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)补充嗅觉神经元数量的能力和 损伤后上皮的再生依赖于干细胞的持久性和维持功能 在成体上皮内。老年人感觉功能减退伴有病理改变 在OE中出现的是因为正常活跃的嗅干和祖细胞，即球状基底细胞 细胞(GBCs)，变得无序并最终耗尽。目前，我们可以构建一个流程图 旨在涵盖各种类别的GBCS并对其进行排序，从具有以下功能的GBCS开始 多能祖细胞，通过充当转运性扩增祖细胞的GBCs进展，并达到 直接分化为神经元的基底节细胞。我们还可以将GBC层次结构中那些所谓的阶段与 各种转录因子的表达。然而，我们对此缺乏全面的认识 关键的干细胞/祖细胞群体。这些阶段是离散的，还是更具流动性的 进展？我们如何解释某些GBCs祖细胞能力的可塑性？老龄化可能会如何 扰乱进程？当前的应用程序提出了两个具体目标，旨在解决关键的 我们对活跃的干细胞和祖细胞群体的了解存在差距。特定目标1将生成 利用转基因小鼠品系对不同功能类别的GBCS转录图谱的研究 表达与转录因子(S)基因(S)-Sox2、Ascl1和Neurog1结合的荧光标记 -用于定义层次结构中的阶段。《特定目标2》将聚焦并描述一个特定的目标-- 已定义的经历长时间有丝分裂静止的GBCs子集，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 通过表达的荧光标记将被FACS分离，作为单个细胞捕获，以及 单独侧写的。对单细胞表达文库的分析将用于无偏聚类 细胞，并定义在不同簇中表达不同的基因。基因表达的模式 区分这些簇的方法将通过Q-PCR和免疫组织化学或原位验证 杂交取决于抗体的可获得性。在分析结束时，我们将实现 全面了解GBC人口，并将澄清现有的流程图- 设想不同的阶段和突然的转变-是对这种茎和 包括祖细胞的种群。反过来，这些进展将为我们提供信息，试图减轻 嗅觉功能障碍，尤指伴随年龄增长的嗅觉功能障碍。