Tissue Engineered Human Neuromuscular Junctions for Modeling Axonal Neuropathy

项目摘要 腓骨肌萎缩症2型（CMT 2）是一种严重衰弱性轴突病性周围神经病变， 以神经肌肉接头（NMJ）断裂、轴突运输缺陷和神经元结构为特征 畸形虽然这种疾病的动物模型是可用的，但已知的广泛的基因突变 导致CMT 2表型（迄今为止鉴定了>30种）使得共同途径缺陷的研究成为问题。 这反过来又使得能够治疗广泛患者的合适治疗靶点的鉴定， 非常困难。人诱导多能干细胞（hiPSC）衍生的运动细胞的成功产生 CMT 2患者的神经元建立了患者特异性人源化测定，用于研究 疾病病因学是一个切实的目标。然而，使用这种细胞在体外有效地模拟CMT 2的能力还没有被证实。 这是由于与在培养中产生稳健和功能成熟的NMJ相关的复杂性。 我们认为，一个文化平台，整合正确的组织水平的结构组织，生理相关的， 细胞密度和正确的电机械条件刺激将促进人类的发育 能够支持成熟突触形成的肌管-运动神经元共培养物。利用我们完善的 纳米图案化细胞片操作技术，我们将产生无支架的3D组织结构，使用 hiPSC衍生的运动神经元和具有高度有序组织结构的原代人成肌细胞。这些 将评估结构促进NMJ形成的能力，并将 然后作为驱动突触发育的手段进行研究。该系统将与 来自四名CMT 2患者的运动神经元。掺入这些细胞的共培养构建体将是 研究了他们在体外准确模拟疾病病理生理学的能力，并强调 不同突变株系的表型相似性。鉴于线粒体在NMJ中的重要作用， 发展，以及在多个CMT 2相关突变中观察到的轴突运输的崩溃，我们 假设突触前末梢线粒体密度降低导致NMJ畸形 突触的发育并最终崩溃。我们将使用我们的CMT 2 hiPSC运动神经元来评估 轴突运输缺陷和结构畸形，并将这些发现与定量 在我们的生物工程共培养平台内NMJ发展的变化。最后，我们将研究是否 在多个CMT 2神经元系中轴突运输性质改善（通过轴突发育的稳定化 与组蛋白去乙酰化酶6抑制剂）导致NMJ发育和稳定性的显着改善， 人体细胞不同患者突变结果的一致性将突出轴突转运缺陷作为一种遗传学机制。 在人类CMT 2表型的发展的主要因果因素，并验证我们的平台作为一个合适的 用于临床前评估旨在改善周围神经病变的新药的工具。

PROJECT SUMMARY Charcot-Marie-Tooth disease type 2 (CMT2) is a severely debilitating axonopathic peripheral neuropathy, characterized by neuromuscular junction (NMJ) breakdown, axonal transport defects, and neuronal structure malformations. Although animal models for this condition are available, the wide array of gene mutations known to cause a CMT2 phenotype (>30 identified to date) makes the study of common pathway deficits problematic. This in turn makes identification of suitable therapeutic targets, capable of treating a wide range of patients, extremely difficult. The successful generation of human induced pluripotent stem cell (hiPSC)-derived motor neurons from patients with CMT2 makes the establishment of patient-specific humanized assays for studying disease etiology a tangible goal. However, the ability to effectively model CMT2 in vitro using such cells has yet to be achieved, due to the complexity associated with generating robust and functionally mature NMJs in culture. We posit that a culture platform integrating correct tissue-level structural organization, physiologically relevant cell densities, and correct electromechanical conditioning stimuli will promote the development of human myotube-motor neuron co-cultures capable of supporting mature synapse formation. Using our well-established nanopatterned cell sheet manipulation techniques, we will generate scaffold-free 3D tissue structures using hiPSC-derived motor neurons and primary human myoblasts with highly ordered tissue structures. These constructs will be assessed for their ability to promote NMJ formation, and electromechanical conditioning will then be investigated as a means to drive synapse development. This system will be developed in conjunction with motor neurons derived from four CMT2 patients. Co-culture constructs incorporating these cells will be investigated for their capacity to accurately model the disease’s pathophysiology in vitro, and to highlight phenotypic similarities across different mutant lines. Given the prominent role of mitochondria in NMJ development, and the observed breakdown in axonal transport in multiple CMT2-related mutations, we hypothesize that reduced mitochondrial density in presynaptic terminals leads to malformations in NMJ development and ultimately breakdown of the synapse. We will use our CMT2 hiPSC-motor neurons to evaluate axon transport deficits and structural malformations in these cells and correlate these findings with quantified changes in NMJ development within our bioengineered co-culture platform. Finally, we will investigate whether improvement in axon transport properties in multiple CMT2 neuron lines (via stabilization of axonal development with histone deacetylase 6 inhibitors) results in significant improvements in NMJ development and stability in human cells. Consistency of results across different patient mutations will highlight axonal transport deficits as a major causal factor in the development of the human CMT2 phenotype, and validate our platform as a suitable tool for use in the preclinical assessment of new drugs designed to ameliorate peripheral neuropathic conditions.