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

代谢重编程增强犬诱导多能干细胞的产生

• 批准号: RGPIN-2019-04027
• 负责人: Betts, Dean
• 金额: $ 4.23万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=690792
• 关键词: Metabolic; reprogramming; enhance; generation; canine

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.**

多能干细胞的特点是具有无限的能力来创造更多的自身副本，以及产生任何特殊细胞类型或身体组织的能力。科学家们已经发现了一些方法，可以获取任何特殊的体细胞，例如皮肤细胞，并通过引入几个将其转化为多能干细胞的基因对其进行“重新编程”。这些所谓的诱导多能干细胞（iPSC）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（ciPSC），然而，目前这些产生的犬 iPSC 的生产效率和质量极低，这表明在将它们应用于狗的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 iPSC。代谢历来被视为根据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参与建立各种多能干细胞状态。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，将其代谢调节为主要的 OXPHOS、糖酵解或混合代谢状态。我们假设，促进体细胞代谢向糖酵解方向发展，将使它们更容易重新编程，并提高犬诱导多能干细胞的生成效率和质量。******我们的 NSERC 发现研究计划将使用确定的燃料来源和代谢物暴露，将狗成纤维细胞引导至特定的代谢状态，以增强其对多能状态的诱导。将使用对假定的 ciPSC 及其分化衍生物进行适当的分子和细胞测定来评估多能性，同时将通过活细胞代谢测量来进行代谢询问，评估糖酵解和 OXPHOS 的各种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，以有效生成高质量的犬 iPSC，并将阐明物种之间的关键基本差异，以共同增强我们关于哺乳动物细胞多能性如何发展的整体知识。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。 **