Modeling Inner Ear Differentiation with Pluripotent Stem Cells

项目总结 人类内耳组织，特别是感觉细胞，对于实验来说是稀缺的，因为活组织检查不是一种 严重听力损失或平衡障碍患者的标准程序。为了绕过这一挑战， 我们最近建立了一个明确的3D培养系统来高效地产生人类内耳感觉上皮细胞 来自人类多能干细胞的聚集体。这些所谓的“人类内耳有机物质”隐藏着一层 由紧密排列的支持细胞和毛细胞组成，这些细胞由感觉神经元支配。基于我们最初的 这些人类干细胞来源的毛细胞表现出结构、生化和功能 特性可与天然感觉毛细胞相媲美。此应用程序的主要目标是定义 时间进程、转录途径、结构变化和蛋白质-蛋白质相互作用 人类内耳器官中的感觉细胞分化。在目标1中，我们将测试PAX2阳性的耳聋 祖细胞在内耳中产生不同类型的细胞。利用单细胞RNA-SEQ、芯片-SEQ的组合 SEQ和谱系追踪分析，我们将确定基因表达、谱系的发育轨迹 规范和转录网络对毛细胞和感觉神经元的规范至关重要 人类的内耳。在目标2中，我们将阐明前庭和耳蜗区的独特转录途径。 腹膜外毛细胞的生化和结构特性的鉴定 祖先。在目标3中，我们将定义毛细胞分化的时间进程(例如，毛束和 在光镜和电子显微镜水平上人类内耳有机体中的带状突触发育)。 此外，利用单细胞电生理学和光遗传学的结合，我们将测试人类 干细胞来源的毛细胞与感觉神经元进行功能性突触连接 产生于文化中。此外，利用酵母双杂交筛选，我们将鉴定出新的蛋白质？蛋白质。 相互作用对毛束的形成至关重要。通过实现这些目标，我们不仅将推进我们的 了解人类内耳发育的生物学，也建立了明确和可扩展的 用于研究各种形式遗传性内耳疾病发病机制的人体模型系统 并确定具有再生人类毛细胞潜力的化合物。

PROJECT SUMMARY Human inner ear tissues, sensory cells in particular, are scarce for experimentation, since biopsy is not a standard procedure for patients with profound hearing loss or balance disorders. To circumvent this challenge, we recently established a defined 3D culture system to efficiently generate human inner ear sensory epithelia from aggregates of human pluripotent stem cells. These so-called “human inner ear organoids” harbor a layer of tightly packed supporting cells and hair cells that are innervated by sensory neurons. Based on our initial characterization, these human stem cell-derived hair cells exhibit structural, biochemical and functional properties comparable to those of native sensory hair cells. The primary goal of this application is to define the temporal progression, transcriptional pathways, structural changes and protein-protein interactions during sensory cell differentiation in the human inner ear organoid. In Aim 1, we will test how PAX2-positive otic progenitors give rise to different cell types in the inner ear. Using a combination of single-cell RNA-seq, ChIP- seq and lineage-tracing analyses, we will determine developmental trajectories of gene expression, lineage specification and transcriptional networks essential for specification of hair cells and sensory neurons in the human inner ear. In Aim 2, we will elucidate the transcriptional pathways distinctive for vestibular vs. cochlear specification and determine biochemical and structural properties of hair cells derived from ventralized otic progenitors. In Aim 3, we will define temporal progression of hair cell differentiation (e.g. hair bundle and ribbon synapse development) in human inner ear organoids at both light and electron microscopic levels. Additionally, using a combination of single-cell electrophysiology and optogenetics, we will test whether human stem cell-derived hair cells make functional synaptic connections with sensory neurons that are concomitantly arising in culture. Moreover, using yeast two-hybrid screening, we will identify novel protein-protein interactions essential for hair bundle formation. By accomplishing these aims, we will not only advance our understanding of the biology of human inner ear development, but also establish a defined and scalable human model system with which to investigate pathogenesis of various forms of hereditary inner ear disorders and identify compounds with the potential of regenerating hair cells in humans.