Generating inner ear organoids from mouse embryonic stem cells

Emma Longworth-Mills, Karl R. Koehler, Eri Hashino

Research output: Chapter in Book/Report/Conference proceedingChapter

16 Scopus citations


This protocol describes a three-dimensional culture method for generating inner ear sensory epithelia, which comprises sensory hair cells and a concurrently arising neuronal population. Mouse embryonic stem cells are initially plated in 96-well plates with differentiation media; following aggregation, Matrigel is added in order to promote epithelialization. A series of small molecule applications is then used over the first 14 days of culture to guide differentiation towards an otic lineage. After 16-20 days, vesicles containing inner ear sensory hair cells and supporting cells arise from the cultured aggregates. Aggregates may be analyzed using immunohistochemistry and electrophysiology techniques. This system serves as a simple and relatively inexpensive in vitro model of inner ear development.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages16
StatePublished - 2016

Publication series

NameMethods in Molecular Biology
ISSN (Print)10643745


  • Cell cultureTechniques
  • Cell differentiation
  • Hair cell
  • Inner ear
  • Neurogenesis
  • Stem cell
  • Three-dimensional cell culture
  • Vestibular

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Longworth-Mills, E., Koehler, K. R., & Hashino, E. (2016). Generating inner ear organoids from mouse embryonic stem cells. In Methods in Molecular Biology (Vol. 1341, pp. 391-406). (Methods in Molecular Biology; Vol. 1341). Humana Press Inc..