A High Throughput, Human, In Vitro Model of Neuronal Stretch Injury

神经元牵张损伤的高通量人体体外模型

• 批准号: 9316304
• 负责人: John D Finan
• 金额: $ 23.4万
• 依托单位: NORTHSHORE UNIVERSITY HEALTHSYSTEM
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9316304
• 关键词: Address; Astrocytes; Automobile Driving; Awareness; Biological Assay; Biological Sciences; Biomedical Research; Cell Culture Techniques; Cell Death; Cell Line; Cell Survival; Cells; Cellular Morphology; Clinic; Clinical; Clinical Sciences; Clinical Trials; Coculture Techniques; Complex; Consensus; Contusions; Custom; Development; Devices; Drug Targeting; Edema; Engineering; Failure; Fostering; Future; Genetic; Genetic Variation; Genetic study; Genome; Genotype; Glucose; Goals; Hematoma; Human; Human Genetics; In Situ; In Vitro; Inflammatory; Influentials; Injury; Light; Measures; Mechanics; Membrane; Methods; Mission; Modeling; Morbidity - disease rate; National Institute of Neurological Disorders and Stroke; Nervous System Trauma; Neurites; Neurons; Outcome; Oxygen; Pathology; Patients; Pharmaceutical Preparations; Pharmacology; Phenotype; Pluripotent Stem Cells; Pre-Clinical Model; Preclinical Drug Evaluation; Public Health; Research; Risk; Role; Science; Silicones; Stretching; Stroke; Techniques; Therapeutic; Therapeutic Effect; Time; Translational Research; Traumatic Brain Injury; United States; Validation; Variant; Work; cytokine; deprivation; design; drug candidate; drug discovery; experimental study; genetic variant; high throughput screening; improved; improved outcome; in vitro Model; induced pluripotent stem cell; innovative technologies; man; microscopic imaging; novel; novel therapeutics; outcome forecast; patient stratification; patient subsets; response; success; tool

More than 100 clinical trials have been conducted in traumatic brain injury (TBI). Nevertheless, this condition, which is the most common killer of young people in the United States, remains without a proven therapy. TBI is hard to treat because it is heterogeneous: every patient has a different combination of pathologies and a different combination of genetic strengths and weaknesses. There may not be a single drug that treats all TBI pathologies in all patients. However, it may be possible to develop a suite of treatments for important pathologies of TBI by studying each in isolation. In the same spirit, treatment may be more effective if it is tailored to common, influential genotypes. Targeting subsets of TBI pathology in subsets of patients may enable piece-wise solution of a problem that seems impossible to solve at a single stroke. However, it requires a new type of pre-clinical model. In this proposal, neuronal stretch injury (NSI) is isolated from other TBI pathologies and applied to human induced pluripotent stem cell-derived neurons (hiPSCNs) for the first time. hiPSCNs can be engineered to contain the genomes of specific patients, or to differ from controls by a single genetic variant (these are known as isogenic cell lines). Homogeneous human neurons can be generated in large numbers, making these cells ideal for high throughput drug discovery. The model applies a biofidelic stretch insult in a 96 well format for the first time. However, a screen cannot be conducted until a very high level of consistency has been achieved and the capacity to detect therapeutic benefit has been verified. High throughput screens make many comparisons with few replicates so they require an extremely rigorous assay. Standard deviations should be 6 times smaller than the difference between positive and negative controls (corresponds to z>0 where z is the standard validation parameter in the field). The long term goal is to discover new treatments for NSI and understand patient-specific, cell autonomous factors driving pathology. The overall objective of this application, which is a vital step towards this goal, is to develop our existing NSI model into a rigorous high throughput drug screening assay. Our central hypothesis is that z will be >0 in the optimized model. hiPSCNs will be cultured on silicone membranes and stretched with a custom-built device to induce NSI. NSI pathology will be measured by quantitative analysis of cell viability and morphology in fluorescent microscopic images. The model will be optimized in 3 steps: optimization of the mechanical insult, optimization of the injury phenotype and optimization of the therapeutic effect of positive control compounds. The rationale for this work is to build a platform for future experiments that discover novel NSI therapies, measure the influence of genetic variants and address other aspects of the in situ condition (e.g. oxygen glucose deprivation, astrocyte activation, inflammatory cytokines etc.). This work will enable the first high throughput screen for an NSI therapy. It will also enable the first isogenic experiment in neurotrauma. These tools hold the promise of incremental clinical success in place of the status quo of total clinical failure.

在创伤性脑损伤（TBI）中进行了100多项临床试验。不过，这个条件， 这是美国年轻人最常见的杀手，仍然没有一种经过验证的治疗方法。TBI为 难以治疗，因为它是异质性的：每个患者都有不同的病理组合， 不同的基因优势和劣势组合。可能没有一种药物可以治疗所有TBI 所有患者的病理。然而，有可能开发一套治疗重要的 TBI的病理学通过研究每一个孤立的。本着同样的精神，治疗可能会更有效，如果它是 针对常见的有影响力的基因型在患者亚群中靶向TBI病理亚群可以 使一个似乎不可能一蹴而就的问题能够逐步得到解决。然而，它需要 一种新型的临床前模型。在该提案中，神经元牵张损伤（NSI）与其他TBI分离 病理学，并首次应用于人类诱导多能干细胞衍生的神经元（hiPSCN）。 hiPSCN可以被工程化以包含特定患者的基因组，或者通过单个突变与对照组不同。 遗传变异（这些被称为同基因细胞系）。同质的人类神经元可以在 大量，使这些细胞成为高通量药物发现的理想选择。该模型应用了生物化学 第一次在96孔格式中拉伸损伤。然而，筛选不能进行，直到一个非常高的 已达到一致性水平，并验证了检测治疗获益的能力。高 通量筛选用很少的重复进行许多比较，因此它们需要极其严格的测定。 标准偏差应小于阳性和阴性对照之间差异的6倍 （对应于z>0，其中z是字段中的标准验证参数）。长期目标是发现 NSI的新疗法，并了解患者特异性，细胞自主因素驱动病理。整体 这个应用程序的目标，这是实现这一目标的重要一步，是把我们现有的NSI模型发展成一个 严格的高通量药物筛选试验。我们的中心假设是在优化的情况下z将>0。 模型hiPSCN将在硅胶膜上培养，并用定制的装置拉伸以诱导 NSI。NSI病理学将通过在荧光显微镜下定量分析细胞活力和形态来测量。 显微图像。该模型将在3个步骤中进行优化：优化机械损伤，优化 的损伤表型和阳性对照化合物的治疗效果的优化。的理由 这项工作的目的是为未来的实验建立一个平台，发现新的NSI疗法，测量 遗传变异的影响，并解决原位条件的其他方面（如氧葡萄糖 剥夺、星形胶质细胞活化、炎性细胞因子等）。这项工作将使第一个高吞吐量 筛查NSI治疗这也将使第一个神经创伤的同基因实验成为可能。这些工具将 逐步取得临床成功的前景，取代了完全临床失败的现状。