Detection of cell type specific effects of pathway manipulation in neural cells

检测神经细胞中通路操纵的细胞类型特异性效应

• 批准号: 8831313
• 负责人: Tracy L YOUNG-PEARSE
• 金额: $ 45.12万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8831313
• 关键词: Adult; Advanced Development; Affect; Aftercare; Algorithms; Alzheimer' s Disease; Amyloid beta-Protein; Animal Model; Antibodies; Autopsy; Award; Brain; Candidate Disease Gene; Cell Line; Cells; Collaborations; Complex; Data Analyses; Detection; Development; Disease; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Enzymes; Excision; Exploratory/Developmental Grant for Diagnostic Cancer Imaging; Funding; Gene Expression; Gene Expression Profiling; Generations; Genes; Genetic; Genetic Transcription; Glass; Glial Differentiation; Health; Human; Immune system; Incubated; Individual; Institutes; Intervention; Laboratories; Lead; Life; Love; Measures; Mental disorders; Methodology; Molecular; Molecular Profiling; Mus; Mutation; National Institute of Mental Health; Neurodegenerative Disorders; Neuroglia; Neuronal Differentiation; Neurons; Outcome Measure; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Physiological; Population Heterogeneity; Presenile Alzheimer Dementia; Process; Production; Protocols documentation; RNA Sequences; Research Personnel; Rodent; Scanning; Slide; Stem cells; Subfamily lentivirinae; System; Techniques; Technology; antibody conjugate; brain cell; brain tissue; cell type; cellular transduction; density; extracellular; genetic risk factor; in vivo; induced pluripotent stem cell; instrumentation; interest; knock-down; mouse model; nano-string; nervous system disorder; new technology; novel; overexpression; primary outcome; protein expression; response; risk variant; seal; small molecule; stem cell biology; stem cell technology; tool; treatment strategy

DESCRIPTION: Recent advances in stem cell biology provide a unique opportunity for researchers to investigate the molecular mechanisms underlying psychiatric and neurological diseases in living neurons derived from the cells of the affected patient. Several laboratories around the world are generating induced pluripotent stem (iPS) cell lines from hundreds of individuals with neurological disease. However, neuronal and glial differentiation protocols may yield heterogeneous cultures, and there may be variability between lines. Furthermore, for many neurological diseases, it is not clear which neuronal or glial subtype(s) to interrogate. We have established and optimized methodologies for directing hiPSCs to a variety of neuronal and glial fates, and we have developed a high throughput methodology to study secretion of analytes from iPSC-derived neuronal and glial cells at the single cell level in a process called microengraving. In this technique, differentiated neurons and glia are plated in nanowells at a density that favors a single cell per well. Wells are sealed from their neighbors with a glass slid coated with antibodies to the analytes of interest. After analyte capture, the slides are incubated with a detection antibody conjugated to a fluorescent tag to detect each, similar to a traditional "sandwich ELISA". Slides are scanned and analyzed using standard microarray instrumentation. After removal of the slides, cells remain in their original nanowells and are either fixed and immunostained or else retrieved for gene expression profiling. Here, we aim to advance the development of this technology through the expansion of the platform to allow for the examination of cell-fate specific responses to small molecule treatments (aim 1) and to genetic perturbations (aim 2). If successful, the development of the methodology outlined herein would increase the overall power of the study of iPSC-derived human neurons and glia by allowing for the detection of meaningful results in a subtype of neurons and glia that could otherwise be missed by solely studying a heterogeneous population. One caveat to this methodology is that cells that are isolated from one another may not behave as they would in vivo. In aim 3, we propose to validate the existence and physiological relevance of the subpopulations identified in aims 1 and 2 through targeted proof-of-principle genetic and small molecule interventions performed in vivo in the adult rodent brain. If successful, the developed technology and associated analysis platforms can be readily applied to the study of the secretion of other analytes of interest as well as to other primary and stem cell-derived cell fates.

描述：干细胞生物学的最新进展为研究人员提供了一个独特的机会，以研究来自受影响患者细胞的活神经元中精神和神经疾病的分子机制。世界各地的多个实验室正在从数百名神经系统疾病患者身上培育诱导多能干 (iPS) 细胞系。然而，神经元和神经胶质分化方案可能会产生异质培养物，并且细胞系之间可能存在差异。此外，对于许多神经系统疾病，尚不清楚要询问哪种神经元或神经胶质亚型。我们已经建立并优化了引导 hiPSC 走向各种神经元和神经胶质细胞命运的方法，并且开发了一种高通量方法，用于在单细胞水平上研究 iPSC 衍生的神经元和神经胶质细胞分析物的分泌，这一过程称为微雕刻。在这项技术中，分化的神经元和神经胶质细胞以有利于每孔单个细胞的密度铺在纳米孔中。使用涂有目标分析物抗体的载玻片将孔与相邻孔密封。捕获分析物后，将载玻片与缀合有荧光标签的检测抗体一起孵育以检测每种分析物，类似于传统的“夹心 ELISA”。使用标准微阵列仪器扫描和分析载玻片。取出载玻片后，细胞保留在原来的纳米孔中，并进行固定和免疫染色，或者检索进行基因表达谱分析。在这里，我们的目标是通过扩展平台来推进这项技术的发展，以检查细胞命运对小分子治疗（目标 1）和遗传扰动（目标 2）的特异性反应。如果成功，本文概述的方法的开发将增加 iPSC 衍生的人类神经元和神经胶质细胞研究的整体能力，允许检测神经元和神经胶质细胞亚型中有意义的结果，否则仅研究异质人群。这种方法的一个警告是，彼此分离的细胞可能不会像在体内那样表现。在目标 3 中，我们建议通过在成年啮齿动物大脑体内进行有针对性的原理验证遗传和小分子干预来验证目标 1 和 2 中确定的亚群的存在和生理相关性。如果成功，所开发的技术和相关分析平台可以轻松应用于其他感兴趣分析物的分泌以及其他原代细胞和干细胞衍生的细胞命运的研究。