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Item 3D mouse embryonic stem cell culture for generating inner ear organoids(Springer Nature, 2014) Koehler, Karl R.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of MedicineThis protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 96-well plates in medium containing extracellular matrix proteins to promote epithelialization. During the first 14 d, a series of precisely timed protein and small-molecule treatments sequentially induce epithelia that represent the mouse embryonic non-neural ectoderm, preplacodal ectoderm and otic vesicle epithelia. Ultimately, these tissues develop into cysts with a pseudostratified epithelium containing inner ear hair cells and supporting cells after 16-20 d. Concurrently, sensory-like neurons generate synapse-like structures with the derived hair cells. We have designated the stem cell-derived epithelia harboring hair cells, supporting cells and sensory-like neurons as inner ear organoids. This method provides a reproducible and scalable means to generate inner ear sensory tissue in vitro.Item Modulation of the Notch Signaling Pathway in 3D Stem-Cell Derived Culture of Inner Ear Organoids(2016-05-10) Elghouche, Alhasan Najib; Hashino, Eri; Nelson, Rick F.; Koehler, Karl RussellHearing loss and vestibular dysfunction are inner ear disease states that arise from an array of diverse etiologies that interfere with mechanosensory hair cell function, including: congenital syndromes, noise-induced trauma, ototoxic drugs, and aging. The investigation of normal inner ear development and the pathological aberrations that cause inner ear disease has been previously advanced through formation of an easily generated, scalable, accurate in vitro model system that readily facilitates experimental applications. This model utilizes a 3D floating cell culture protocol which guides differentiation of stem cell aggregates into inner ear organoids, which are vesicles containing a sensory epithelium with functioning mechanosensory hair cells. Inner ear organoid formation enables studying the effects of modulating the signaling pathways that guide developing inner ear structure and function. The Notch signaling pathway heavily influences the formation of the inner ear through two major mechanisms: lateral induction of sensory progenitor cells and lateral inhibition to determine which of those progenitors differentiate into mechanosensory hair cells. The effects of inhibiting Notch signaling within the inner ear organoid system were explored through application of the ɣ-secretase inhibitor MDL28170 (MDL) at a concentration of 25μM on day 8 of organoid culture. Aggregates were harvested on day 32, fixed, sectioned, and stained according to a standard immunohistochemistry protocol. Sections were stained for the mechanosensory hair cell markers Myosin7a (Myo7a) and Sox2. MDL-treated aggregates demonstrated statistically significant reductions in the total number of vesicles and the number of vesicles containing hair cells compared to control aggregates. In contrast to control aggregates which demonstrated two distinct organoid variants (protruding and embedded), MDL-treated aggregates only formed the embedded variant. Differences in the expression pattern of Sox2, which is also a marker of stemness and neural progenitor cells were also noted between the two conditions. MDL-treated aggregates demonstrated regions of ‘ectopic’ Sox2 expression whereas Sox2 expression in control aggregates was consistently expressed within Myo7a+ regions.