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

产品说明：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，可以在来自受影响患者细胞的活神经元中研究精神和神经疾病的分子机制。世界各地的几个实验室正在从数百名患有神经系统疾病的个体中产生诱导多能干细胞（iPS）细胞系。然而，神经元和神经胶质细胞分化方案可能产生异质性培养物，并且细胞系之间可能存在变异性。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将hiPSC导向各种神经元和神经胶质细胞命运的方法，并且我们已经开发了一种高通量方法，以在称为微雕刻的过程中在单细胞水平上研究来自iPSC衍生的神经元和神经胶质细胞的分析物的分泌。在该技术中，将分化的神经元和神经胶质以有利于每孔单个细胞的密度接种在多细胞中。威尔斯用涂有目标分析物抗体的载玻片将孔与其相邻孔密封。在分析物捕获之后，载玻片与缀合至荧光标签的检测抗体一起孵育以检测每一种，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。在移除载玻片后，细胞保留在其原始细胞中，并被固定和免疫染色，或者被回收用于基因表达谱分析。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，以允许检查对小分子治疗（目标1）和遗传扰动（目标2）的细胞命运特异性反应。如果成功的话，本文概述的方法的开发将通过允许检测神经元和神经胶质的亚型中的有意义的结果来增加iPSC衍生的人神经元和神经胶质的研究的总体能力，否则仅研究异质群体可能会错过这些结果。这种方法的一个警告是，彼此分离的细胞可能不会像它们在体内那样表现。在目标3中，我们建议通过在成年啮齿动物大脑中进行体内靶向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。如果成功的话，所开发的技术和相关的分析平台可以容易地应用于研究其他感兴趣的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。

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.