Molecular mechanisms regulating motility and migration of muscle satellite cells

描述(申请人提供)：修复和替换脊椎动物受损的骨骼肌需要一群常驻的成体干细胞(卫星细胞)的活动。急性损伤后，这些相对稀少且高度分散的细胞需要做出反应，迅速激活，增殖产生大量替代成肌细胞池，重新定位到损伤部位，并分化形成新的肌肉纤维。成体肌肉生成(再生)所需的分子和细胞过程与肌肉最初形成时胚胎中活跃的过程重叠，但在很大程度上不同。在细胞与细胞外环境相互作用的水平上，卫星细胞在组织内运动和迁移的要求是肌肉再生的一个关键方面，但尚未得到很好的探索。与George Davis博士合作，我们将在可编程3D胶原基质中对宿主肌纤维进行的原代卫星细胞培养与时间推移视频显微镜相结合，开发了一种新的系统，用于定性和定量检测卫星细胞在其天然底物上的迁移。可以通过添加外源刺激来改变条件，例如潜在的诱变剂，用抗体或肽模拟物阻断可溶性或细胞表面蛋白，添加特定信号通路或细胞骨架重构体的药物抑制剂，通过构建病毒表达载体对卫星细胞进行特定感染，或者使用来自靶向突变的纤维和卫星细胞；所有这些也可以单独或联合进行检测。我们已经使用这个系统评估了可溶性因子的作用，如生长因子、趋化因子和信号脂；细胞外基质和黏附因子；跨膜信号受体和整合素；以及细胞骨架的细胞内调节剂。我们这项短期探索性赠款的目标是扩展和完善这些结果，以在这些已定义的信号因子和效应器类别的背景下建立卫星细胞迁移的工作模型。一旦我们确定了关键的相互作用和控制点，我们将继续询问这些活动如何整合到单个卫星细胞中，以实现增殖的成肌细胞群体向损伤区域的一致迁移。我们广泛的长期目标是了解卫星细胞如何在时间和空间上对构成体内肌肉损伤的瞬时和动态信号环境做出适当的检测、整合和反应，以及在健康肌肉组织中被视为理所当然的生物健壮性，在营养不良的肌肉中受到损害。 公共卫生相关性：除了解决成人肌肉发生机制的基础研究中未被充分探索的方面外，该项目还具有很高的潜力，有助于开发基于卫星细胞的治疗方法来治疗肌肉营养不良等疾病。在当前成人成肌细胞和肌源性干细胞植入过程中，一个关键的令人担忧的领域是，注射细胞至少要从注射部位广泛分散，或者充其量是积极地聚集在研究人员定义的一个或多个损伤最严重的部位。通过深入了解在系统(单纤维培养)中迁移的卫星细胞所使用的运动刺激、首选的迁移底物和特定的指导信号，有望概括体内发现的许多影响，这项工作将理想地提出修改当前成肌细胞植入方案以提高其治疗效果的潜在途径。

DESCRIPTION (provided by applicant): Repair and replacement of damaged skeletal muscle in vertebrates requires the activity of a population of resident adult stem cells (satellite cells.) Following acute injury, these relatively rare and highly dispersed cells are required to respond by quickly becoming `activated', multiplying to produce a large pool of replacement myoblasts, relocating to the site of the injury, and differentiating to form new muscle fibers. The molecular and cellular processes required for adult myogenesis (regeneration) overlap with, but are largely distinct from, those active in the embryo when muscle is first formed. At the level of the cell's interactions with the extracellular environment, the requirements for satellite cell motility and migration within the tissue are a critical aspect of muscle regeneration that has not been well explored. In collaboration with Dr. George Davis, we have combined primary satellite cell culture on their host myofibers in a programmable 3D collagen matrix with timelapse videomicroscopy to develop a novel system for qualitatively and quantitatively examining satellite cell migration on their native substrate. Conditions can be altered by addition of exogenous stimuli such as potential mutagens, blocking of soluble or cell-surface proteins with antibodies or peptide mimetics, addition of pharmacological inhibitors of specific signaling pathways or cytoskeletal remodelers, specific infection of satellite cells with viral expression constructs, or use of fibers and satellite cells derived from targeted mutations; all of these can also be assayed both individually and in combination. We have used this system to assess the roles of soluble factors such as growth factors, chemokines and signaling lipids; extracellular matrix and adhesion factors; transmembrane signaling receptors and integrins; and intracellular modulators of the cytoskeleton. Our goal for this short-term exploratory grant is to extend and refine these results to build a working model of satellite cell migration in the context of these defined classes of signaling factors and effectors. Once we have identified critical interactions and points of control, we will continue on to ask how such activities are integrated within individual satellite cells to effect a coherent migration of the population of proliferating myoblasts towards an area of injury. Our broad, long-term goal is to understand how satellite cells detect, integrate, and respond appropriately in time and space to the transient and dynamic signaling environment that would constitute a muscle injury in vivo, with the biological robustness that is taken for granted in healthy muscle tissue and compromised in dystrophic muscle. PUBLIC HEALTH RELEVANCE: In addition to addressing an underexplored facet of basic research into the mechanisms of adult myogenesis, this project has high potential to contribute to the development of satellite cell- based therapies for diseases such as the muscular dystrophies. A critical area of concern in current adult myoblast and muscle-derived stem cell engraftment procedures is the unmet requirement for injected cells to, at least, disperse broadly from the injection site or, at best, actively home to either investigator-defined sites or sites of maximum damage. By providing insight into the motogenic stimuli, preferred migration substrate, and specific guidance cues used by satellite cells migrating in a system (single fiber culture) that could be expected to recapitulate many of the influences found in vivo, this work will ideally suggest potential avenues to modify current myoblast engraftment protocols to enhance their therapeutic effectiveness.