Metabolic reprogramming in pluripotent induction and cardiac lineage specificatio

描述(由申请人提供)：定义干细胞命运的调节者在为定制的再生治疗派生特定血统的人群中至关重要。除了已确立的对细胞身份的遗传/表观遗传控制之外，新出现的证据表明，干细胞使用能量和代谢物的方式决定了它们的命运。为了保持组织再生能力，干细胞必须在不同的状态之间转换，从活跃的增殖/自我更新到分化，每一种状态都施加了独特的生物能量需求。为了能够在不同的状态之间转换，代谢可塑性优先考虑生化途径，以建立分解代谢和合成代谢之间的平衡。分解代谢是分解底物以产生能量的过程，合成代谢是构建大分子的过程。这项拟议研究的总体目标是了解能量代谢的可塑性如何调节细胞命运的决定，以及特定的代谢途径如何支持特定阶段的干细胞功能。该奖项的K99阶段将重点定义特定的线粒体依赖成分和代谢途径，这些成分和代谢途径对于体细胞核重新编程回到多能性状态至关重要。这将通过使用基本的线粒体生物学技术和高通量代谢组学/代谢通量分析比较真正诱导的多能干细胞与其亲代成纤维细胞，并验证所确定的途径对使用线粒体缺陷细胞和酶特异性抑制进行核重新编程的影响来实现。在发展了线粒体生物学和高通量代谢组学和代谢流量分析方面的专业知识后，该奖项的R00阶段将专注于将这些新技术应用于心脏分化方案，该方案允许分离心脏发生期间的发育阶段，以便定义特定的线粒体复合体和代谢重新编程，以心脏分化为模型系统，促进谱系规范。总而言之，K99和R00通过定义在多能性诱导和谱系指定期间启动代谢重编程所需的线粒体功能的不同组成部分，将从根本上促进我们目前对干细胞代谢及其如何影响细胞命运的知识。这一知识的应用将使代谢策略的发展成为可能，以产生用于再生治疗的组织特异性细胞群，并有可能转化为基于能量代谢的新靶点，以防止慢性疾病的退化或引发返老还童。候选人的研究经验和突出的当前环境的协同作用，为目前在干细胞新陈代谢和细胞命运决策领域的应用奠定了基础。他的研究生工作建立在最初的生物化学本科培训的基础上，提供了能量代谢和心血管病理生理学的全面知识基础，并在最初的博士后培训期间通过干细胞生物学和代谢组学方面的培训加强了这一基础。该奖项的K99阶段将设在梅奥诊所，那里的核心使命侧重于旨在促进医学科学、改进疾病预防和治疗并培养下一代卫生保健专业人员的研究和教育。科学提案将受益于梅奥诊所的尖端技术平台和核心资源，而职业发展将通过导师建立的定制课程来促进，利用梅奥诊所的职业发展工具。候选人的短期目标包括出版当前和拟议的关于核重新编程的线粒体/代谢决定因素的手稿(K99阶段)，建立拟议项目所需的模型系统，并获得在解码干细胞命运决定的背景下剖析线粒体和代谢功能的综合技能集。完成这些目标将有助于候选人的长期职业目标，即建立一个独立的和外部资助的研究计划，以检查能量代谢的可塑性如何调节细胞命运决定，以及特定的代谢途径如何支持特定阶段的干细胞功能(R00阶段)。职业发展将通过与导师A.Terzic博士(梅奥诊所再生医学中心主任)和由J.Burnett Jr.博士组成的咨询委员会的互动来促进。他们是：梅奥临床心肺研究中心前主任S.Nair、古根海姆生物化学和分子生物学教授F.Prendergast和生物化学和分子生物学系主任J.van Deursen。重点将放在获得新的技能和方法上，特别是在线粒体生物学、代谢组学、代谢通量分析和系统生物学领域，以补充干细胞生物学和基于核磁共振的代谢组学的现有专门知识。这种技术熟练程度与科学提案中概述的概念创新相结合，为在新兴的干细胞代谢领域开始独立的研究生涯提供了一个发射台。

DESCRIPTION (provided by applicant): Defining the regulators of stem cell fate is critical in deriving lineage-specified populations for tailored regenerative therapy. Beyond established genetic/epigenetic control of cell identity, emerging evidence indicates that the way stem cells use energy and metabolites determine their fate. To maintain tissue regenerative capacity, stem cells must transit between distinct states from active proliferation/self-renewal to differentiatio, each imposing unique bioenergetic demands. To enable transition between distinct states, metabolic plasticity prioritizes biochemical pathways to establish a balance between catabolism, the process of breaking down substrates to produce energy, and anabolism, the process of constructing macromolecules. The overall goal of this proposed research is to understand how plasticity in energy metabolism regulates cell fate decisions and how specific metabolic pathways support stage-specific stem cell function. The K99 phase of this award will focus on defining the specific mitochondrial dependent components and metabolic pathways essential for nuclear reprogramming of somatic cells back to the pluripotent state. This will be accomplished by comparing bona fide induced pluripotent stem cells versus their parental fibroblasts using fundamental mitochondrial biology techniques and high throughput metabolomics/metabolic flux analysis, and validating the impact of identified pathways on nuclear reprogramming using mitochondrial deficient cells and enzyme- specific inhibition. Having developed this expertise in mitochondrial biology and high throughput metabolomics and metabolic flux analysis, the R00 phase of the award will focus on applying these new technologies to a cardiac differentiation protocol that allows isolation of developmental stages during cardiogenesis in order to define the specific mitochondrial complexes and metabolic reprogramming that fuels lineage specification, using cardiac differentiation as a model system. Collectively the K99 and R00, by defining the distinct components of mitochondrial function necessary for initiation of metabolic reprogramming during induction of pluripotency and lineage specification, will fundamentally advance our current knowledge of stem cell metabolism and how it impacts cell fate. Application of this knowledge will enable development of metabolic strategies for generation of tissue-specific cell populations for regenerative therapy, with potential translation to identify new targets based upon energy metabolism to prevent degeneration or elicit rejuvenation in chronic disease. The synergy of the candidate's research experience and outstanding current environment has laid the foundation for the present application in the field of stem cell metabolism and cell fate decisions. His graduate work built upon initial undergraduate training in biochemistry that provided a comprehensive knowledge base in energy metabolism and cardiovascular pathophysiology, which was enhanced with training in stem cell biology and metabolomics profiling during the initial post-doctoral training. The K99 phase of this award will be based at Mayo Clinic, where the core mission focuses on research and education aimed to advance the science of medicine, improve disease prevention and treatment, and prepare next generations of health care professionals. The scientific proposal will benefit from Mayo Clinic's cutting edge technical platforms and core resources, while career development will be facilitated by tailored curriculum established by the mentor, leveraging the career development instruments of Mayo Clinic. The candidate's short-term goals include publication of current and proposed manuscripts on the mitochondrial/metabolic determinants of nuclear reprogramming (K99 phase), establishment of model systems required for the proposed projects and acquisition of comprehensive skill sets to dissect mitochondrial and metabolic function in the setting of decoding stem cell fate decisions. Completion of these goals will facilitate the candidate's long-term career goal of establishing an independent and extramurally funded research program to examine how plasticity in energy metabolism regulates cell fate decisions and how specific metabolic pathways support stage-specific stem cell function (R00 phase). Career development will be fostered through interaction with the mentor, Dr. A. Terzic (Director, Mayo Clinic Center for Regenerative Medicine) and the advisory committee composed of Drs. J. Burnett Jr. (Former Director, Mayo Clinic Heart and Lung Research Center), S. Nair (Director, Mayo Clinic Comprehensive Metabolomics Center), F. Prendergast (Guggenheim Professor of Biochemistry and Molecular Biology), and J. van Deursen (Chair, Department of Biochemistry and Molecular Biology). Emphasis will be placed on acquisition of new skills and methodology, specifically in the areas of mitochondrial biology, metabolomics, metabolic flux analysis and systems biology to complement the current expertise in stem cell biology and NMR based metabolomics. This technical proficiency paired with the conceptual innovation outlined in the scientific proposal provides a launchpad to embark on an independent research career in the emerging domain of stem cell metabolism.