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受到特化小生境、结构复杂的微环境的调节，所述微环境以可溶性因子、细胞外基质（ECM）和来自邻近细胞的促肾上腺素因子的形式向其驻留干细胞呈现许多线索。例如，在成年哺乳动物脑内，海马体中的神经干细胞（NSC）不断分裂以产生在学习和记忆中发挥作用的新神经元，并且这些细胞受到来自邻近星形胶质细胞的生长因子、形态发生素、ECM和肾上腺素信号的调节。深入了解这些小生境调节干细胞的机制可以帮助开发用于在转化医学应用中扩增和分化干细胞的体外生物材料培养系统。现在广泛认识到，除了其生物化学性质之外，壁龛的机械性质（例如，刚度）也可以有力地调节干细胞的行为，事实上，我们最近表明，这对神经干细胞是正确的。然而，一般来说，该领域缺乏关键的分子和机制的信息，干细胞如何处理这样的机械线索在细胞-ECM界面，以引起细胞命运的变化，这些mechanotransductive信号如何与传统上理解为控制过程，如神经发生的转录事件接口，以及mechanotransductive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研究机械力对细胞粘附受体和细胞骨架的作用。具体来说，我们将进行生物物理和生化测量，以分析如何从细胞微环境的机械信息是通过细胞传播-包括粘附受体，粘着斑蛋白，和非肌肉肌球蛋白II -作为细胞命运的决定。此外，目标2将研究底物硬度如何影响NeuroD的激活以控制神经元分化。我们将通过量化动力学来实现这一点，其中编码关键前神经转录因子的基因接收并整合上游机械信号，因为细胞致力于神经元的命运。在这个目标中的创新工具，新的修改后的申请，是使用合成ECM，其刚度可以动态和可逆地切换。这一修订后的申请还包括新的数据，证明我们有能力通过在大鼠模型中遗传操纵机械转导信号来控制体内神经发生。因此，在这两个目标中，我们将应用这种能力来确定是否在体外机械敏感的命运选择中涉及的信号转导效应子也在体内调节这一过程。总之，该提案融合了干细胞生物学，机械生物学和材料合成，以开发定量，机械的干细胞机械调节的见解，与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的影响。

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.