TISSUE ENGINEERED HUMAN NEUROMUSCULAR JUNCTIONS FOR MODELING AXONAL NEUROPATHY

用于轴突神经病建模的组织工程人体神经肌肉接头

基本信息

批准号：
10054199
负责人：
Deok-Ho Kim
金额：
$ 35.64万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-11-15 至 2023-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10054199
关键词：
3-Dimensional Address Animal Model Autopsy Axon Axonal Neuropathy Axonal Transport Beds Biochemical Biological Assay Biological Models Biomedical Engineering Biophysics Biopsy Cell Density Cell Line Cells Charcot-Marie-Tooth Disease Clinical Treatment Coculture Techniques Critical Pathways Defect Development Disease Disease model Drug Design Drug Screening Electrophysiology (science)Engineering Etiology Evaluation Exhibits Functional disorder Future Gene Mutation Generations Genes Goals HDAC6 gene Hereditary Motor and Sensory-Neuropathy Type II Human Human Engineering Imaging Device In Vitro Investigation Knowledge Limb structure Methods Mitochondria Modeling Molecular Motor Neurons Muscle Muscle Fibers Mutation Myoblasts Neuromuscular Junction Neurons Neuropathy Pathologic Pathology Pathway interactions Patients Performance Peripheral Peripheral Nervous System Diseases Pharmaceutical Preparations Phenotype Physiological Presynaptic Terminals Property Role Sampling Severities Signal Transduction Skeletal Muscle Source Stimulus Structural defect Structure Study models Symptoms Synapses System Techniques Testing Therapeutic Therapeutic Intervention Tissue Engineering Tissues Validation base conditioning density disorder subtype experimental group human model human tissue improved in vitro activity in vivo induced pluripotent stem cell inhibitor/antagonist insight malformation mouse model mutant nanopattern novel novel therapeutics organizational structure pre-clinical pre-clinical assessment presynaptic prevent restoration scaffold screening stem cells synaptic function synaptogenesis targeted treatment theories therapeutic target three-dimensional modeling tool trait

项目摘要

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.

项目摘要 Charcot-Marie-Tooth疾病2型（CMT2）是一种严重令人衰弱的轴突性周围神经病，以神经肌肉连接（NMJ）分解，轴突传输缺陷和神经元结构为特征畸形。尽管可用于这种情况的动物模型，但已知的广泛基因突变已知引起CMT2表型（迄今为止确定的30个表型）使得对公共途径的研究定义了问题。反过来，这可以识别合适的治疗靶标，能够治疗各种患者极其困难。人类诱导的多能干细胞（HIPSC）衍生的电动机的成功产生来自CMT2患者的神经元可以建立患者特异性人性化评估以进行研究疾病病因是一个切实的目标。但是，使用此类细胞在体外有效地对CMT2进行建模的能力尚未可以实现，由于与在培养中产生健壮和功能成熟的NMJ相关的复杂性。我们认为一个文化平台整合正确的组织级结构组织，与物理相关细胞密度和正确的机电调节刺激将促进人类的发展 Myotube运动神经元共培养能够支持成熟的突触形成。使用我们的公认纳米图案的细胞表操纵技术，我们将使用无支架的3D组织结构生成 HIPSC衍生的运动神经元和具有高度有序组织结构的原发性成肌细胞。这些将评估结构的促进NMJ形成的能力，机电条件将然后将其作为推动突触发展的手段进行调查。该系统将结合开发来自四名CMT2患者的运动神经元。结合这些细胞的共培养结构将是研究了其在体外准确对疾病的病理生理学进行建模的能力，并突出显示不同突变线的表型相似性。鉴于线粒体在NMJ中的重要作用开发以及在多个与CMT2相关突变中观察到的轴突转运的分解，我们假设在突触前末端的线粒体密度降低会导致NMJ的畸形发展并最终破坏了突触。我们将使用CMT2 HIPSC-MOTOR神经元评估轴突的运输定义和结构性畸形在这些细胞中，并将这些发现与量化相关我们生物工程联合文化平台内NMJ开发的变化。最后，我们将调查是否多个CMT2神经元线中轴突传输性能的改善（通过轴突发育的稳定借助组蛋白脱乙酰基酶6抑制剂）导致NMJ发育和稳定性的显着改善人类细胞。跨不同患者突变的结果的一致性将突出显示轴突运输定义为人CMT2表型的发展的主要因素，并验证我们的平台作为合适的平台用于临床前评估的工具，用于改善周围神经性疾病的新药。

项目成果

期刊论文数量（19）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

NanoMEA: A Tool for High-Throughput, Electrophysiological Phenotyping of Patterned Excitable Cells.

DOI：
10.1021/acs.nanolett.9b04152
发表时间：
2020-03-11
期刊：
Nano letters
影响因子：
10.8
作者：
Smith AST;Choi E;Gray K;Macadangdang J;Ahn EH;Clark EC;Laflamme MA;Wu JC;Murry CE;Tung L;Kim DH
通讯作者：
Kim DH

HDAC6 Inhibition Corrects Electrophysiological and Axonal Transport Deficits in a Human Stem Cell-Based Model of Charcot-Marie-Tooth Disease (Type 2D).

DOI：
10.1002/adbi.202101308
发表时间：
2022-03
期刊：
Advanced biology
影响因子：
3.7
作者：
Smith AST;Kim JH;Chun C;Gharai A;Moon HW;Kim EY;Nam SH;Ha N;Song JY;Chung KW;Doo HM;Hesson J;Mathieu J;Bothwell M;Choi BO;Kim DH
通讯作者：
Kim DH

A biomaterial approach to cell reprogramming and differentiation.