Profiling the transcriptome of globose basal cells of the olfactory epithelium at the single cell level

在单细胞水平上分析嗅上皮球状基底细胞的转录组

• 批准号: 9226320
• 负责人: JAMES E. SCHWOB
• 金额: $ 24.75万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9226320
• 关键词: Acceleration; Activities of Daily Living; Address; Adult; Age; Aging; Antibodies; Area; Basal Cell; Bioinformatics; Biological; CRISPR/Cas technology; Categories; Cell Lineage; Cell Proliferation; Cell physiology; Cells; Chimeric Proteins; Chronic; Data; Development; Disease; Doxycycline; Elderly; Environment; Epithelial; Epithelium; Exhibits; Exploratory/Developmental Grant; Expression Library; Functional disorder; Future; Gene Expression Profile; Genes; Goals; Grant; Hand; Harvest; Histones; Homeostasis; Immunohistochemistry; In Situ Hybridization; Individual; Injury; Kinetics; Knock-in; Knock-out; Knowledge; Label; Learning; Liquid substance; Mediating; Metaplasia; Mission; Mitotic; Molecular; Molecular Profiling; Mouse Strains; Multipotent Stem Cells; Mus; National Institute on Deafness and Other Communication Disorders; Natural regeneration; Nature; Neurons; Nose; Nutritional status; Odors; Olfactory Basal Cell; Olfactory Epithelium; Pathologic; Pathology; Population; Positioning Attribute; Proliferating; Quality of life; Recovery; Role; Safety; Sensory; Smell Perception; Stem cells; Structure of respiratory epithelium; TCF3 gene; Testing; Thymidine; Tissues; Transgenic Mice; Transgenic Organisms; adult stem cell; aged; analog; cell type; design; exhaustion; experience; homologous recombination; insight; molecular marker; neurogenesis; neuron loss; olfactory sensory neurons; progenitor; reconstitution; respiratory; single cell analysis; stem; stem cell biology; stemness; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

The capacity of the olfactory epithelium (OE) for replenishing the population of olfactory sensory neurons and for regenerating the epithelium after injury depends on the persistence and maintained function of stem cells within that adult epithelium. Decline in sensory function in the elderly is accompanied by pathological changes in the OE that emerge because the normally active olfactory stem and progenitor cells, namely globose basal cells (GBCs), become disordered and eventually depleted. At present, we can construct a flow diagram designed to encompass and sequence the various categories of GBCs, beginning with GBCs that function as multipotent progenitors, progressing through GBCs that act as transit amplifying progenitors, and reaching GBCs that give rise directly to neurons. We can also align those purported stages in the GBC hierarchy with the expression of various transcription factors. However, we lack a comprehensive understanding of this critical stem/progenitor cell population. Are these stages discrete or are they snapshots of a more fluid progression? How do we explain the plasticity in the progenitor capacity of some GBCs? How might aging disorder the progression? The current application proposes two specific aims designed to address the critical gaps in our knowledge of the active stem and progenitor cell population. Specific Aim 1 will generate transcriptomic profiles of the various functional categories of GBCs using transgenic mouse strains that express a fluorescent marker in conjunction with the transcription factor(s) gene(s) – Sox2, Ascl1, and Neurog1 – used to define the stages in the hierarchy. Specific Aim 2 will focus on and profile a specific kinetically- defined subset of GBCs that experience prolonged mitotic quiescence, as shown by the retention of the tagged histone fusion protein H2B/GFP, which is a feature common to many stem cell types. In both cases, cells labeled by expression of the fluorescent tags will be isolated by FACS, captured as single cells, and individually profiled. Analysis of the single cell expression libraries will be used for unbiased clustering of the cells and defining the genes whose expression differs across the clusters. The patterns of gene expression that differentiate the clusters will be validated by Q-PCR and either immunohistochemistry or in situ hybridization depending on antibody availability. At the conclusion of the analysis we will have achieved a comprehensive understanding of the GBC population and will clarify whether the existing flow diagram – which envisions distinct stages and abrupt transitions – is an accurate representation of the biology of this stem and progenitor cell-encompassing population. These advances, in turn, will inform our attempts to alleviate olfactory sensory dysfunction, particularly that which accompanies aging.

嗅觉上皮 (OE) 补充嗅觉感觉神经元数量的能力 损伤后上皮的再生取决于干细胞的持久性和维持功能 在成人上皮内。老年人感觉功能下降伴有病理变化 在 OE 中，由于正常活跃的嗅觉干细胞和祖细胞（即球状基底细胞）而出现 细胞（GBC）变得紊乱并最终耗尽。目前我们可以构建一个流程图 旨在涵盖和排序不同类别的 GBC，从具有以下功能的 GBC 开始： 多能祖细胞，通过充当转运放大祖细胞的 GBC 进展，并达到 GBC 直接产生神经元。我们还可以将 GBC 层次结构中的那些所谓的阶段与 各种转录因子的表达。但我们对此缺乏全面的认识 关键干/祖细胞群。这些阶段是离散的还是更流畅的快照？ 进展？我们如何解释某些 GBC 祖细胞能力的可塑性？怎么会衰老 扰乱进展？当前的申请提出了两个旨在解决关键问题的具体目标 我们对活性干细胞和祖细胞群的了解存在差距。具体目标 1 将生成 使用转基因小鼠品系对 GBC 的各种功能类别进行转录组分析 与转录因子基因（Sox2、Ascl1 和 Neurog1）结合表达荧光标记 – 用于定义层次结构中的阶段。具体目标 2 将重点关注并描述特定的动力学- 定义的 GBC 子集经历长期有丝分裂静止，如标记的保留所示 组蛋白融合蛋白 H2B/GFP，这是许多干细胞类型的共同特征。在这两种情况下，细胞 通过荧光标签的表达进行标记的将通过FACS分离，捕获为单细胞，并且 单独分析。单细胞表达文库的分析将用于对单细胞表达文库进行无偏聚类。 细胞并定义其表达在簇中不同的基因。基因表达模式 区分簇的方法将通过 Q-PCR 和免疫组织化学或原位验证 杂交取决于抗体的可用性。在分析结束时，我们将获得 全面了解 GBC 群体，并将澄清现有的流程图 – 哪个 设想了不同的阶段和突然的转变 - 是该茎的生物学的准确表示 包含祖细胞的群体。这些进步反过来又将为我们减轻压力的尝试提供信息 嗅觉感觉功能障碍，尤其是伴随衰老的嗅觉功能障碍。