Modeling Inner Ear Differentiation with Pluripotent Stem Cells

用多能干细胞模拟内耳分化

• 批准号: 9916726
• 负责人: Eri Hashino
• 金额: $ 57.34万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916726
• 关键词: Adult; Affect; Afferent Neurons; Americas; Auditory Receptor Cell; Biochemical; Biological Models; Biopsy; Cell Differentiation process; Cell Lineage; Cells; ChIP-seq; Child; Cochlea; Data; Development; Dorsal; Drug Modelings; Drug Screening; ES Cell Line; Electrons; Electrophysiology (science); Epithelial; Epithelium; Equilibrium; Exhibits; Expression Profiling; Foundations; Funding; Future; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Goals; Hair; Hair Cells; Health; Human; Human Biology; Immunofluorescence Immunologic; In Vitro; Inherited; Investigation; Labyrinth; Light; Microscopic; Modeling; Natural regeneration; Neuronal Differentiation; Organoids; Otic Placodes; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Pluripotent Stem Cells; Population; Preclinical Testing; Procedures; Property; Proteins; Reporter; SHH gene; Sensory; Sensory Hair; Signal Transduction; Structure; Supporting Cell; Synapses; System; Testing; Time; Tissues; Vestibular Hair Cells; WNT Signaling Pathway; Yeasts; base; cell type; deaf; electron tomography; equilibration disorder; experimental study; hearing impairment; human embryonic stem cell; human model; human pluripotent stem cell; human stem cells; inner ear development; inner ear diseases; nerve supply; novel; novel strategies; optogenetics; progenitor; protein protein interaction; ribbon synapse; screening; single-cell RNA sequencing; smoothened signaling pathway; stem cells; therapeutic target; therapy development; three dimensional cell culture; transcriptome; yeast two hybrid system

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.

项目摘要 人类内耳组织，特别是感觉细胞，在实验中是稀缺的，因为活组织检查不是一种有效的方法。 严重听力损失或平衡障碍患者的标准程序。为了规避这一挑战， 我们最近建立了一个定义的三维培养系统，以有效地产生人内耳感觉上皮细胞 从人类多能干细胞的聚集体中。这些所谓的“人类内耳类器官”拥有一层 由感觉神经元支配的紧密排列的支持细胞和毛细胞组成。根据我们最初的 这些人干细胞衍生的毛细胞表现出结构、生物化学和功能特性， 与天然感觉毛细胞的性质相当。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， 人内耳类器官中感觉细胞的分化。在目标1中，我们将测试PAX 2阳性耳 祖细胞在内耳中产生不同的细胞类型。使用单细胞RNA-seq、ChIP- seq和谱系追踪分析，我们将确定基因表达的发展轨迹， 在毛细胞和感觉神经元的特化和转录网络必不可少的， 人的内耳在目标2中，我们将阐明前庭与耳蜗不同的转录途径， 对源自腹侧化耳毛细胞的生物化学和结构特性进行鉴定和测定 祖先在目标3中，我们将定义毛细胞分化的时间进程（例如毛束和毛细胞分化）。 带状突触发育）在人类内耳类器官中的光镜和电镜水平。 此外，使用单细胞电生理学和光遗传学的组合，我们将测试人类是否 干细胞衍生的毛细胞与感觉神经元形成功能性突触连接， 在文化中兴起。此外，利用酵母双杂交筛选，我们将确定新的蛋白质-蛋白质 毛束形成的关键相互作用。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，而且还建立了一个定义和可扩展的 用于研究各种形式遗传性内耳疾病发病机制的人类模型系统 并鉴定出具有再生人类毛细胞潜力的化合物。