Matrix-induced Myogenesis & Pharmaco-Screens of MSCs

间充质干细胞（MSC）能够分化成许多组织谱系，包括肌肉，导致人们相信这些细胞有助于体内平衡修复过程。在许多疾病中，包括肌肉萎缩症，组织基质异常，祖细胞对修复的贡献不足。我们的目标是建立正常和病变肌生成的模拟组织细胞培养模型，重点研究正常和异常基质弹性的影响，以便评估已知药物(如：强的松龙)，并最终在NIH文库中筛选引导基质偶联干细胞肌生成的候选药物。作为一种特殊的疾病，我们也关注核营养不良症，因为它与肌肉有关，也为了更深入地了解分化机制。核层与染色质相互作用，并通过跨越包膜的连接蛋白与细胞骨架相互作用。我们最近的研究表明，骨骼肌细胞肌动-肌球蛋白条纹中的细胞骨架受到基质弹性的强烈影响[Engler J. Cell Biology 2004]，以及MSCs的多系分化[Engler Cell 2006]。在这些细胞和大多数其他组织细胞中，与基质的粘附在物理上与肌球蛋白的收缩性结合在一起，因此形成了从基质穿过细胞骨架进入正常细胞核的联系。这种从细胞核~细胞骨架~基质的连续联系使基质弹性成为一个重要的生物学输入。因此，R21的“开发和探索”目标将(1)使我们的弹性基质适应低通量和高通量筛选格式，并最终在异常基质上的正常人类MSC（机械模仿纤维化）上筛选新的肌分化诱导剂，并将(2)开发肌源性疾病模型，用于筛选和测试核缺陷强烈影响肌源性分化和核可塑性的假设。仅在血清培养基中，我们将使用基质弹性来控制正常间充质干细胞的肌源性分化，而且，重要的是，模拟疾病中典型的刚性和纤维化组织。同样的基质系统允许比较刚性基质上的成骨和柔软的模拟脑基质上的神经发生。可溶性诱导因子(如；糖皮质激素)增强或抑制谱系将被筛选和研究的安全性和有效性，以促进肌肉生成的特定目标。我们还将设计人类MSC来表达已知的引起营养不良的突变体，如Lamin-A/C中的突变体，同时敲除内源性蛋白以尽量减少过表达产物。随着筛选的分化状态和核突变体用先导药物处理，完整细胞内核成分（如层粘连蛋白）的蛋白质折叠和结合状态将通过一种新的蛋白质组学规模的Cys Shotgun标记策略（利用定量质谱分析方法）进行探测[Johnson 2007]。

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.