Engineering High-Fidelity Human Cochlear Organoids

项目总结 耳蜗区感觉毛细胞和/或神经细胞的丧失可导致不可逆性听力损失 人类。然而，实现听力生物恢复的研究进展受到了应有的阻碍 人类耳蜗组织的稀缺性。我的实验室最近开发了一种新的有机系统来 从人的集合体中培养出含有功能性感觉毛细胞的内耳感觉上皮 3D培养中的多能干细胞。虽然这些第一代有机化合物是研究 人类内耳发育，它们只产生具有天然结构和功能特性的毛细胞 前庭毛细胞，不能产生任何类型的耳蜗细胞。我们原始系统的另一个限制是 缺乏中枢神经系统的组成部分。为了克服这些限制，我们的目标是开发下一代- 生成更真实地概括听觉发育的人类微生理系统 外周和脑干。在目标1中，我们将进行遗传编程和小规模CRISPR筛查，以 增加耳蜗器中出现的外毛细胞的数量。衍生毛细胞的身份将是 经单细胞电生理学、电子显微镜和单细胞RNA测序验证。另外， 甲状腺激素治疗将促进衍生毛细胞的成熟。在目标2中，我们将确立 新型人类耳蜗脑-后脑集合体及其传入神经回路发育的研究 集合水滴。在目标3中，我们将开发动态/可调水凝胶，并测试是否在 类有机物形成过程中微环境的僵硬会影响组织构型或细胞 差异化。信号分子的空间梯度也将被引入水凝胶中，以测试是否DOSO- 腹轴的形成可以在类器分化的过程中重现。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范式转换的方法 细胞和它们的上升神经回路。

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.