Tympanic membrane progenitor cells in homeostasis in injury

鼓膜祖细胞在损伤中的稳态

• 批准号: 10599161
• 负责人: Aaron Tward
• 金额: $ 56.16万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-25 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10599161
• 关键词: Ablation; Adult; Air; Algorithms; Architecture; Behavior; Bioinformatics; Biological; Biological Models; Cell Differentiation process; Cell Separation; Cell physiology; Cells; Cholesteatoma; Chronic; Cluster Analysis; Connective Tissue; Data; Defect; Development; Disease; Dissection; Dissociation; Dyes; Ear; Ear Diseases; Environment; Epithelium; External auditory canal; Foundations; Gene Expression Profile; Hearing; Heterogeneity; Homeostasis; Human; Immunohistochemistry; In Situ Hybridization; Individual; Injury; Keratosis; Knockout Mice; Knowledge; Label; Labyrinth; Learning; Liquid substance; LoxP-flanked allele; Maintenance; Malleus; Mammals; Mesenchymal; Modeling; Molecular; Movement; Mucous Membrane; Mus; Operative Surgical Procedures; Organoids; Otitis; PDGFRB gene; Pathologic; Patients; Perforation; Physiologic pulse; Physiology; Platelet-Derived Growth Factor alpha Receptor; Population; Procedures; Process; Proliferating; Radial; Reconstructive Surgical Procedures; Reporter; Research; Signal Pathway; Stains; Stratified Squamous Epithelium; Structure; System; Therapeutic; Traction; Tympanic Membrane Perforation; Tympanic membrane; Tympanostomy; biophysical properties; cell type; design; experimental study; external ear auricle; healing; improved; in vivo; inhibitor; injured; keratinocyte; live cell imaging; middle ear; migration; monomer; mouse model; novel; pharmacologic; progenitor; repaired; response to injury; restoration; single-cell RNA sequencing; small molecule; small molecule inhibitor; sound; stem; stem cells; tool; translation to humans; vibration

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.

项目摘要 哺乳动物的听力取决于有效地进行声音振动的能力 内耳的环境。该传导设备包括耳膜，外部 听觉运河，鼓膜膜（TM），中耳空间和骨链。虽然 已知的大量研究已针对耳朵的生物物理特性。 关于这些结构的细胞组成以及如何组成这些结构的各种细胞 结构在病理状态中形成，维持和相互作用。 TM有三层： 分层鳞状上皮的外层，结缔组织的中间层和一个 粘膜上皮的内层。未知在 这些层的茎或祖细胞种群驻留在这些层中，或 这些层如何保持的动力。经典染料研究表明外部 TM的上皮从碎石附着到TM上径向向外迁移。我们和 其他人则表明，在TM中，绝大多数扩散发生在附近 麦龙，然后该细胞向外迁移。这意味着至少两个人群： 疟疾附近的茎/祖先种群，径向部分的后代人口 TM。我们将首先从小鼠和人类，分类细胞中解散正常和受伤的TMS， 并执行单细胞RNA测序分析与最近的邻居聚类结合 使用新型算法CellFindR分析，以鉴定细胞亚群 TM并预测它们之间的血统关系。我们将通过 使用小鼠和人类TMS的免疫荧光染色。然后，我们将执行脉搏练习 使用EDU以及与转基因小鼠的谱系追踪进行标记实验 验证通过suedotime分析预测的谱系层次结构，并明确识别 穿孔修复过程中TM的茎和祖细胞种群。最后，我们会扰动 使用诱导和 细胞特异性敲除小鼠体内以及新型的离体中定义的小分子抑制剂 为了定义epplanted小鼠和人类TM的空气液体接口模型 这些人群分化和维护并提供的机制 治疗概念证明。我们希望一旦我们深入了解 TM中的功能性蜂窝结构和生理学，然后我们可以了解这些 过程出现了，并为疾病的疾病创造更好的生物学和外科治疗方法 TM。