Acquisition of Embryonic Stem Cell Metabolic Signature

胚胎干细胞代谢特征的获取

• 批准号: 8083859
• 负责人: Hannele RUOHOLA-BAKER
• 金额: $ 30.43万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8083859
• 关键词: Affect; Back; Binding; Bioenergetics; Carbon; Cell surface; Cells; Characteristics; Citric Acid Cycle; Commit; Data; Development; Electron Transport; Exposure to; Future; Goals; Grant; Hypoxia; Hypoxia Inducible Factor; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Medicine; Messenger RNA; Metabolic; Metabolic Pathway; Metabolism; Methods; MicroRNAs; Mitochondria; Mitochondrial DNA; Morphology; Outcome; Oxidation-Reduction; Oxidative Phosphorylation; Pathway interactions; Pattern; Phenotype; Play; Regenerative Medicine; Role; Small Interfering RNA; Staging; Stem cells; Teratoma; Testing; cancer stem cell; defined contribution; embryonic stem cell; genome-wide; high throughput screening; human embryonic stem cell; metabolomics; novel strategies; pluripotency; promoter; regenerative therapy; response; self-renewal; small molecule; stem; stem cell fate; stemness; transcription factor

DESCRIPTION (provided by applicant): The goal of this grant is to test the hypothesis that hESCs and iPSCs acquire specific metabolic signatures important for stemness through a common means, possibly by an exposure to stabilized HIF activity. Recent data support this hypothesis; HIFs have shown to play a role in ESC fate and ESCs have shown to sustain highly unsaturated metabolome. However, it is not yet clear how ESCs acquire their metabolic state, how the state is maintained and further, how the metabolic state of differentiation is obtained. We have showed that hypoxia is involved specifically in the acquisition state of stemness since hypoxia alone can de-differentiate committed cells back to stem cell fate. The de-differentiated cells re-activate Oct4 promoter analyzed by Oct4- GFP. These hypoxia induced stem like cells also mimic hESCs by their colony morphology, long-term self- renewal capacity, capability to replicate both in hypoxia and normoxia, genome wide mRNA and miRNA profiles, cell surface marker, TRA1 and SSEA expression and capacity to form teratomas. Furthermore, we have observed a specific metabolic signature in ESCs. We will now test whether hypoxia induced transcription factor, HIF is sufficient to induce the ESC specific metabolic pattern. We will first analyze glycolytic and oxidative phosphorylation responses to hypoxia using a panel of stem cells with different stages of pluripotency (piSC, diSC, EpiSC, HiPSC) and differentiated cells, followed by detailed assessment of redox poise within the mitochondrial electron transport chain (ETC) and metabolic pathway flux. Secondly, we will define the contribution of HIF activity for induction of unique stemness signatures (metabolism, mtDNA copy number) using hypoxia de-differentiation paradigms (HiPSC). Paradoxically, hypoxia has shown to affect both stemness and differentiation, suggesting that HIF might be differentially regulated to obtain the desired cellular outcomes. Therefore, we will devise high throughput screens to identify molecules that specifically affect HIF- induced stemness by promoting or interfering with stem cell-associated metabolic pathways. These studies will beget a new level of understanding of the acquisition of stemness and cancer and as such are bound to result in new approaches both in re-generative and cancer medicine. PUBLIC HEALTH RELEVANCE: The goal of this grant is to test the hypothesis that stem cells (in normal or cancer development) acquire a common characteristic metabolic signature through exposure to stabilized hypoxia-inducible factor (HIF) activity. Further, this metabolic signature may be determinative for stemness. Metabolites that are enriched in stem cells will be tested for their ability to reverse ESC differentiation in a high throughput format. These studies will spur improvements in regenerative medicine and potential therapies against cancer stem cells.

描述（由申请人提供）：本次资助的目的是检验以下假设：hESC 和 iPSC 通过常见方式（可能是通过暴露于稳定的 HIF 活性）获得对干性重要的特定代谢特征。最近的数据支持这一假设； HIF 已被证明在 ESC 命运中发挥作用，并且 ESC 已被证明能够维持高度不饱和的代谢组。然而，目前尚不清楚ESCs如何获得其代谢状态、如何维持该状态以及进一步如何获得分化的代谢状态。我们已经表明，缺氧特别参与干性的获得状态，因为仅缺氧就可以使定型细胞去分化回到干细胞命运。通过 Oct4-GFP 分析，去分化细胞重新激活 Oct4 启动子。这些缺氧诱导的干细胞样细胞还通过其集落形态、长期自我更新能力、在缺氧和常氧下复制的能力、全基因组mRNA和miRNA谱、细胞表面标记、TRA1和SSEA表达以及形成畸胎瘤的能力来模仿hESC。此外，我们在 ESC 中观察到了特定的代谢特征。我们现在将测试缺氧诱导的转录因子 HIF 是否足以诱导 ESC 特定的代谢模式。我们将首先使用一组具有不同多能阶段的干细胞（piSC、diSC、EpiSC、HiPSC）和分化细胞来分析对缺氧的糖酵解和氧化磷酸化反应，然后详细评估线粒体电子传递链（ETC）内的氧化还原平衡和代谢途径通量。其次，我们将使用缺氧去分化范例 (HiPSC) 定义 HIF 活性对诱导独特干性特征（代谢、mtDNA 拷贝数）的贡献。矛盾的是，缺氧已被证明会影响干性和分化，这表明 HIF 可能受到差异性调节以获得所需的细胞结果。因此，我们将设计高通量筛选来识别通过促进或干扰干细胞相关代谢途径来特异性影响 HIF 诱导的干细胞性的分子。这些研究将使人们对干性获得和癌症的理解达到一个新的水平，因此必将在再生医学和癌症医学方面产生新的方法。 公共健康相关性：这项资助的目的是检验以下假设：干细胞（正常或癌症发展中）通过暴露于稳定的缺氧诱导因子 (HIF) 活性而获得共同特征的代谢特征。此外，这种代谢特征可能是干性的决定因素。将以高通量形式测试干细胞中富集的代谢物逆转 ESC 分化的能力。这些研究将促进再生医学和针对癌症干细胞的潜在疗法的改进。