Strategy of Isolating ‘Primed’ Tumor Initiating Cells Based on Mitochondrial Transmembrane Potential
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Author:
Date:
2021-03-05
[Abstract] Various stem cells have been found to be dependent on mitochondrial energetics. The role of mitochondria in regulating the self-renewal of normal stem cells and stem-like tumor initiating cells (TICs) is increasingly being appreciated. We proposed that TIC populations have a sub population of cells that are “primed” by mitochondria for self-renewal. Using ovarian cancer model, we have developed a protocol to identify and isolate these “primed” cells using Fluorescence-Assisted Cell Sorting (FACS). We combined live cell stains for a functional marker of TICs and for mitochondrial transmembrane potential to enrich TICs with higher mitochondrial potential that form in vitro spheroids 10-fold more than the other TICs with lower mitochondrial potential. This protocol ...
[摘要] [摘要]已经发现各种干细胞都依赖于线粒体的能量学。线粒体在调节正常干细胞和干细胞样肿瘤起始细胞(TICs)自我更新中的作用日益受到人们的重视。我们提出,TIC种群具有由线粒体“引发”自我更新的亚细胞群。使用卵巢癌模型,我们开发了一种协议,可以使用荧光辅助细胞分选(FACS)识别和分离这些“初免”细胞。我们结合活细胞染色剂作为TIC的功能标记物和线粒体跨膜电位,以富集具有更高线粒体电位的TIC(在体外形成)球状体比线粒体电位较低的其他TIC高10倍。该协议可以直接使用或修改以用于各种小区类型。因此,预期该方案对于干细胞群体中线粒体和能量异质性的基本理解是不可侵犯的,并且在再生医学和癌症生物学的转化研究中也可能被证明是有价值的。
背景技术肿瘤中的细胞异质性对癌症治疗提出了严峻的挑战(Magee等,2012)。当某些肿瘤通过自身更新和分化维持肿瘤时,其与肿瘤抗性的肿瘤起始细胞(也称为癌症干细胞)分层地排列在细胞层次的底部(Magee et ...
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Investigating Neural Stem Cell and Glioma Stem Cell Self-renewal Potential Using Extreme Limiting Dilution Analysis (ELDA)
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Author:
Date:
2018-09-05
[Abstract] Glioma stem cells (GSC) grown as neurospheres exhibit similar characteristics to neural stem cells (NSC) grown as neurospheres, including the ability to self-renew and differentiate. GSCs are thought to play a role in cancer initiation and progression. Self-renewal potential of GSCs is thought to reflect many characteristics associated with malignancy, including tumor recurrence following cytotoxic therapy due to their proliferative dormancy and capacity to allow for the development of resistant tumor cell sub-clones due to mutations acquired during their differentiation. Here, we demonstrate that using extreme limiting dilution analysis (ELDA), subtle differences in the frequency of sphere-forming potential between PI3K-mutant oncogenic NSCs and non-oncogenic NSCs can be measured, in ...
[摘要] 作为神经球生长的神经胶质干细胞(GSC)表现出与作为神经球生长的神经干细胞(NSC)相似的特征,包括自我更新和分化的能力。 GSC被认为在癌症的发生和发展中起作用。 GSC的自我更新潜力被认为反映了与恶性肿瘤相关的许多特征,包括细胞毒性治疗后的肿瘤复发,这是由于它们的增殖性休眠和由于在其分化期间获得的突变而允许产生抗性肿瘤细胞亚克隆的能力。在这里,我们证明使用极限稀释分析(ELDA),可以测量PI3K-突变致癌NSCs和非致癌NSCs之间的球形成潜力频率的细微差异体外。我们进一步展示了ELDA如何在强制分化之前和之后用于细胞,以放大突变体和对照NSCs之间的球形成潜力的固有差异。最终,ELDA利用单个或少数种子干细胞自我更新,分裂和形成神经球的能力差异。重要的是,该测定还允许在不同条件下在遗传上不同的细胞之间或相同细胞之间进行比较,其中可以测试靶特异性药物或其他新型癌症干细胞疗法的影响。
【背景】胶质母细胞瘤(GBM)是最常见的脑癌之一,预后极差(Kaye和Morokoff,2014)。 ...
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FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand
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Author:
Date:
2017-12-20
[Abstract] Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma samples, exploiting the ability of cells to bind the Epidermal Growth Factor (EGF) ligand in fluorescence-activated cell sorting (FACS). We demonstrated that FACS-isolated EGF-bound neural and glioblastoma populations encompass the sphere-forming colonies in vitro, and are capable of both self-renewal and multilineage differentiation. Here we describe in detail the purification methodology of EGF-bound (i.e., EGFR+) human neural and ...
[摘要] 从新鲜组织中直接分离人类神经和胶质瘤干细胞允许其在没有事先培养的情况下进行生物学研究,并且可以在其天然状态中捕获其分子表型的新方面。最近,我们展示了前瞻性地从新鲜人类生发基质和胶质母细胞瘤样品中分离干细胞群的能力,利用细胞在荧光激活细胞分选(FACS)中结合表皮生长因子(EGF)配体的能力。我们证明FACS分离的EGF结合的神经和成胶质细胞瘤细胞群体在体外包含球体形成的集落,并且能够自我更新和多向分化。在此我们详细描述了具有来自新鲜死亡和手术组织的干细胞特性的EGF-结合(即EGFR +)人类神经和胶质瘤细胞的纯化方法。利用天然配体结合能力前瞻性分离干细胞群的能力为了解非培养条件下的正常和肿瘤细胞生物学打开了新的门,并且适用于在种群和单细胞分辨率下的各种下游分子测序研究。
【背景】由于缺乏通用的神经和神经胶质瘤干细胞标志物(Lathia et al。,2015)以及频繁依赖于培养的细胞,理解人神经和胶质瘤干细胞的内在生物学一直是一个挑战比那些直接从组织分离的。跨膜糖蛋白Prominin或CD133是分离神经(Uchida等,2000)和神经胶质瘤干细胞(GSC)(Singh等,2000)的最好描述和经常使用的干细胞标记物之一。等人,2003; Singh等人,2004; ...
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