Mechanisms of Neural Stem Cell Mechanoregulation

描述（由申请人提供）：干细胞自我更新和分化的过程在很大程度上是由专门的壁龛、结构复杂的微环境调节的，这些微环境以可溶性因子、细胞外基质（ECM）和邻近细胞的近胞因子的形式向其驻留的干细胞提供大量线索。例如，在成年哺乳动物大脑中，海马中的神经干细胞（NSCs）不断分裂产生新的神经元，这些神经元在学习和记忆中发挥作用，这些细胞受生长因子、形态因子、ECM和邻近星形胶质细胞的近胞嘧啶信号的调节。深入了解这些小生境调节干细胞的机制有助于开发体外生物材料培养系统，以扩大和分化干细胞在转化医学中的应用。现在人们普遍认识到，除了其生化特性外，生态位的机械特性（例如，硬度）也可以有效地调节干细胞的行为，事实上，我们最近证明了NSCs也是如此。然而，总的来说，该领域缺乏关于干细胞如何在细胞- ecm界面处理这些机械信号以引起细胞命运变化的关键分子和机制信息，这些机械转导信号如何与传统上理解的转录事件相结合以控制神经发生等过程，以及机械转导信号是否也可以控制体内神经发生。在本提案中，我们将解决所有这些悬而未决的问题。目的1将研究细胞粘附受体和细胞骨架的机械转导动力学。具体来说，我们将进行生物物理和生化测量来分析来自细胞微环境的机械信息是如何通过细胞传播的——包括粘附受体、局灶粘附蛋白和非肌肉肌球蛋白II——作为细胞命运的决定。此外，Aim 2将研究底物硬度如何影响NeuroD的激活以控制神经元分化。我们将通过量化编码关键前神经转录因子的基因在细胞承诺神经元命运时接收和整合上游机械信号的动力学来做到这一点。在这方面的一个创新工具，新修订的应用，是使用合成ecm，其刚度可以动态和可逆地切换。这个修订后的应用程序还包括新的数据，证明了我们在大鼠模型中通过基因操纵机械传导信号来控制体内神经发生的能力。因此，在这两个目的中，我们将应用这种能力来确定与体外机械敏感性命运选择有关的信号效应物是否也在体内调节这一过程。综上所述，本研究将干细胞生物学、机械生物学和材料合成相结合，对干细胞机械调控进行定量的、机械的研究，对基础干细胞生物学和再生医学先进生物材料系统的未来发展具有重要意义。

DESCRIPTION (provided by applicant): The processes of stem cell self-renewal and differentiation are regulated in large part by specialized niches, structurally complex microenvironments that present their resident stem cells with numerous cues in the form of soluble factors, extracellular matrix (ECM), and juxtacrine factors from neighboring cells. Within the adult mammalian brain, for example, neural stem cells (NSCs) in the hippocampus continuously divide to give rise to new neurons that play roles in learning and memory, and these cells are regulated by growth factors, morphogens, ECM, and juxtacrine signals from neighboring astrocytes. Gaining deeper insights into the mechanisms through which these niches regulate stem cells can aid in the development of in vitro biomaterials culture systems for expanding and differentiating stem cells in translational medicine applications. It is now widely appreciated that in addition to its biochemical properties, the mechanical properties of the niche (e.g., stiffness) can also powerfully regulate stem cell behavior, and indeed we recently showed that this true of NSCs. However, in general the field lacks key molecular and mechanistic information about how stem cells process such mechanical cues at the cell-ECM interface to give rise to changes in cell fate, how these mechanotransductive signals interface with transcriptional events traditionally understood to control processes such as neurogenesis, and whether mechanotransductive signaling can also control neurogenesis in vivo. In this proposal we will address all of these open questions. Aim 1 will investigate the dynamics of mechanotransduction to cellular adhesion receptors and the cytoskeleton. Specifically, we will conduct biophysical and biochemical measurements to analyze how mechanical information from the cellular microenvironment is propagated through cells - including adhesion receptors, focal adhesion proteins, and nonmuscle myosin II - as cell fate decisions are made. In addition, Aim 2 will investigate how substrate stiffness impacts the activation of NeuroD to control neuronal differentiation. We will do this by quantifying the dynamics by which the gene encoding a key proneural transcription factor receives and integrates upstream mechanical signals as cells commit to a neuronal fate. An innovative tool in this aim, new to this revised application, i the use of synthetic ECMs whose stiffness may be dynamically and reversibly switched. This revised application also includes new data demonstrating our ability to control neurogenesis in vivo by genetically manipulating mechanotransductive signals in a rat model. Thus, in both aims we will apply this capability to determine whether signaling effectors implicated in mechanosensitive fate choice in vitro also regulate this process in vivo. In summary, this proposal blends stem cell biology, mechanobiology, and materials synthesis to develop quantitative, mechanistic insights into stem cell mechanoregulation, with implications for both basic stem cell biology and the future development of advanced biomaterials systems for regenerative medicine.