Label-free, live-cell classification of neural stem cell activation state and dynamics

神经干细胞激活状态和动力学的无标记活细胞分类

• 批准号: 10863309
• 负责人: Darcie Leann Moore
• 金额: $ 56.76万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10863309
• 关键词: Acute; Address; Aging; Agreement; Alzheimer' s Disease; Behavior; Binding; Brain; Breeding; Cell Cycle; Cell Fractionation; Cell Separation; Cells; Chemicals; Classification; Data; Detection; Dimensions; Disease; Epilepsy; Fluorescence; Functional disorder; Future; GLAST Protein; Genetic Recombination; Glial Fibrillary Acidic Protein; Hippocampus; Image; Impaired cognition; In Vitro; Injections; Intrinsic factor; Knowledge; Label; Life; LoxP-flanked allele; Lysosomes; Measures; Mental Depression; Metabolic Pathway; Metabolism; Methods; Molecular; Mus; Optics; Organelles; Pathway interactions; Photons; Population; Process; Production; Proliferating; Property; Proteins; Quality of life; Regulation; Reporter; Resolution; Signal Transduction; Societies; Sorting; Tamoxifen; Techniques; Testing; Time; Transcript; Transgenic Mice; Viral; Visualization; adult neurogenesis; cell type; cognitive enhancement; cognitive function; fluorescence lifetime imaging; fluorophore; imaging modality; improved; indexing; insight; lipid metabolism; lipidomics; live cell imaging; metabolomics; nerve stem cell; nervous system disorder; nestin protein; neurogenesis; neuroregulation; newborn neuron; novel; prospective; prototype; single-cell RNA sequencing; stem cell proliferation; stem cells; tool

PROJECT SUMMARY Neural stem cells (NSCs) in the brain proliferate and generate newborn neurons throughout life. Dysfunctions in neurogenesis have been associated with neurological diseases such as epilepsy, depression, and Alzheimer’s Disease. A significant rate-limiting step in adult neurogenesis is NSC quiescence exit, when a non-dividing quiescent NSC (qNSC) enters the cell cycle prior to population expansion and differentiation. Further, during aging and disease, extrinsic and intrinsic factors drive NSCs deeper into quiescence, reducing neurogenesis, and contributing to cognitive decline. Therefore, identifying factors controlling NSC quiescence and quiescence exit are critical to improving neurogenesis and enhancing cognitive function. Currently our understanding of NSC quiescence is incomplete due to technical limitations imposed by the bias of markers used to isolate each population of NSCs and the lack of live cell labeling strategies. However, recently we observed distinct optical signatures separating activated NSCs (aNSCs) from qNSCs using fluorescence-lifetime imaging (FLIM) and the relative abundance of two signals: 1) the metabolite NAD(P)H, and 2) autofluorescence within lysosomes (LAF), a technique we refer to as optical cell state imaging (OCSI). OCSI is a non-invasive tool capable of tracking NSC cell state in living cells over time, without exogenous label. OCSI collects 2 types of data from each cell: the relative abundance of NAD(P)H and LAF through fluorescence intensity, and a decay rate of fluorescent photons from NAD(P)H and LAF using FLIM. This decay rate can change based on fluorophore binding to protein partners or chemical state, which is dependent on the metabolic pathways used by a given cell. Importantly, many studies have shown that qNSCs and aNSCs preferentially rely on different types of cellular metabolism for generating energy. Using dimension reduction analyses of the 8 measures collected with OCSI in young mouse NSCs, we have not only identified distinct signatures separating qNSCs and aNSCs and tracked the dynamic changes of these measures through live cell imaging during quiescence exit, but also prospectively sorted NSCs based on this autofluorescent signal to successfully predict their proliferative behavior and identity from in vitro cultures and acutely isolated NSCs. These results reveal OCSI as a novel tool that uses the energetics of a cell to define its cell state, allowing us to unbiasedly address unanswered questions about NSC quiescence and activation to advance our understanding of these processes. We here propose to 1) identify the molecular signal associated with LAF, one of the primary contributors to OCSI’s predictive ability, 2) determine which quiescent populations current methods of NSC identification target, and 3) develop and validate a FLIM-based cell sorter to increase the throughput for future studies while maintaining the high-resolution separation of quiescent to activated cell states. Completion of these Aims will provide a novel tool and establish OCSI as a method to answer critical questions regarding the mechanisms and regulators underlying NSC quiescence and activation that can be targeted to drive NSC proliferation.

项目摘要 大脑中的神经干细胞（NSC）在一生中增殖并产生新生神经元。功能障碍 在神经发生中，与癫痫，抑郁症和阿尔茨海默氏症等神经系统疾病有关 疾病。成人神经发生的显着限速步骤是NSC静止出口，当 静态NSC（QNSC）在种群扩张和分化之前进入细胞周期。此外，期间 衰老和疾病，外在和内在因子将NSC逐渐深入静止，减少神经发生， 并导致认知能力下降。因此，识别控制NSC静止和静止的因素 出口对于改善神经发生和增强认知功能至关重要。 目前，由于技术限制，我们对NSC静止的理解是不完整的 标记的偏差用于隔离NSC的每个群体和缺乏现场细胞标记策略。然而， 最近，我们观察到使用QNSC分隔活化的NSC（ANSC）的独特光学特征 荧光成像（FLIM）和两个信号的相对抽象：1）代谢物NAD（P）H，并且 2）溶酶体（LAF）内的自动荧光，我们称为光细胞态成像（OCSI）。 OCSI 是一种无创的工具，能够随着时间的推移在活细胞中跟踪NSC细胞状态，而无需外源标签。 OCSI 从每个细胞中收集2种数据类型：通过荧光的NAD（P）H和LAF的相对抽象 使用FLIM的强度和来自NAD（P）H和LAF的荧光照片的衰减速率。这个衰减率可以 基于荧光团结合与蛋白质伴侣或化学状态的变化，这取决于代谢 给定单元使用的途径。重要的是，许多研究表明，QNSC和ANSC优先依赖 在不同类型的细胞代谢上产生能量。使用8尺寸分析8 用OCSI收集的年轻小鼠NSC的措施，我们不仅确定了分开的不同签名 QNSC和ANSC并通过活细胞成像跟踪这些度量的动态变化 静止出口，但也基于此自动荧光信号的前瞻性排序NSC，以成功预测 他们从体外培养物和急性分离的NSC中的增殖行为和身份。这些结果揭示了 OCSI是一种新颖的工具，它使用细胞的能量来定义其细胞状态，从而使我们无偏处理 关于NSC静止和激活的未解决问题，以提高我们对这些过程的理解。 我们在这里提出提示1）确定与LAF相关的分子信号，这是造成的主要贡献者之一 OCSI的预测能力，2）确定NSC识别目标的当前方法， 3）开发和验证一个基于FLIM的细胞分辨率，以增加未来研究的吞吐量 维持静止分离到活化细胞态的高分辨率分离。这些目标的完成将 提供一种新颖的工具，并建立OCSI作为回答有关机制和机制的关键问题的方法 可以针对驱动NSC增殖的NSC静止和激活的调节器。