bastidas F, Allende ML, Mayor R (2004) Role of BMP signaling and the homeoprotein Iroquois in the specification of the cranial placodal field. Dev Biol 272(1):89–103CrossRefPubMedGoogle Scholar
  22. Groves A, Bronner-Fraser M (2000) Competence, specification and commitment in otic placode induction. Development 139:3489–3499Google Scholar
  23. Groves A, Fekete D (2012) Shaping sound in space: the regulation of inner ear patterning. Development 139:1175–1187CrossRefGoogle Scholar
  24. Kalinec F (2005) High-throughput screening of ototoxic and otoprotective pharmacologic drugs. Volta Rev 105(3):383–406Google Scholar
  25. Kalinec G, Webster P, Lim D, Kalinec F (2003) A cochlear cell line as an in vitro system for drug ototoxicity screening. Audiol Neurootol 8:177–189CrossRefPubMedGoogle Scholar
  26. Koehler K, Hashino E (2014) 3D mouse embryonic stem cell culture for generating inner ear organoids. Nat Protoc 9(6):1229–1244CrossRefPubMedGoogle Scholar
  27. Koehler K, Mikosz A, Molosh A, Patel D, Hashino E (2013) Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500:217–223CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kwon H, Bhat N, Sweet E, Cornell R, Riley B (2010) Identification of early requirements for preplacodal ectoderm and sensory organ development. PLoS Genet 6(9):e1001133CrossRefPubMedPubMedCentralGoogle Scholar
  29. Ladher R, Anakwe K, Gurney A, Schoenwolf G, Francis-West P (2000) Identification of synergistic signals initiating inner ear development. Science 290:1965–1967CrossRefPubMedGoogle Scholar
  30. Ladher R, O'Neill P, Begbie J (2010) From shared lineage to distinct functions: the development of the inner ear and epibranchial placodes. Development 137:1777–1785CrossRefPubMedGoogle Scholar
  31. Lang D, Powell SK, Plummer RS, Young KP, Ruggeri BA (2007) PAX genes: Roles in development, pathophysiology, and cancer. Biochem Pharmacol 73(1):1–14CrossRefPubMedGoogle Scholar
  32. Leung A, Morest D, Li J (2013) Differential BMP signaling controls formation and differentiation of multipotent preplacodal ectoderm progenitors from human embryonic stem cells. Dev Biol 379(2):208–220CrossRefPubMedPubMedCentralGoogle Scholar
  33. Li A, Xue J, Peterson E (2008) Architecture of the mouse utricle: macular organization and hair bundle heights. J Neurophysiol 99:718–733CrossRefPubMedGoogle Scholar
  34. Li H, Roblin G, Liu H, Heller S (2003) Generation of hair cells by stepwise differentiation of embryonic stem cells. PNAS 100(23):13,495–13,500CrossRefGoogle Scholar
  35. Li M, Suzuki K, Kim N, Liu G, Belmonte J (2013) A cut above the rest: targeted genome editing technologies in human pluripotent stem cells. J Biol Chem 289(8):4594–4599CrossRefPubMedPubMedCentralGoogle Scholar
  36. Litsiou A, Hanson S, Streit A (2005) A balance of FGF, BMP and WNT signalling positions the future placode territory in the head. Development 132:4051–4062CrossRefPubMedGoogle Scholar
  37. Lysakowski A, Gaboyard-Niay S, Calin-Jageman I, Chatlani S, Price S, Eatock R (2011) molecular microdomains in a sensory terminal, the vestibular calyx ending. J Neurosci 31(27):10,101–10,114CrossRefGoogle Scholar
  38. Meyers J, MacDonald R, Duggan A et al (2003) Lighting up the senses: FM1–43 loading of sensory cells through nonselective ion channels. J Neurosci 23(10):4054–4065PubMedGoogle Scholar
  39. Misui K, Tokuzawa Y, Itoh H, Segawa K et al (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 30(113):631–642Google Scholar
  40. Muguruma K, Nishiyama A, Ono Y et al (2010) Ontogeny-recapitulating generation and tissue integration of ES cell–derived Purkinje cells. Nat Neurosci 13:1171–1180CrossRefPubMedGoogle Scholar
  41. Müller U, Barr-Gillespie P (2015) New treatment options for hearing loss. Nat Rev Drug Discov 14:346–365CrossRefPubMedGoogle Scholar
  42. Nakano T, Ando S, Takata N (2012) Self-formation of optic cups and storable stratifed neural retina from humans ESCs. Cell Stem Cell 10:771–785CrossRefPubMedGoogle Scholar
  43. Oesterle E, Campbell S, Taylor R, Forge A, Hume C (2008) Sox2 and Jagged1 expression in normal and drug-damaged adult mouse inner ear. J Assoc Res Otolaryngol 9:65–89CrossRefPubMedGoogle Scholar
  44. Ohyama T, Mohamed O, Taketo M, Dufort D, Groves A (2006) Wnt signals mediate a fate decision between otic placode and epidermis. Development 133:865–875CrossRefPubMedGoogle Scholar
  45. Oshima K, Shin K, Diensthuber M, Peng A, Ricci A, Heller S (2010) Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. Cell 141(4):704–716CrossRefPubMedPubMedCentralGoogle Scholar
  46. Park Y-H (2015) Stem cell therapy for sensorineural hearing loss, still alive? J Audiol Otol 19(2):63–67CrossRefPubMedPubMedCentralGoogle Scholar
  47. Pieper M, Ahrens K, Rink E, Peter A, Schlosser G (2012) Differential distribution of competence for panplacodal and neural crest induction to non-neural and neural ectoderm. Development 139(6):1175–1187CrossRefPubMedGoogle Scholar
  48. Reichert S, Randall R, Hill C (2013) A BMP regulatory network controls ectodermal cell fate decisions at the neural plate border. Development 140:4435–4444CrossRefPubMedGoogle Scholar
  49. Ruf R, Xu P, Silvius D, Otto E, Beekmann F, Muerb U et al (2004) SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1–SIX1–DNA complexes. Proc Natl Acad Sci 101:8090–8095CrossRefPubMedPubMedCentralGoogle Scholar
  50. Sacheli R, Delacroix L, Vandenackerveken P, Nguyen L, Malgrange B (2013) Gene transfer in inner ear cells: a challenging race. Gene Ther 20:237–247CrossRefPubMedGoogle Scholar
  51. Sai X, Ladher R (2015) Early steps in inner ear development: induction and morphogenesis of the otic placode. Front Pharmacol 6(19):1–8Google Scholar
  52. Sai X, Yonemura S, Ladher R (2014) Junctionally restricted RhoA activity is necessary for apical constriction during phase 2 inner ear placode invagination. Dev Biol 394:206–216CrossRefPubMedGoogle Scholar
  53. Saint-Jeannet J, Moody S (2014) Establishing the pre-placodal region and breaking it into placodes with distinct identities. Dev Biol 389(1):13–27CrossRefPubMedPubMedCentralGoogle Scholar
  54. Sasai Y, Eiraku M, Hidetaka S (2012) In vitro organogenesis in three dimensions: self-organising stem cells. Development 139(22):4111–4121CrossRefPubMedGoogle Scholar
  55. Sato S, Ikeda K, Shioi G, Ochi H, Ogino H, Yajima H et al (2010) Conserved expression of mouse Six1 in the pre-placodal region (PPR) and iden- tification of an enhancer for the rostral PPR. Dev Biol 344:158–171CrossRefPubMedGoogle Scholar
  56. Sato T, Vries R, Snippert H (2009) Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459:262–265CrossRefPubMedGoogle Scholar
  57. Schimmang T (2007) Expression and functions of FGF ligands during early otic development. Int J Dev Biol 51:473–481CrossRefPubMedGoogle Scholar
  58. Schlosser G (2006) Induction and specification of cranial placodes. Dev Biol 294:303–351CrossRefPubMedGoogle Scholar
  59. Schlosser G (2014) Early embryonic specification of vertebrate cranial placodes. Dev Biol 3:349–363Google Scholar
  60. Schlosser G, Ahrens K (2004) Molecular anatomy of placode development in Xenopus laevis. Dev Biol 271(2):439–466CrossRefPubMedGoogle Scholar
  61. Seiler M, Aramant R, Thomas B, Peng Q, Sadda S, Keirstead H (2010) Visual restoration and transplant connectivity in degernerate rats implanted with retinal progenitor sheets. Eur J Neurosci 31:508–520CrossRefPubMedPubMedCentralGoogle Scholar
  62. Streit A (2007) The preplacodal region: an ectodermal domain with multipotential progenitors that contribute to sense organs and cranial sensory ganglia. Int J Dev Biol 51(6–7):447–461. doi: 10.1387/ijdb.072327as CrossRefPubMedGoogle Scholar
  63. Suga H (2011) Self-formation of functional adenohypophysis in three-dimensional culture. Nature 480:57–62CrossRefPubMedGoogle Scholar
  64. Urness L, Paxton C, Wang X, Schoenwolf G, Mansour S (2010) FGF signaling regulates otic placode induction and refinement by controlling both ectodermal target genes and hindbrain Wnt8a. Dev Biol 340:595–604CrossRefPubMedPubMedCentralGoogle Scholar
  65. Vendrell V, Vázquez-Echeverría C, López-Hernández I, Alons B, Marinez S, Pujades C et al (2013) Roles of Wnt8a during formation and patterning of the mouse inner ear. Mech Dev 130:160–168CrossRefPubMedGoogle Scholar
  66. Warchol M, Richardson G (2009) Expression of the pax2 transcription factor is associated with vestibular phenotype in the avian inner ear. Dev Neurobiol 69:191–202CrossRefPubMedPubMedCentralGoogle Scholar
  67. Wright T, Mansour S (2003) Fgf3 and Fgf10 are required for mouse otic placode induction. Development 130:3379–3390CrossRefPubMedGoogle Scholar
  68. Wu W, Sha S, McLaren J, Kawamoto K, Raphael Y, Schacht J (2001) Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague-Dawley rat. Hear Res 158(1–2):165–178CrossRefPubMedGoogle Scholar
  69. Xinaris C, Brizi V, Remuzzi G (2015) Organoid models and applications in biomedical research. Nephron 130:191–199CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2017

Authors and Affiliations

  • Alhasan N. Elghouche
    • 1
  • Rick F. Nelson
    • 1
  • Eri Hashino
    • 1
    Email author
  1. 1.Department of Otolaryngology-Head & Neck SurgeryIndiana University School of MedicineIndianapolisUSA

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