Profiling of cortical cells by microengraving and mass spectrometry imaging

描述（由申请人提供）：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，可以研究来自受影响受试者细胞的活神经元中神经系统发育疾病的分子机制。世界各地的几个实验室正在从数百名患有神经退行性疾病和精神疾病的人身上产生诱导多能干细胞（iPS）细胞系。在某些情况下，受试者有已知的因果基因突变，而另一些人表达遗传风险等位基因，还有一些人没有已知的遗传易感性。虽然iPS细胞技术为研究感兴趣的细胞中遗传改变的影响提供了令人兴奋的进展，但该领域需要高通量的分析，以便分析对神经元和神经胶质亚群的影响。纳米技术的进步提供了一种高通量分析单细胞的方法。利用微雕刻和单细胞培养技术研究免疫系统细胞已经发展出了强大的检测方法。虽然神经细胞的类似方法的发展面临着独特的挑战，但成功地分析单个神经元和神经胶质亚型将使研究人员能够询问特定细胞类型中的基因功能和功能障碍，并有助于识别受特定遗传变化影响最大的细胞类型。在这里，我们建议优化一个系统，通过实时捕获分泌分析物，多重免疫染色和质谱成像来建立大脑皮层单个细胞亚型在发育时间内的蛋白质表达谱。此外，我们将研究分化为神经细胞和胶质细胞的iPS细胞的身份，并确定使用该系统来确定iPS系之间基因变化的影响的可行性。

DESCRIPTION (provided by applicant): Recent advances in stem cell biology provide a unique opportunity for researchers to investigate the molecular mechanisms underlying developmental diseases of the nervous system in living neurons derived from the cells of the affected subject. Several laboratories around the world are generating induced pluripotent stem (iPS) cell lines from hundreds of individuals with neurodegenerative and psychiatric disease. In some cases the subject has a known causal genetic mutation, while others express genetic risk alleles, and still others have no known genetic predisposition. While iPS cell technology provides an exciting inroad to study the effects of genetic alterations in the cells of interest, the field is in need of high throughput assays that allow for the analyses of effects in neuronal and glial subpopulations. Advances in nanotechnologies have provided a method to analyze single cells in a high throughput manner. Powerful assays have been developed using microengraving and single cell culture for the study of cells of the immune system. While the development of a similar method for neural cells presents unique challenges, successful profiling of individual neuronal and glial subtypes would allow researchers to interrogate gene function and dysfunction in specific cell types, and aid in the identification of the cell types most affected by specific genetic changes. Here, we propose to optimize a system to establish protein expression profiles for individual cell subtypes of the cerebral cortex over developmental time via real-time capture of secreted analytes, multiplexed immunostaining, and mass spectrometry imaging. In addition, we will investigate the identity of iPS cells differentiated to neuronal and glial cell fates, and determine the feasibility of using this system to determine effects of gene changes between iPS lines. PUBLIC HEALTH RELEVANCE: Understanding how genetic alterations lead to molecular changes that ultimately lead to disease is key for the identification of novel therapeutic targets. These studies aim to develop a novel technology through a cross- disciplinary collaborative effort that will enable neuroscientists to examine how genetic differences between people lead to an increased risk for developmental diseases of the nervous system.