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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In vitro Explant Cultures to Interrogate Signaling Pathways that Regulate Mouse Lung Development
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Author:
Date:
2018-05-20
[Abstract] Early mouse lung development, including specification of primordia, patterning of early endoderm and determination of regional progenitor cell fates, is tightly regulated. The ability to culture explanted embryonic lung tissue provides a tractable model to study cellular interactions and paracrine factors that regulate these processes. We provide up-to-date protocols for the establishment of this culture model and its application to investigate hedgehog signaling in the developing lung.
[摘要] 早期鼠肺发育,包括原基的规范,早期内胚层的构图和区域祖细胞命运的确定,受到严格的调控。 培养移植胚胎肺组织的能力提供了一种易处理的模型来研究调节这些过程的细胞相互作用和旁分泌因子。 我们提供最新的协议,以建立这种文化模式及其应用来研究肺部发育中的刺猬信号。
【背景】小鼠肺发育起始于前肠前内胚层的内胚层憩室(E9.5),随后关闭近端气管食管中隔以形成不同的气管和食道管(Minoo和King,1994)。原始内胚层管的随后分支通过E12.5产生平面肺结构,随后正交分支产生成熟肺的三维结构特征(Metzger等人,2008)。在E12.5之前分离的肺遗传的平面结构适合于在空气液体界面进行体外培养(Carraro等人,2010; Del Moral和Warburton, 2010)。胚胎肺通过解剖使用立体显微镜在亮场照明下或通过与谱系追踪和荧光报道分子偶联时的荧光照明进行分离。在这里,我们描述了使用Shh Cre / Rosa mTmG报告小鼠,其允许Cre介导的从约E8.75起在前部前肠内胚层内的膜定位的GFP的激活(Montgomery <等人,2007;高斯等人,2009; Yao等人,2017)。因此,肺内胚层通过红色荧光由绿色荧光和周围组织显现,从而允许清晰识别和显微切割包括肺在内的发育内胚层结构,并在体外培养期间成像。
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