Modeling Inner Ear Differentiation with Pluripotent Stem Cells

用多能干细胞模拟内耳分化

基本信息

批准号：
10615050
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
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-03-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10615050
关键词：
Adult Affect Afferent Neurons 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)Epithelium Equilibrium Exhibits 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 Specific qualifier value Supporting Cell Synapses System Testing Time Tissues Vestibular Hair Cells WNT Signaling Pathway Yeasts cell type deaf electron tomography equilibration disorder experimental study hair cell regeneration 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 presynaptic progenitor protein protein interaction regeneration potential 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

项目摘要

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.

项目概要人类内耳组织，特别是感觉细胞，很少用于实验，因为活组织检查不是患有严重听力损失或平衡障碍的患者的标准程序为了规避这一挑战，我们最近建立了一个明确的 3D 培养系统，以有效生成人类内耳感觉上皮细胞来自人类多能干细胞聚集体，这些所谓的“人类内耳类器官”含有一层。基于我们最初的研究，由感觉神经元支配的紧密排列的支持细胞和毛细胞组成。通过表征，这些人类干细胞衍生的毛细胞表现出结构、生化和功能与天然感觉毛细胞的特性相当本应用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类器官中的感觉细胞分化在目标 1 中，我们将测试 PAX2 阳性耳部如何分化。利用单细胞 RNA-seq、ChIP- 的组合，祖细胞在内耳中产生不同的细胞类型。 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于毛细胞和感觉神经元的规范至关重要在目标 2 中，我们将阐明前庭与耳蜗的独特转录途径。规范并确定腹侧耳毛细胞的生化和结构特性在目标 3 中，我们将定义毛细胞分化的时间进程（例如发束和毛细胞）。在光和电子显微镜水平上人类内耳类器官中的带状突触发育）。此外，结合单细胞电生理学和光遗传学，我们将测试人类是否干细胞衍生的毛细胞与感觉神经元建立功能性突触连接，同时此外，利用酵母双杂交筛选，我们将鉴定新的蛋白质-蛋白质。对于发束形成至关重要的相互作用通过实现这些目标，我们不仅会提高我们的发束形成。了解人类内耳发育的生物学，还建立一个明确的和可扩展的人体模型系统，用于研究各种形式的遗传性内耳疾病的发病机制并鉴定具有再生人类毛细胞潜力的化合物。