Metabolic reprogramming to enhance the generation of canine induced pluripotent stem cells

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Pluripotent stem cells are characterized by their unlimited ability to create more copies of themselves and their knack for producing any specialized cell type or tissue of the body. Scientists have discovered ways to take any specialized somatic cell, such as a skin cell, and “reprogram” it by introducing several genes that convert it into a pluripotent stem cell. These so-called induced pluripotent stem cells (iPSCs) closely resemble embryonic stem cells but are genetically matched to the somatic cell donor. Several research groups have generated canine induced pluripotent stem cells (ciPSCs), however, the production efficiency and quality of these resultant canine iPSCs are extremely low at this time indicating that greater research is required before their clinical applications in dogs occurs. ******Somatic cell reprogramming requires a shift from differentiated cells producing energy via oxidative phosphorylation (OXPHOS) to iPSCs producing energy primarily by glycolysis. Metabolism has historically been viewed as a process that changes in response to the needs of cells, but now has emerged as a “driver” of stem cell function. Increasing evidence implicates cellular metabolism in establishing various pluripotent stem cell states. We can modulate the metabolism of specialized somatic cells to either a primarily OXPHOS, glycolytic or mixed metabolic state by culturing them in defined media containing defined fuel sources and metabolites. We hypothesize that promoting somatic cell metabolism towards glycolysis will make them more amendable to reprogramming and will increase the generation efficiency and quality of canine induced pluripotent stem cells.******Our NSERC Discovery research program will direct dog fibroblasts towards specific metabolic states using defined fuel sources and metabolite exposure to enhance their induction towards a pluripotent state. Pluripotency will be assessed using appropriate molecular and cellular assays on putative ciPSCs and their differentiated derivatives, while interrogation of metabolism will be carried out by live cell metabolic measurements, assessing various markers of glycolysis and OXPHOS and analyzing various metabolic signaling pathways. These studies will develop a simple and reliable culture protocol to modulate the metabolic state of somatic cells to efficiently generate high quality canine iPSCs and will elucidate key fundamental differences across species to collectively enhance our overall knowledge regarding how cellular pluripotency develops within mammals. Support for these studies will place Canada in the forefront of these emerging biotechnologies, providing high-tech training for numerous highly qualified personnel (HQP) while enhancing the biomedical and commercial potential for implementing stem cell technologies in the dog.**