Tympanic membrane progenitor cells in homeostasis in injury

项目摘要 哺乳动物的听力取决于有效传导声音振动的能力， 环境到内耳。该传导装置包括耳廓、外 耳道、鼓膜（TM）、中耳腔和听骨链。虽然 大量的研究已经指向耳朵的生物物理特性， 关于这些结构的细胞组成以及组成这些结构的不同细胞 结构在病理状态下形成、维持并相互作用。技术备忘录有三层： 外层为复层鳞状上皮，中层为结缔组织， 粘膜上皮内层。目前还不清楚有多少不同的细胞类型存在于 这些层中的每一层，其中这些层的干细胞或祖细胞群驻留，或 这些层是如何维持的。经典的染料研究表明， TM的上皮从踝关节附着处径向向外迁移到TM。我们和 其他人已经表明，在TM内，绝大多数增殖发生在TM附近， 锤骨，然后细胞径向向外迁移。这意味着至少有两个群体： 锤骨附近的茎/祖细胞群体，以及径向部分内的后代群体 的TM。我们将首先从小鼠和人类中分离出正常和受损的TM，对细胞进行分类， 并结合最近邻聚类进行单细胞RNA测序分析 分析使用一种新的算法，CellfindR，以确定细胞亚群内 并预测它们之间的谱系关系。我们将确认这些人口， 使用小鼠和人TM的免疫荧光染色。然后我们将执行脉冲追踪 使用EdU的标记实验以及用遗传修饰的小鼠进行谱系追踪， 验证伪时间分析预测的谱系层次结构，并明确识别 穿孔修复期间TM的干细胞和祖细胞群。最后，我们将扰动 使用可诱导的， 细胞特异性敲除小鼠体内以及定义的小分子抑制剂在一种新的离体 空气-液体界面模型，以定义 这些种群分化和维持的机制，并提供一个 概念的治疗验证。我们希望，一旦我们更深入地了解了 功能性细胞结构和TM内的生理学，然后我们可以了解这些 过程出错，并创造更好的生物和外科治疗疾病的 TM.

Project Summary Hearing in mammals is dependent upon the ability to efficiently conduct sound vibrations from the environment to the inner ear. This conduction apparatus includes the auricle, external auditory canal, tympanic membrane (TM), middle ear space, and ossicular chain. Although a great deal of research has been directed to the biophysical properties of the ear less is known about the cellular composition of these structures and how the diverse cells that make up these structures are formed, maintained, and interact in pathological states. The TM has three layers: an outer layer of stratified squamous epithelium, a middle layer of connective tissue, and an inner layer of mucosal epithelium. It is unknown how many different cell types are present in each of these layers, where the stem or progenitor cell populations of these layers reside, or the dynamics of how these layers are maintained. Classic dye studies indicated that the outer epithelium of the TM migrates radially outward from the malleal attachment to the TM. We and others have shown that within the TM the vast majority of the proliferation is occurring near the malleus, and that cells then migrate radially outward. This implies at least two populations: a stem/progenitor population near the malleus, and a progeny population within the radial portions of the TM. We will first dissociate normal and injured TMs from mice and humans, sort cells, and perform single cell RNA sequencing analysis combined with nearest-neighbor clustering analysis using a novel algorithm, CellfindR, in order to identify the cellular subpopulations within the TM and to predict lineage relationships between them. We will confirm these populations by using immunofluorescent staining of mouse and human TMs. We will then perform pulse-chase labelling experiments using EdU as well as lineage tracing with genetically modified mice to validate the lineage hierarchies predicted by the psuedotime analysis, and definitively identify the stem and progenitor populations of the TM during perforation repair. Finally, we will perturb the PDGFR and BMP signalling pathway in defined populations of the TM using inducible and cell specific knockout mice in vivo as well as defined small molecule inhibitors in a novel ex vivo air-liquid interface model of explanted mouse and human TMs in order to define the mechanisms by which these populations differentiate and are maintained and to provide a therapeutic proof of concept. We hope that once we gain a deeper understanding of the functional cellular architecture and physiology within the TM, we can then learn how these processes go awry in and create better biological and surgical treatments for disorders of the TM.