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

多能干细胞的特点是它们有无限的能力创造更多的自我复制，以及产生任何特定类型的细胞或身体组织的诀窍。科学家们已经发现了一种方法，可以提取任何特定的体细胞，比如皮肤细胞，并通过引入几个基因来对其进行重新编程，将其转化为多能干细胞。这些所谓的诱导多能干细胞(IPSCs)与胚胎干细胞非常相似，但在基因上与体细胞捐赠者匹配。几个研究小组已经培养出了犬诱导多能干细胞(CiPSCs)，然而，目前这些合成的犬IPSCs的生产效率和质量都非常低，这表明在其临床应用于狗之前需要更多的研究。 体细胞重新编程需要从分化的细胞通过氧化磷酸化(OXPHOS)产生能量，转变为IPSCs主要通过糖酵解产生能量。新陈代谢历来被认为是随着细胞需求而变化的过程，但现在已成为干细胞功能的“驱动力”。越来越多的证据表明，细胞代谢与多种多能干细胞状态的建立有关。我们可以通过在含有特定燃料来源和代谢物的特定培养基中培养特化体细胞，将它们的代谢调节到主要是氧磷酸盐、糖酵解或混合代谢状态。我们推测，促进体细胞代谢向糖酵解方向发展将使它们更易于重新编程，并将提高犬诱导的多能干细胞的生成效率和质量。 我们的NSERC发现研究计划将使用特定的燃料来源和代谢物暴露来引导狗成纤维细胞进入特定的代谢状态，以增强它们向多能性状态的诱导。多能性将通过对可能的ciPSCs及其分化的衍生物进行适当的分子和细胞分析来评估，而新陈代谢的询问将通过活细胞代谢测量、评估糖酵解和OXPHOS的各种标记物以及分析各种代谢信号通路来进行。这些研究将开发一种简单而可靠的培养方案来调节体细胞的代谢状态，以高效地产生高质量的犬类IPSCs，并将阐明关键的基本差异，以共同增强我们对哺乳动物体内细胞多能性发展的全面了解。对这些研究的支持将使加拿大走在这些新兴生物技术的前沿，为许多高素质的人员(HQP)提供高科技培训，同时增强在狗身上实施干细胞技术的生物医学和商业潜力。

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.