Tympanic membrane progenitor cells in homeostasis in injury

项目摘要 哺乳动物的听力依赖于有效地传导声音振动的能力 环境对内耳的影响。该传导装置包括耳廓、外 耳道、鼓膜、中耳间隙和听骨链。尽管一个 很多研究都是针对耳朵的生物物理特性进行的，但知之甚少 关于这些结构的细胞组成，以及组成这些结构的不同细胞 结构是在病理状态下形成、维持和相互作用的。TM有三层： 复层鳞状上皮的外层，结缔组织的中间层，以及 粘膜上皮内层。目前尚不清楚有多少不同类型的细胞存在于 这些层中的每一层，这些层的干细胞或祖细胞群体所在的地方，或者 这些层如何维护的动态。经典染料研究表明，外层 小腿上皮从踝部向外迁移到小腿上。我们和 其他人则表明，在TM内部，绝大多数的扩散发生在 然后这些细胞径向向外迁移。这意味着至少有两个群体：a 锤部附近的茎/祖代群体和放射部内的子代群体 在TM中。我们将首先从小鼠和人身上分离出正常和受损的TM，对细胞进行分类， 结合最近邻聚类进行单细胞RNA测序分析 使用一种新的算法CellfindR进行分析，以确定 并预测它们之间的血统关系。我们将通过以下方式确认这些人口 用小鼠和人TMS进行免疫荧光染色。然后我们将进行脉搏追逐 使用EDU的标记实验以及对转基因小鼠的血统追踪 验证PsuedoTime分析预测的世系等级，并最终确定 穿孔修复期间TM的茎和祖细胞种群。最后，我们将扰乱 在TM的特定种群中，使用可诱导的和 体内细胞特异性基因敲除小鼠以及新的体外定义的小分子抑制物 移植小鼠和人TMS的气液界面模型 这些种群分化和维持的机制，并提供一种 概念的治疗性证明。我们希望，一旦我们对此有了更深入的了解 在TM内的功能细胞架构和生理学，然后我们可以了解这些 过程出了问题，创造了更好的生物和外科治疗方法 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.