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

多能干细胞的特点是它们具有无限的复制能力，并且能够产生任何特殊的细胞类型或身体组织。科学家们已经发现了一些方法，可以将任何特殊的体细胞，比如皮肤细胞，通过引入几个基因将其转化为多能干细胞，对其进行“重新编程”。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供体相匹配。几个研究小组已经产生了犬诱导多能干细胞（ciPSCs），然而，目前这些犬诱导多能干细胞的生产效率和质量都非常低，这表明在犬临床应用之前需要进行更多的研究。体细胞重编程需要从通过氧化磷酸化（OXPHOS）产生能量的分化细胞转变为主要通过糖酵解产生能量的iPSCs。新陈代谢历来被认为是一个响应细胞需要而发生变化的过程，但现在已成为干细胞功能的“驱动因素”。越来越多的证据暗示细胞代谢在建立各种多能干细胞状态。我们可以通过在含有特定燃料源和代谢物的特定培养基中培养特定体细胞，将其代谢调节为主要的氧化磷酸化、糖酵解或混合代谢状态。我们推测，促进体细胞糖酵解代谢将使它们更容易被重编程，从而提高犬诱导多能干细胞的生成效率和质量。我们的NSERC发现研究项目将引导狗成纤维细胞进入特定的代谢状态，使用确定的燃料来源和代谢物暴露，以增强它们对多能状态的诱导。多能性将通过对假定的ciPSCs及其分化衍生物进行适当的分子和细胞分析来评估，而代谢的调查将通过活细胞代谢测量来进行，评估各种糖酵解和OXPHOS标记物，并分析各种代谢信号通路。这些研究将开发一种简单可靠的培养方案来调节体细胞的代谢状态，以有效地产生高质量的犬类多能干细胞，并将阐明物种之间的关键基本差异，以共同提高我们对哺乳动物细胞多能性如何发展的总体认识。对这些研究的支持将使加拿大处于这些新兴生物技术的前沿，为众多高素质人员（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.