Proteomic characterization of molecular pathways at the single-cell level in human heterogeneous systems

生物基础科学的最新进展强调了组织异质性和细胞层次结构对于理解调节器官发育、稳态和对损伤的反应的生理机制的重要性。例如，研究体干细胞（SC）或原代神经元群体具有相当大的技术挑战，因为它们通常代表组织异质性的罕见成分。因此，对这样复杂的生物系统的分子研究必须依赖于单细胞分辨率技术，以捕获细胞特异性的生理相关的情况下的现象。uOttawa的Yannick Benoit、Mireille Khacho、William斯坦福大学和Barbara Vanderhyden博士正在研究不同分子机制对干细胞可塑性和线粒体动力学的影响。作为一个共同的目标，这组研究人员正在努力更好地了解特定的微环境线索如何影响表型变化，如自我更新（Benoit），命运和分化（斯坦福大学），上皮-间质转化（Vanderhyden）以及稀有细胞群体中的应激反应（Khacho）。这项研究需要经常利用基于单细胞分辨率的蛋白质组学评估的最新技术。现在可以使用流式细胞术和高含量成像从复杂的异质样品中鉴定、定量、表征和分离离散的细胞群。然而，以可持续的成本进行日常单细胞蛋白质组学研究的选择很少。单细胞Western（scWestern）分析为科学家提供了一个独特的机会，可以在异质细胞群中分析分子信号级联、亚型表达、蛋白水解加工和翻译后蛋白修饰。这种称为米洛的新技术由ProteinSimple（BioTechne）开发，并由Hughes等人（Nature Methods，2014）描述。与其他方法如CyTOF相比，该系统与大多数市售抗体兼容，并且能够进行具有成本效益的常规单细胞蛋白质组学分析实验，每次运行具有4种不同抗体的多路复用能力。当前的研究工具和仪器应用程序正在申请必要的资金来购买完整的米洛scWestern系统，该系统还提供5年延长保修计划和专业培训包。该基础设施将成为渥太华医院研究所StemCore设施的一部分，并将满足渥太华社区几名研究人员的迫切需求，而不仅限于本提案的主要和共同申请人，以进行干细胞生物学，神经科学和其他自然科学领域的尖端研究。

Recent advances in biological basic sciences highlighted the importance of tissue heterogeneity and cellular hierarchies for the understanding of physiological mechanisms regulating organ development, homeostasis, and response to injury. For instance, studying somatic stem cell (SC) or primary neuron populations comes with considerable technical challenges, since they often represent rare constituents of tissue heterogeneity. Thus, molecular investigations on such complex biological systems must rely on single-cell resolution techniques to capture cell--specific phenomenon in a physiologically relevant context. The groups of Drs. Yannick Benoit, Mireille Khacho, William Stanford, and Barbara Vanderhyden at uOttawa are studying the impact of diverse molecular mechanisms on stem cell plasticity and mitochondrial dynamics. As a common goal, this group of researchers is working to gain a better understanding on how specific microenvironmental cues influence phenotypic changes such as self--renewal (Benoit), fate and differentiation (Stanford), epithelial--mesenchymal transition (Vanderhyden), as well as stress response (Khacho) in rare cell populations. This research necessitates the frequent utilization of state--of--the--art technologies based on proteomic assessments at the single--cell resolution. It is now possible to identify, quantify, characterize, and isolate discrete cell populations from complex, heterogeneous samples, using flow cytometry and high content imaging. However, very few options are available for day--to--day single--cell proteomic investigations at a sustainable cost. Single--cell western (scWestern) analysis represents a unique opportunity for scientists to profile molecular signaling cascades, isoform expression, proteolytic processing, and posttranslational protein modifications across heterogeneous cell populations. This novel technology called MILO was developed by ProteinSimple (BioTechne) and described by Hughes et al. (Nature Methods, 2014). In contrast with other methods such as CyTOF, this system is compatible with most commercially available antibodies and enables cost efficient, routine single--cell proteomic profiling experiments, with a multiplexing capacity of 4 different antibodies per run. This current Research Tools and Instruments application is requesting the necessary funding to acquire a complete MILO scWestern system, which also comes with a 5-year extended warranty plan, and a specialized training package. This infrastructure will be part of the StemCore facility of the Ottawa Hospital Research Institute and will meet an urgent need from several investigators across the community at uOttawa, and not limited to main and co-applicants on this proposal, to conduct cutting- -edge research in stem cell biology, neurosciences, and other fields of natural sciences.