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

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来研究精神和神经疾病的分子机制，这些分子机制来自受影响患者的细胞的活神经元。世界各地的几个实验室正在从数百名神经系统疾病患者身上产生诱导多能干细胞（iPS）细胞系。然而，神经元和胶质细胞分化方案可能产生异质培养，并且在系之间可能存在可变性。此外，对于许多神经系统疾病，尚不清楚该询问哪种神经元或胶质亚型。我们已经建立并优化了将ipsc导向各种神经元和胶质细胞的方法，并且我们已经开发了一种高通量的方法来研究ipsc衍生的神经元和胶质细胞在单细胞水平上的微雕刻过程中分析物的分泌。在这项技术中，分化的神经元和胶质细胞以有利于每孔单个细胞的密度被镀在纳米孔中。用涂有抗体的玻璃片将孔与相邻的孔隔离。在分析物捕获后，将载玻片与与荧光标签结合的检测抗体孵育以检测每种抗体，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。除去载玻片后，细胞保留在原来的纳米孔中，要么进行固定和免疫染色，要么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，以允许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，本文概述的方法的发展将增加ipsc衍生的人类神经元和神经胶质研究的整体力量，允许在神经元和神经胶质亚型中检测有意义的结果，否则单独研究异质群体可能会错过这些结果。对这种方法的一个警告是，相互分离的细胞的行为可能与它们在体内的行为不同。在目标3中，我们建议通过在成年啮齿动物大脑中进行有针对性的原理证明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及其生理相关性。如果成功，所开发的技术和相关的分析平台可以很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.