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

项目摘要 脑中的神经干细胞（NSC）在整个生命过程中增殖并产生新生神经元。功能障碍 在神经发生中的作用与神经系统疾病如癫痫、抑郁症和阿尔茨海默氏症有关 疾病在成体神经发生中，一个重要的限速步骤是NSC退出静止期，当非分裂的神经元进入静止期时， 静止NSC（qNSC）在群体扩增和分化之前进入细胞周期。此外，在 衰老和疾病，外在和内在因素驱使NSC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， 出口对改善神经发生和增强认知功能至关重要。 目前，我们对NSC静止的理解是不完整的，这是由于技术上的限制。 用于分离每个NSC群体的标记物的偏差和缺乏活细胞标记策略。然而，在这方面， 最近，我们观察到不同的光学特征分离激活的神经干细胞（aNSC）从qNSC，使用 荧光寿命成像（FLIM）和两种信号的相对丰度：1）代谢物NAD（P）H，和 2)溶酶体内的自发荧光（LAF），一种我们称为光学细胞状态成像（OCSI）的技术。OCSI 是一种非侵入性工具，能够随时间跟踪活细胞中的NSC细胞状态，而无需外源性标记。OCSI 从每个细胞收集两种类型的数据：通过荧光检测NAD（P）H和LAF的相对丰度 强度和使用FLIM的来自NAD（P）H和LAF的荧光光子的衰减速率。这种衰减率可以 基于荧光团与蛋白质伴侣或化学状态结合的变化，这取决于代谢 特定细胞使用的途径。重要的是，许多研究表明，qNSC和aNSC优先依赖于 不同类型的细胞代谢来产生能量。利用8维的降维分析， 在年轻小鼠NSC中用OCSI收集的测量，我们不仅确定了分离NSC的不同特征， qNSC和aNSC，并通过活细胞成像跟踪这些措施的动态变化， 静止退出，而且还基于该自体荧光信号前瞻性地分选NSC，以成功地预测 它们的增殖行为和身份来自体外培养物和急性分离的NSC。这些结果揭示 OCSI作为一种新的工具，它使用细胞的能量来定义其细胞状态，使我们能够无偏见地解决 关于NSC静止和激活的未回答的问题，以促进我们对这些过程的理解。 我们在这里提出：1）识别与LAF相关的分子信号，LAF是导致LAF的主要因素之一。 OCSI的预测能力，2）确定目前NSC鉴定方法的目标是哪些静止群体， 以及3）开发和验证基于FLIM的细胞分选仪，以增加未来研究的吞吐量， 保持静止与活化细胞状态的高分辨率分离。实现这些目标将 提供了一种新的工具，并建立了OCSI作为一种方法来回答有关机制的关键问题， NSC静止和激活的潜在调节因子，其可以被靶向以驱动NSC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.