Matrix-induced Myogenesis & Pharmaco-Screens of MSCs

描述(申请人提供)：基质诱导的肌肉发生和MSCs间充质干细胞(MSC)的药物筛选能够分化为许多组织谱系，包括肌肉，导致相信这些细胞有助于内环境平衡修复过程。在许多疾病中，包括肌营养不良，组织基质异常，祖细胞对修复的贡献不足。我们的目标是开发正常和患病的肌肉发生的组织模拟细胞培养模型，重点关注正常和异常基质弹性的影响，以便评估已知的药物(例如。强的松龙)，并最终筛选NIH文库中引导干细胞基质偶联肌肉发生的候选药物。作为一种特定的疾病，我们也关注核营养不良，因为它与肌肉有关，也是为了更深入地了解分化机制。核层与染色质相互作用，并通过横跨被膜的连接蛋白与细胞骨架相互作用。我们最近发现，在骨骼肌细胞的细胞肌球蛋白横纹过程中，细胞骨架受到基质弹性的强烈影响[Enger J.Cell Biology 2004]，以及在MSCs的多系分化过程中[Enger Cell 2006]。在这些细胞和大多数其他组织细胞中，与基质的黏附在物理上与基于肌球蛋白的收缩有关，因此从基质通过细胞骨架形成了一种连接，进入了正常的细胞核。这种从核-细胞骨架-基质的连续连接使基质弹性成为关键的生物输入。因此，这个R21‘S“开发和探索”的目标将(1)使我们的弹性矩阵适应低通量和高通量筛选模式，并最终在异常基质(机械地模仿纤维化)的正常人MSC上筛选新型的肌肉分化诱导剂，以及(2)为筛选和验证核缺陷强烈影响肌源性分化和核可塑性的假说而建立肌源性疾病模型。在血清培养液中，我们将使用基质弹性来控制正常MSC的肌源性分化，重要的是，我们还将模拟疾病中典型的僵硬和纤维化组织。同样的基质系统可以与刚性基质上的成骨作用和软的、模拟大脑的基质上的神经发生进行比较。可溶性诱导因子(如增强或抑制血统的糖皮质激素)的安全性和有效性将被筛选和研究，其特定目标是促进肌肉生成。我们还将设计人类MSC来表达已知的营养不良引起的突变，如Lamin-A/C中的突变，同时击倒内源性蛋白以减少过度表达的伪影。随着筛选出用先导药物治疗的特定分化状态和核突变，对完整细胞内蛋白质折叠和核组件(如层粘连蛋白)结合状态的洞察将通过一种新的蛋白质组级半胱氨酸鸟枪标记策略来探索，该策略利用了定量质谱学方法[Johnson 2007]。 公共卫生相关性：我们建议开发一种弹性基质培养格式，用于多孔筛选影响成人来源的间充质干细胞肌肉生成的可溶性因素。不同的弹性矩阵模拟了正常和疾病状态下组织力学性能的影响变化。我们将把筛选应用于异常基质(机械模拟纤维化)上的正常人类干细胞，并最终应用于表达已知的肌肉营养不良导致突变的细胞，如Lamin-A/C中的突变。目标将是了解分化的机制块，还将筛选已知的化合物，并最终筛选覆盖基质信号并促进疾病细胞分化的文库。

DESCRIPTION (provided by applicant): Matrix-induced Myogenesis & Pharmaco-Screens of MSCs Mesenchymal Stem Cells (MSC) are capable of differentiating into many tissue lineages, including muscle, leading to the belief that such cells contribute to homeostatic repair processes. In many diseases, including muscular dystrophies, tissue matrix is abnormal and progenitor contributions to repair are inadequate. Our goal is to develop tissue-mimetic cell culture models of normal and diseased myogenesis, focused on the influence of normal and abnormal matrix elasticity, in order to assess known drugs (eg. Prednisolone) and ultimately screen an NIH library for drug candidates that steer matrix-coupled myogenesis of stem cells. As a specific disease, we also focus on nucleo-dystrophies because of relevance to muscle and also for deeper insight into differentiation mechanisms. The nuclear lamina interacts with chromatin and through linker proteins that span the envelope the lamina also interacts with the cytoskeleton. We have shown recently that the cytoskeleton is strongly influenced by matrix elasticity in acto-myosin striation of skeletal muscle cells [Engler J. Cell Biology 2004] as well as in multi-lineage differentiation of MSCs [Engler Cell 2006]. Adhesion to matrix physically couples to myosin-based contractility in these cells and most other tissue cells, and so a linkage is formed from the matrix through the cytoskeleton and into the normal nucleus. This continuous linkage from nucleus~cytoskeleton~matrix makes MATRIX ELASTICITY A CRITICAL BIOLOGICAL INPUT. This R21's "develop and explore" objectives will therefore (1) adapt our elastic matrices to both low & high throughput screening formats and eventually screen for novel myo-differentiation inducers on normal human MSC on abnormal matrix (that mechanically mimics fibrosis), and will (2) develop a myogenic disease model both for the screen and to test the hypothesis that nuclear defects strongly influence myogenic differentiation and nuclear plasticity. In just serum media, we will use matrix elasticity to control myogenic differentiation of normal MSC and also, importantly, to mimic rigid and fibrotic tissues typical in disease. The same matrix system allows comparisons to osteogenesis on rigid matrices and neurogenesis on soft, brain- mimetic matrices. Soluble induction factors (eg. glucocorticoids) that enhance or inhibit the lineages will be screened and studied for safety & efficacy with a particular goal to promote myogenesis. We will also engineer human MSC to express known dystrophy causing mutants such as those in Lamin-A/C, while knocking down endogenous protein to minimize over-expression artifacts. With select differentiated states and nuclear mutants treated with lead drugs from the screen, insights into protein folding and association state of nuclear components such as lamins within the intact cell will be probed by a novel proteomic-scale Cys Shotgun labeling strategy that exploits quantitative Mass Spectrometry methods [Johnson 2007]. PUBLIC HEALTH RELEVANCE: We propose to develop an elastic matrix culture format for multi-well screens of soluble factors that influence myogensis of adult-derived mesenchymal stem cells. The various elastic matrices mimic the influential variation of tissue mechanical properties in normal and diseased states. We will apply the screen to normal human stem cells on abnormal matrices (that mechanically mimic fibrosis), and ultimately to re- engineered cells expressing known muscular dystrophy-causing mutants such as those in Lamin-A/C. A goal will be to understand mechanistic blocks to differentiation and also to screen known compounds and eventually libraries that over-ride matrix signals and promote differentiation in diseased cells.