Engineering High-Fidelity Human Cochlear Organoids

项目摘要 耳蜗感觉毛细胞和/或神经支配神经元的丧失会导致不可逆的听力损失， 人类然而，实现生物学听力恢复的研究进展受到阻碍， 人类耳蜗组织的缺乏。我的实验室最近开发了一种新的类器官系统， 从人的聚集体产生含有功能性感觉毛细胞的内耳感觉上皮细胞 多能干细胞在3D培养中的应用虽然这些第一代类器官是研究 在人类内耳发育过程中，它们只产生具有天然毛细胞结构和功能特性的毛细胞。 前庭毛细胞和不能产生任何耳蜗细胞类型。我们原始系统的另一个局限性是 缺乏中枢神经系统成分。为了克服这些限制，我们的目标是开发下一个- 一代人类微生理系统，更忠实地重演发展的听觉 外周和脑干。在目标1中，我们将进行遗传编程和小规模CRISPR筛选， 增加耳蜗类器官中产生的外毛细胞的数量。衍生毛细胞的身份将是 通过单细胞电生理学、电子显微镜和单细胞RNA测序验证。此外，本发明还 通过甲状腺激素处理将促进衍生毛细胞的成熟。在目标2中，我们将建立 新的人类耳蜗-后脑神经胶质瘤，并评估这些神经元传入神经回路的发展， 类人猿在目标3中，我们将开发动态/可调水凝胶，并测试是否引入空间梯度 在类器官形成期间微环境中的硬度可影响组织图案化或细胞形态。 分化信号分子的空间梯度也将被引入水凝胶中，以测试背- 腹轴的形成可以在类器官分化过程中重演。这项研究的结果将 为研究耳蜗毛的正常和病理发育提供了一种改变范式的方法 细胞及其上行神经回路

PROJECT SUMMARY Loss of sensory hair cells and/or innervating neurons in the cochlea causes irreversible hearing loss in humans. However, progress on research for realizing biological restoration of hearing has been hampered due to the paucity of human cochlear tissues. My laboratory recently developed a novel organoid system to generate inner ear sensory epithelia containing functional sensory hair cells from aggregates of human pluripotent stem cells in 3D culture. While these first-generation organoids are a valuable tool for studying human inner ear development, they only generate hair cells with structural and functional properties of native vestibular hair cells and fail to produce any cochlear cell types. Another limitation with our original system is the lack of central nervous system components. To overcome these limitations, we aim at developing a next- generation human microphysiological system that more faithfully recapitulates development of the auditory periphery and brainstem. In Aim 1, we will carry out genetic programming and a small-scale CRISPR screen to increase the number of outer hair cells arising in cochlear organoids. The identity of derived hair cells will be validated by single-cell electrophysiology, electron microscopy and single-cell RNA-sequencing. Additionally, maturation of derived hair cells will be promoted by thyroid hormone treatments. In Aim 2, we will establish novel human cochlear-hindbrain assembloids and assess afferent neural circuit development in these assembloids. In Aim 3, we will develop dynamic/tunable hydrogels and test if introducing a spatial gradient of stiffness in the microenvironment during organoid formation can affect tissue patterning or cellular differentiation. Spatial gradients of signaling molecules will be also introduced in hydrogels to test if the dorso- ventral axis formation can be recapitulated during organoid differentiation. The outcome of this study will provide a paradigm-changing approach for studying normal and pathological development of cochlear hair cells and their ascending neural circuits.