Longworth-Mills, EmmaKoehler, Karl R.Hashino, Eri2019-07-262019-07-262016Longworth-Mills, E., Koehler, K. R., & Hashino, E. (2016). Generating Inner Ear Organoids from Mouse Embryonic Stem Cells. Methods in molecular biology (Clifton, N.J.), 1341, 391–406. doi:10.1007/7651_2015_215https://hdl.handle.net/1805/19965This 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.en-USPublisher PolicyCell culture techniquesCell differentiationHair cellInner earNeurogenesisStem cellThree-dimensional cell cultureVestibularArticle