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

项目总结 大脑中的神经干细胞(NSCs)在一生中增殖并生成新生的神经元。功能障碍 神经发生与癫痫、抑郁症和阿尔茨海默氏症等神经系统疾病有关 疾病。在成年神经发生中，一个重要的限速步骤是NSC静止退出，当一个非分裂 静止的神经干细胞(QNSC)在种群扩张和分化之前进入细胞周期。此外，在 衰老和疾病，外在和内在因素促使神经干细胞更深地进入静止状态，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 退出对于改善神经发生和增强认知功能至关重要。 目前我们对NSC静止的理解还不完整，这是由于 用于分离每个NSCs群体的标记的偏差以及缺乏活细胞标记策略。然而， 最近，我们观察到不同的光学特征分离激活的神经干细胞(ANSCs)和qNSCs 荧光寿命成像(FLIM)和两个信号的相对丰度：1)代谢物NAD(P)H，和 2)溶酶体内的自体荧光(LAF)，我们称之为光学细胞状态成像(OCSI)。OCSI 是一种非侵入性工具，能够随着时间的推移跟踪活细胞中的NSC细胞状态，而不需要外源标记。OCSI 从每个细胞收集两种数据：NAD(P)H和LAF的相对丰度通过荧光 强度和来自NAD(P)H和LaF的荧光光子的衰减率。这个衰减率可以 基于荧光团与蛋白质配对的结合或化学状态的变化，这取决于代谢 给定细胞使用的路径。重要的是，许多研究表明qNSCs和aNSCs优先依赖于 不同类型的细胞新陈代谢来产生能量。使用降维分析方法对8种数据进行降维 用OCSI在幼年小鼠神经干细胞中收集的测量结果，我们不仅识别出区分 QNSCs和aNSCs，并通过活细胞成像跟踪这些指标的动态变化 静止期退出，而且还根据这种自动荧光信号对神经干细胞进行前瞻性分类，以成功预测 它们的增殖行为和来自体外培养和急性分离的神经干细胞的身份。这些结果揭示了 OCSI是一种新的工具，它使用细胞的能量学来定义其细胞状态，使我们能够公正地解决 关于NSC静止和激活的悬而未决的问题，以促进我们对这些过程的理解。 我们在这里建议1)确定与LAF相关的分子信号，LAF是 OCSI的预测能力，2)确定当前NSC识别目标的方法中哪些静止群体， 3)开发并验证了基于FLIM的细胞分选器，以提高未来研究的吞吐量 保持静止细胞状态到激活细胞状态的高分辨率分离。完成这些目标将 提供一种新的工具，并建立OCSI作为一种方法来回答有关机制和 NSC静止和激活的基础调控因子，可以靶向驱动NSC的增殖。

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.