Department of Otolaryngology - Head and Neck Surgery, and Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA.
Methods Mol Biol. 2022;2520:135-150. doi: 10.1007/7651_2021_448.
The sensory epithelia of the inner ear contain mechanosensitive hair cells that detect sound and head acceleration. This protocol details a 3D differentiation method to generate inner ear organoids containing sensory epithelia with hair cells. Human pluripotent stem cells are aggregated in low-binding 96-well plates and treated in chemically defined media with extracellular matrix to promote epithelialization. Small molecules and recombinant proteins are applied in a stepwise manner to recapitulate the morphogenic cues (BMP, TGF-β, FGF, and WNT) present during inner ear development in vivo. These treatments induce the sequential formation of nonneural ectoderm, otic-epibranchial progenitor domain, and otic placodes. The derived otic placodes then undergo self-guided morphogenesis to form otic vesicles, which eventually give rise to sensory epithelia containing hair cells and supporting cells, as well as neurons with synaptic formations to hair cells. This human stem cell-derived inner ear organoid system provides an ideal platform to study human inner ear development and disease in vitro.
内耳的感觉上皮包含机械敏感的毛细胞，可检测声音和头部加速度。本方案详细介绍了一种 3D 分化方法，可生成包含毛细胞的感觉上皮的内耳类器官。将人多能干细胞聚集在低结合 96 孔板中，并在含有细胞外基质的化学定义培养基中进行处理，以促进上皮化。小分子和重组蛋白以逐步的方式施加，以再现体内内耳发育过程中存在的形态发生线索（BMP、TGF-β、FGF 和 WNT）。这些处理诱导非神经外胚层、耳前-鳃弓祖细胞域和耳基板的顺序形成。衍生的耳基板随后进行自我引导的形态发生，形成耳泡，最终产生包含毛细胞和支持细胞的感觉上皮，以及具有与毛细胞突触形成的神经元。这种人干细胞衍生的内耳类器官系统为体外研究人类内耳发育和疾病提供了理想的平台。
