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，导致相信这样的细胞有助于稳态修复过程。在许多疾病中，包括肌营养不良症，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病变肌发生的组织模拟细胞培养模型，重点是正常和异常基质弹性的影响，以评估已知的药物（例如，泼尼松龙），并最终筛选NIH文库中的候选药物，指导干细胞的基质偶联肌发生。作为一种特定的疾病，我们也关注核营养不良，因为它与肌肉相关，也是为了更深入地了解分化机制。核纤层与染色质相互作用， 通过跨越包膜的连接蛋白 纤层还与细胞骨架相互作用。我们最近已经表明，在骨骼肌细胞的肌动蛋白肌球蛋白条纹中[Engler J. Cell Biology 2004]以及在MSC的多谱系分化中[Engler Cell 2006]，细胞骨架受到基质弹性的强烈影响。在这些细胞和大多数其他组织细胞中，与基质的粘附与基于肌球蛋白的收缩性物理偶联，因此形成从基质通过细胞骨架进入正常细胞核的连接。这种从细胞核到细胞骨架到基质的连续连接使得基质弹性成为一种关键的生物输入。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形式，并最终在异常基质上的正常人MSC上筛选新型肌分化诱导剂。（机械模拟纤维化），将（2）开发一种肌源性疾病模型，用于筛选和测试核缺陷强烈影响肌源性分化的假设，核可塑性仅在血清培养基中，我们将使用基质弹性来控制正常MSC的肌源性分化，并且重要的是，模拟疾病中典型的刚性和纤维化组织。相同的基质系统允许与刚性基质上的骨发生和软的模拟脑基质上的神经发生进行比较。可溶性诱导因子（如将筛选增强或抑制谱系的激素（例如糖皮质激素），并研究其安全性和功效，特别是促进肌生成的目标。我们还将设计人MSC以表达已知的引起营养不良的突变体，例如Lamin-A/C中的突变体，同时敲低内源性蛋白质以最大限度地减少过度表达的假象。通过筛选筛选出的经先导药物处理的选择分化状态和核突变体，将通过利用定量质谱法的新型蛋白质组学规模Cys Shotgun标记策略探索完整细胞内核组分（如核纤层蛋白）的蛋白质折叠和缔合状态[约翰逊2007]。 公共卫生相关性：我们建议开发一种弹性基质培养形式，用于多孔筛选影响成体间充质干细胞成肌作用的可溶性因子。各种弹性基质模拟正常和患病状态下组织机械特性的影响性变化。我们将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，并最终重新设计表达已知肌营养不良症引起突变体（如Lamin-A/C中的突变体）的细胞。目标将是了解分化的机制块，并筛选已知的化合物和最终的文库，这些化合物和文库可以覆盖基质信号并促进病变细胞的分化。

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.