| 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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| Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures
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Author:
Date:
2020-07-05
[Abstract] MYC family members, MYC, MYCN, and MYCL, are oncogenic transcription factors that regulate the expression of genes involved in normal development, cell growth, proliferation, metabolism, and survival. While MYC is amplified and/or overexpressed across a variety of tissue types, MYCN is often overexpressed in tumors of the nervous system (neuroblastoma and medulloblastoma) or with neuroendocrine features (neuroendocrine prostate cancer). Given recent reports that MYCN expression is also deregulated in a variety of non-neuronal tissue types, we investigated whether MYCN was also deregulated in triple-negative breast cancer (TNBC). In contrast to previous individual immuno-fluorescence (IF) stains against higher expressing MYC family isoform protein, we developed an IF stain to ...
[摘要] [摘要] MYC家族成员MYC、MYCN和MYCL是一类致癌转录因子，它们调节与正常发育、细胞生长、增殖、代谢和生存有关的基因的表达。虽然MYC在多种组织类型中扩增和/或过度表达，但MYCN通常在神经系统肿瘤（神经母细胞瘤和髓母细胞瘤）或具有神经内分泌特征（神经内分泌前列腺癌）中过度表达。鉴于最近的报道，MYCN在多种非神经元组织中的表达也被解除了调控，我们研究了MYCN在三阴性乳腺癌（TNBC）中是否也被解除了调控。与以往针对高表达MYC家族亚型蛋白的个体免疫荧光（IF）染色不同，我们开发了一种IF染色法来同时检测同一肿瘤细胞群中MYCN-和MYC表达细胞。我们的方法允许检测低水平的MYCN和MYC表达，并且可以与额外的蛋白质探针复合。在此，我们利用酪酰胺信号放大（TSA），提出了两种检测MYCN和MYC的方案，用于在福尔马林固定石蜡包埋（FFPE）肿瘤切片和生长后原位固定的细胞系中检测MYCN和MYC。
[背景] ...
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| Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration
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Author:
Date:
2020-06-20
[Abstract] Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to design cell lines stably expressing fluorophore proteins shuttling between the nucleus and cytoplasm by karyopherins of choice. To validate this protocol, we measured an ALS-associated nucleocytoplasmic transport pathway in the presence of the disease-associated peptide poly-PR. This technique allows measuring a particular active nucleocytoplasmic transport pathway in intact cells in a neurodegenerative disease-associated context. Moreover, these ...
[摘要] [摘要]核细胞质运输缺陷被认为在神经退行性疾病中发挥作用，包括肌萎缩性侧索硬化症（ALS）。鉴于这一过程的重要性和复杂性，了解这些畸变何时发生以及涉及哪些途径是非常重要的。在这里，我们利用CRISPR-Cas9技术来设计细胞系，稳定地表达荧光素蛋白在细胞核和细胞质之间穿梭的选择的卡里菲林。为了验证这个协议，我们测量了一个ALS相关的核细胞质运输途径，在疾病相关的多肽poly-PR的存在。这种技术允许测量在神经退行性疾病相关的背景下，在完整细胞中的一个特定的活性核细胞质运输途径。此外，这些实验可以在不需要昂贵的设备的情况下进行，并有可能升级为高通量筛选目的。
[背景]核细胞质运输对细胞稳态至关重要，并通过形成水通道的大型多蛋白复合物穿过核膜，称为核孔复合物（NPCs）（Stoffler et al., 1999; Ryan and Wente, 2000)。虽然这些通道允许小蛋白被动地平衡穿过核膜（被动运输），但大多数蛋白质似乎是由核素主动运输的，即进口蛋白或出口蛋白（主动运输）。含有核定位信号(NLS)的货物蛋白被导入素所定位。已经确定了各种受体介导的导入途径，但最有特色的途径涉及导入素-β1/导入素-α(KPNB1/KPNAx)，其中货蛋白含有经典的NLS(cNLS)，以SV40大T抗原NLS为例(Lange et al., ...
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