An In Vitro Model of Stem Cell Innervation of Myotubes

描述(申请人提供)：反射弧是脊髓的基本功能单位。该单元中任何细胞元素的疾病或损伤都可能导致严重的运动障碍。对这一系统的工程方法将允许在受控的、定义的环境中在体外系统中评估这些问题和紊乱。该项目计划开发一种生物集成的微制硅设备，以研究运动神经元和肌管之间的突触通讯、发育和再神经支配。我们的假设是，干细胞和生长因子的结合将使肌肉纤维重新支配，而人类干细胞将在体外对大鼠肌肉进行神经支配，从而能够在体内对上述结果进行评估。以胚胎大鼠运动神经元为对照，与成年大鼠运动神经元和人干细胞运动神经元进行比较。肌管最初将来自胚胎大鼠，然后是成年大鼠，最后是成年人类组织。该项目的主要目标是建立人类干细胞对成年大鼠肌肉的神经支配能力，以确定使用体外系统在大鼠脊髓损伤模型中实现体内神经再支配和功能恢复的最佳条件。首先，在目标1中，运动神经元到肌肉的部分将在单细胞水平上进行研究，以评估由带有图案的表面与MEMS构建物组成的系统中的体外神经再支配。模拟PNS和CNS环境的腔体将使用MEMS制造方法制造。目标1b检查了目标1中创建的系统，其中运动神经元的目标是来自人类组织的成年肌管。目标2在存在生长因子的情况下对系统进行评估。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单神经元对肌节功能的影响。目的4介绍体内实验，这些实验将评估生长因子和在AIMS 1-3中优化的人类干细胞的组合，以允许大鼠肌肉的神经再支配和研究功能恢复。将综合基础神经科学、细胞生物学、微系统工程和表面化学来构建和测试这种混合设备，最终设计出预防、诊断和治疗发育异常和慢性神经/肌肉疾病的方案。还展望了假体和矫形器设计和评估的新策略，以及从活动依赖的组织神经支配的角度进行脊柱修复的新方法。最后，如果这项技术成功，可以用来创建功能分析，在动物模型可能还不存在的情况下，研究人类细胞的疾病。中佛罗里达大学纳米科学技术中心、南卡罗来纳医科大学神经科学研究所、海军研究实验室(NRL)和神经干细胞(一家致力于人类干细胞技术的生物技术公司)已合作开发这项技术。

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.