| In vitro Differentiation of Human iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs)
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Author:
Date:
2020-09-20
[Abstract] Induced pluripotent stem cell derived cardiovascular progenitor cells (iPSC-CVPCs) provide an unprecedented platform for examining the molecular underpinnings of cardiac development and disease etiology, but also have great potential to play pivotal roles in the future of regenerative medicine and pharmacogenomic studies. Biobanks like iPSCORE ( Stacey et al., 2013; Panopoulos et al., 2017), which contain iPSCs generated from hundreds of genetically and ethnically diverse individuals, are an invaluable resource for conducting these studies. Here, we present an optimized, cost-effective and highly standardized protocol for large-scale derivation of human iPSC-CVPCs using small molecules and purification using metabolic selection. We have successfully applied this protocol ...
[摘要] [摘要 ] 诱导性多能干细胞衍生的心血管祖细胞（iPSC-CVPCs）为检查心脏发育和疾病病因的分子基础提供了前所未有的平台，但在再生医学和药物基因组学的未来中也具有重要作用。像iPSCORE这样的生物库（Stacey 等，2013 ；Panopoulos 等，2017）， 其中包含由数百个遗传和种族不同的个体产生的iPSC，是进行这些研究的宝贵资源。在这里，我们为小分子大规模衍生人iPSC-CVPCs和代谢选择纯化提供了一种优化，具有成本效益和高度标准化的方案。我们已经成功地应用了该协议，从154种不同的iPSCORE iPSC品系中获得了iPSC-CVPC，从而获得了大量的高纯度心脏细胞。一个重要的我们的协议的组成部分是Ç ELL Ç onfluency 估计S（ccEstimate ），用于估计当iPSC集单层将达到80％汇合，这是用于发起的iPSC-CVPC推导最佳的时间的自动方法，并且使得协议为易于在具有不同增长率的iPSC系列中使用。此外，我们发现跨iPSC-CVPC的细胞异质性是由于两种截然不同的心脏细胞类型（心肌细胞（CMs）和心外膜衍生细胞（EPDCs））的比例不同导致的，这两种细胞在心脏再生中均具有关键作用。该协议消除了iPSC线到线优化的需要，并且可以轻松地进行调整和扩展，以进行高通量研究或生成大量适用于再生医学应用的细胞。
[背景 ] ...
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| Assessment of Brown Adipocyte Thermogenic Function by High-throughput Respirometry
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Author:
Date:
2015-11-05
[Abstract] Brown adipose tissue (BAT) has the unique ability to dramatically increase mitochondrial uncoupled fuel oxidation for thermogenesis in response to adrenergic stimulation. A key parameter in assessing brown adipocyte thermogenic capacity is mitochondrial uncoupling as determined by respiration. Measuring mitochondrial oxygen consumption rate (OCR) therefore provides valuable information to study the regulation and dysregulation of fuel metabolism and energy expenditure. Adding measurements of mitochondrial membrane potential allows for more in-depth interpretation of the respirometry data. Here we provide protocols for measuring respiration in adherent intact and plasma membrane permeabilized brown adipocytes using the Seahorse XF Analyzer. In the protocol Part I, a combination of ...
[摘要] 棕色脂肪组织（BAT）具有显着增加线粒体解偶联燃料氧化以响应肾上腺素刺激的热生成的独特能力。评估棕色脂肪细胞产热能力的关键参数是通过呼吸确定的线粒体解偶联。因此，测量线粒体氧消耗率（OCR）为研究燃料代谢和能量消耗的调节和失调提供了有价值的信息。添加线粒体膜电位的测量允许更加深入地解释呼吸数据。在这里我们提供使用Seahorse XF分析仪测量贴壁完整和质膜透性棕色脂肪细胞呼吸的协议。在方案部分I中，去甲肾上腺素和游离脂肪酸的组合用于诱导解偶联呼吸。然后使用ATP合酶抑制剂寡霉素，化学去偶联剂FCCP和复合物III抑制剂抗霉素A分别测量偶联的，最大的和非线粒体的氧消耗。在方案第II部分中，质膜用重组perfringolysin O透化，胆固醇依赖性细胞溶解素寡聚化成在质膜中专门的孔。这允许代谢物可用性的实验性控制，而不从天然细胞环境中分离线粒体。
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| Chromatin Immunoprecipitation (ChIP), Streptavidin and ATP-agarose Mediated Pull-down Analyses
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Author:
Date:
2013-09-20
[Abstract] Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) induces expression of both viral and cellular genes in virus infected B cells by mimicking activated Notch receptors (Notch-IC) that mediate transcription activation through binding to the repressing domain of the recombining binding protein suppressor of hairless (RBP-Jκ). In general, chromatin immunoprecipitation (ChIP) assays, electrophoresis mobility shift assays (EMSA), streptavidin-agarose mediated DNA pull-down assays, together with cell-based transcription reporter assays were conducted to verify whether the query protein is involved in EBNA2-dependent transcription. The ATP-bound state of nuclear chaperone nucleophosmin (NPM1) has been implicated in pleiotropic biological processes. An ATP-agarose-mediated pull-down protocol was ...
[摘要] EB病毒（EBV）核抗原2（EBNA2）通过模拟激活的Notch受体（Notch-IC）诱导病毒感染的B细胞中病毒和细胞基因的表达，所述Notch受体通过结合重组结合的抑制结构域介导转录激活无毛蛋白抑制剂（RBP-Jκ）。通常，进行染色质免疫沉淀（ChIP）测定，电泳迁移率变动测定（EMSA），链霉亲和素 - 琼脂糖介导的DNA下拉测定以及基于细胞的转录报道基因测定，以验证查询蛋白是否参与EBNA2依赖性转录。核伴侣核蛋白（NPM1）的ATP结合状态已涉及多效生物过程。开发ATP-琼脂糖介导的下拉方案以监测由ATP结合的NPM1诱导的引发前复合物的形成。根据EBNA2和Notch-IC已经显示出在B细胞系中靶基因的活化方面是部分可互换的，可以想象EBNA2是活化的Notch IC的生物学等价物。
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