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

点击翻译按钮获取中文摘要

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.