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，可以在源自受影响受试者细胞的活神经元中研究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个体中产生诱导多能干（iPS）细胞系。在某些情况下，受试者具有已知的因果基因突变，而其他人表达遗传风险等位基因，还有一些人没有已知的遗传倾向。虽然iPS细胞技术提供了一个令人兴奋的进展，研究基因改变的影响，在感兴趣的细胞，该领域是在需要的高通量分析，允许在神经元和神经胶质细胞亚群的影响的分析。纳米技术的进步提供了一种以高通量方式分析单细胞的方法。使用微刻和单细胞培养已经开发出了强大的检测方法，用于免疫系统细胞的研究。虽然开发用于神经细胞的类似方法提出了独特的挑战，但成功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，以建立蛋白质表达谱的个别细胞亚型的大脑皮层在发育过程中，通过实时捕获分泌的分析物，多重免疫染色，和质谱成像。此外，我们将研究分化为神经元和神经胶质细胞命运的iPS细胞的身份，并确定使用该系统来确定iPS系之间的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.