Acquisition of Embryonic Stem Cell Metabolic Signature

胚胎干细胞代谢特征的获取

• 批准号: 8442879
• 负责人: Hannele RUOHOLA-BAKER
• 金额: $ 29.36万
• 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/8442879
• 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; public health relevance; 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.

描述（由申请人提供）：该赠款的目的是检验以下假设：hESC和IPSCS通过共同手段获得对茎的特定代谢特征，可能是通过暴露于稳定的HIF活动的情况下。最近的数据支持这一假设； HIF已显示在ESC命运中发挥作用，ESC已证明可以维持高度不饱和代谢。但是，目前尚不清楚ESC如何获得其代谢状态，如何维持状态以及如何获得分化的代谢状态。我们已经表明，缺氧专门参与了干性的采集状态，因为仅缺氧就可以将差异化的细胞解散回干细胞的命运。脱不同的细胞重新激活OCT4-GFP分析的OCT4启动子。这些缺氧像细胞一样诱导了茎，还通过其集落形态，长期自我更新能力，在缺氧和正常氧中复制的能力，基因组宽mRNA和miRNA谱，细胞表面标记，TRA1和SSEA表达以及形成TeratoMas的能力。此外，我们已经观察到ESC中的特定代谢特征。现在，我们将测试缺氧是否引起转录因子，HIF足以诱导ESC特异性代谢模式。我们将首先使用具有不同阶段的多能性阶段（PISC，DISC，EPISC，HIPSC）和分化细胞的干细胞和分化细胞来分析对缺氧的糖酵解和氧化磷酸化反应，然后对线粒体电子传输链（等）内的氧化还原po进行详细评估。其次，我们将使用缺氧de-Differentiation范式（HIPSC）来定义HIF活性对诱导独特的干性特征（代谢，mtDNA拷贝数）的贡献。矛盾的是，缺氧已证明会影响干性和分化，这表明HIF可能受到差异调节以获得所需的细胞结局。因此，我们将设计高吞吐量筛选，以鉴定通过促进或干扰与干细胞相关的代谢途径的特异性影响的分子。这些研究将有一个新的了解对干性和癌症的获取的新水平，因此必将导致重新产生和癌症医学的新方法。