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Tissue Engineered Human Neuromuscular Junctions for Modeling Axonal Neuropathy

用于模拟轴突神经病的组织工程人类神经肌肉接头

基本信息

批准号：
9235682
负责人：
Deok-Ho Kim
金额：
$ 34.73万
依托单位：
UNIVERSITY OF WASHINGTON
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 2021-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9235682
关键词：
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 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 Preclinical Drug Evaluation Presynaptic Terminals Property Role Sampling Severities Signal Transduction Skeletal Muscle Source Stem cells Stimulus Structural defect Structure Study models Symptoms Synapses System Techniques Testing Therapeutic Therapeutic Intervention Tissue Engineering Tissues Validation base conditioning density disorder subtype human tissue improved in vitro activity in vivo induced pluripotent stem cell inhibitor/antagonist insight malformation mouse model mutant nanopattern novel novel therapeutics pre-clinical presynaptic prevent restoration scaffold screening 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.

项目总结