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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=714324
• 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它们无限的能力，以创造更多的副本，他们的诀窍，以产生任何专门的细胞类型或组织的机构。科学家们已经发现了一种方法，可以将任何特殊的体细胞，如皮肤细胞，通过引入几个基因将其转化为多能干细胞来“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，但与体细胞供体在基因上匹配。几个研究小组已经产生了犬诱导多能干细胞（ciPSC），然而，目前这些犬诱导多能干细胞的生产效率和质量极低，这表明在其在犬中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。代谢在历史上被视为一个响应细胞需求而变化的过程，但现在已成为干细胞功能的“驱动器”。越来越多的证据表明，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确定的燃料源和代谢物的确定的培养基中培养特化体细胞来将其代谢调节为主要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将使它们更容易重编程，并将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定义的燃料来源和代谢物暴露来指导狗成纤维细胞朝向特定的代谢状态，以增强它们对多能状态的诱导。多能性将使用适当的分子和细胞测定法对推定的ciPSC及其分化的衍生物进行评估，而代谢的询问将通过活细胞代谢测量进行，评估糖酵解和OXPHOS的各种标志物并分析各种代谢信号传导途径。这些研究将开发一种简单可靠的培养方案，以调节体细胞的代谢状态，有效地产生高质量的犬iPSC，并将阐明不同物种之间的关键基本差异，以共同增强我们对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最前沿，为众多高素质人员提供高技术培训，同时提高在狗身上实施干细胞技术的生物医学和商业潜力。