An In Vitro Model of Stem Cell Innervation of Myotubes

描述（由申请人提供）：反射弧是脊髓的基本功能单位。单位中任何细胞成分的疾病或损伤都可能导致严重的运动障碍。该系统的工程方法将允许在受控的、限定的环境中的体外系统中评价这些问题和病症。本计画拟发展一种生物整合的微加工矽元件，以研究运动神经元与肌管之间的突触通讯发展与神经再支配。我们的假设是，干细胞和生长因子的组合将使肌纤维的神经再支配和人干细胞将神经支配大鼠肌肉在体外，使上述结果的体内评价。运动神经元将来源于作为对照的胚胎大鼠，并与成年大鼠运动神经元和来源于人干细胞的运动神经元进行比较/对比。肌管最初将来源于胚胎大鼠，然后是成年大鼠，然后是成年人组织。该项目的主要目标是建立人类干细胞神经支配成年大鼠肌肉的能力，以确定使用体外系统在大鼠SCI模型中实现体内神经再支配和功能恢复的最佳条件。最初，在目标1中，将在单细胞水平上研究运动神经元-肌肉段，以评估由与MEMS结构集成的图案化表面组成的系统中的体外神经再支配。将使用MEMS制造方法制造模拟PNS和CNS环境的腔室。目标1b检查了目标1中创建的系统，其中运动神经元的靶是来自人体组织的成人肌管。目的2评价生长因子存在下的系统。目的3研究神经胶质细胞、雪旺细胞和小胶质细胞的存在对肌节单神经元功能的影响。目的4涉及体内实验，其将评价在目的1-3中优化的生长因子和人干细胞的组合，以允许大鼠肌肉的神经再支配并研究功能恢复。基础神经科学，细胞生物学，微系统工程和表面化学的整合将被实施，以建立和测试这种混合设备，最终导致设计方案，以预防，诊断和治疗发育异常和慢性神经/肌肉疾病。假体和矫形器的设计和评估的新策略，以及从组织的活动依赖性神经再支配方面进行脊柱修复的新方法也被设想。最后，这项技术如果成功，可以用来建立功能性测定，以研究人类细胞中可能还不存在动物模型的疾病。中央佛罗里达大学的纳米科学技术中心、南卡罗来纳州医科大学的神经科学研究所、海军研究实验室（NRL）和Neuralstem（一家致力于人类干细胞技术的生物技术公司）已经合作开发这项技术。

DESCRIPTION (provided by applicant): The reflex arc is a fundamental functional unit of the spinal cord. Disease or injury of any of the cellular elements in the unit can result in profound movement disorders. An engineering approach to this system would allow the evaluation of these problems and disorders in an in vitro system in a controlled, defined environment. This project proposes to develop a biologically integrated microfabricated silicon device to study synaptic communication development and reinnervation between motoneurons and myotubes. Our Hypothesis is that a combination of stem cells and growth factors will enable the reinnervation of muscle fibers and that human stem cells will innervate rat muscle in vitro to enable the in vivo evaluation of the above results. The motoneurons will be derived from embryonic rat as a control and compared/contrasted to adult rat motoneurons and motoneurons derived from human stem cells. The myotubes will be derived initially from embryonic rat, then adult rat and later adult human tissue. The primary goal of the project is to establish the ability of human stem cells to innervate adult rat muscle to determine the best conditions using the in vitro system to achieve in vivo reinnervation and functional recovery in a rat SCI model. Initially, in Aim 1 the motoneuron-to-muscle segment will be investigated at the single cell level to assess in vitro reinnervation in a system that is composed of patterned surfaces integrated with a MEMS construct. A chamber to mimic the PNS and CNS environment will be fabricated using MEMS fabrication methodology. Aim 1b examines the system created in Aim 1 where the target for the motoneurons is an adult myotube derived from human tissue. Aim 2 evaluates the system in the presence of growth factors. Aim 3 examines the effect of the presence of glial, Schwann and microglial cells on the functional capacity of the montoneuron to muscle segment. Aim 4 addresses the in vivo experiments that will evaluate the combination of growth factors and human stem cells optimized in Aims 1-3 to allow reinnervation of rat muscle and investigate functional recovery. An integration of fundamental neuroscience, cell biology, microsystem engineering, and surface chemistry will be implemented to build and test this hybrid device, leading eventually to designing schemes to prevent, diagnosis, and treat developmental abnormalities and chronic neurological/muscle disorders. New strategies for prosthetic and orthotic design and evaluation, and new approaches for spinal repair from the aspect of an activity dependent reinnervation of tissue are also envisioned. Finally, this technology if successful could be used to create functional assays to investigate human cells for maladies where animal models may not yet exist. The Nanoscience Technology Center at the University of Central Florida, the Neuroscience Institute at the Medical University of South Carolina, the Naval Research Laboratory (NRL), and Neuralstem (a biotech company devoted to human stem cell technology) have partnered to develop this technology.