Integration of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons into Rat Brain
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Author:
Date:
2020-09-05
[Abstract] Human neuron transplantation offers novel opportunities for modeling human neurologic diseases and potentially replacement therapies. However, the complex structure of the human cerebral cortex, which is organized in six layers with tightly interconnected excitatory and inhibitory neuronal networks, presents significant challenges for in vivo transplantation techniques to obtain a balanced, functional and homeostatically stable neuronal network. Here, we present a protocol to introduce human induced pluripotent stem cell (hiPSC)-derived neural progenitors to rat brains. Using this approach, hiPSC-derived neurons structurally integrate into the rat forebrain, exhibit electrophysiological characteristics, including firing, excitatory and inhibitory synaptic activity, and establish ...
[摘要] [摘要] 人类神经元移植为建模人类神经系统疾病和潜在的替代疗法提供了新的机会。然而,人脑皮层的复杂结构分为六层,具有紧密互连的兴奋性和抑制性神经元网络,这对体内移植技术获得平衡,功能稳定和稳态稳定的神经元网络提出了重大挑战。在这里,我们提出了一项协议,将人类诱导的多能干细胞(hiPSC )衍生的神经祖细胞引入大鼠脑。使用这种方法,hiPSC 衍生的神经元在结构上整合到大鼠前脑中,表现出电生理特性,包括放电,兴奋性和抑制性突触活性,并与宿主电路建立神经元连通性。
[背景] 人类大脑皮层是一个复杂的细胞镶嵌体,在不同的皮质层(I-VI)中包含多样化的神经元亚型,可建立轴突输出和树突状输入的特定模式,提供了皮质电路的基本底物(Rakic,2009; Lodato 等等人,2011; Lui 等人,2011)。特别地,兴奋性和抑制性神经传递的平衡对于适当的脑功能是必需的(Turrigiano和Nelson,2004)。人类诱导的多能干细胞(hiPSC )可以在人类遗传背景下对人类神经系统疾病进行建模(Dolmetsch和Geschwind,2011; Brennand 等,2015; Vera和Studer,2015)。建立体外系统以将hiPSCs ...
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Rapid Generation of Human Neuronal Cell Models Enabling Inducible Expression of Proteins-of-interest for Functional Studies
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Author:
Date:
2020-05-05
[Abstract] CRISPR-Cas9 technology has transformed the ability to edit genomic sequences and control gene expression with unprecedented ease and scale. However, precise genomic insertions of coding sequences using this technology remain time-consuming and inefficient because they require introducing adjacent single-strand cuts through Cas9 nickase action and invoking the host-encoded homology-directed repair program through the concomitant introduction of large repair templates. Here, we present a system for the rapid study of any protein-of-interest in two neuronal cell models following its inducible expression from the human AAVS1 safe harbor locus. With lox-flanked foundation cassettes in the AAVS1 site and a tailor-made plasmid for accepting coding sequences-of-interest in ...
[摘要] [摘要] CRISPR-Cas9技术以前所未有的简便性和规模改变了编辑基因组序列和控制基因表达的能力。但是,由于需要引入相邻的单链,因此使用该技术进行精确的基因组编码插入仍然很耗时且效率低下。通过减少Cas9切口酶的作用并通过同时引入大型修复模板来调用宿主编码的同源性指导的修复程序。在此,我们提出了一种系统,用于在其诱导后的两个神经元细胞模型中快速研究任何目的蛋白该系统可从人类AAVS1 安全港基因座表达,在AAVS1 位点具有lox侧翼的基础盒和定制的质粒以接受感兴趣的编码序列,该系统使研究人员能够为诱导型产生自己的神经元细胞模型任何编码表达序列不到一个月的时间。由于可用性Preinserted 增强型绿色的Fluo 可以与目标蛋白质融合的最新蛋白质(EGFP)编码序列,该系统可帮助功能研究通过活细胞显微镜以及利用非常有效的可用性进行的相互作用组分析来跟踪目标蛋白质EGFP捕获矩阵。
[背景] ...
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In vivo Tumor Growth and Spontaneous Metastasis Assays Using A549 Lung Cancer Cells
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Author:
Date:
2020-04-05
[Abstract] Metastasis accounts for the majority of cancer related deaths. The genetically engineered mouse (GEM) models and cell line-based subcutaneous and orthotopic mouse xenografts have been developed to study the metastatic process. By using lung cancer cell line A549 as an example, we present a modified protocol to establish the cell line-based xenograft. Our protocol ensures sufficient establishment of the mouse xenografts and allows us to monitor tumor growth and spontaneous metastasis. This protocol could be adapted to other types of established cancer cell lines or primary cancer cells to study the mechanism of metastatic process as well as to test the effect of the potential anti-cancer agents on tumor growth and metastatic capacity.
[摘要] [摘要 ] 中号etastasis应收大多数癌症相关死亡。已经开发了基因工程小鼠(GEM)模型以及基于细胞系的皮下和原位小鼠异种移植物,以研究转移过程。以肺癌细胞系A549为例,我们提出了一种改进的方案来建立基于细胞系的异种移植物。我们的协议可确保小鼠异种移植物的充分建立,并允许我们监测肿瘤的生长和自发转移。该方案可适用于其他类型的已建立癌细胞系或原发性癌细胞,以研究转移过程的机制以及测试潜在抗癌药对肿瘤生长和转移能力的影响。
[背景 ] 的5年生存率速率为患者与晚期阶段肺癌症是少大于15%时,与在大多数的患者死于从转移性疾病(斯特和恩格尔曼,2012 ; 惠特塞特,等人,2013 ; D'安东尼奥等人。,2014 ; Steeg的,2016) 。因此,使用鼠标模型来调查的机制的肺肿瘤侵袭和转移可能促进了发展的新的战略来控制的转移过程。
转移是一个多步骤过程是包括本地侵袭,血管内,并且生存在的循环,外渗,一个d建立macrometastasis 在遥远的站点。小鼠异种移植和基因工程小鼠(GEM)模型均已用于研究肺癌转移。GEM模型模仿了人类肺癌的遗传标记,例如TP53 ,K- ras 和LKB1,为研究转移过程的机制以及研究新型抗癌剂如何影响转移过程提供了免疫系统(Dutt和Wong ,2006年)。 ; Zheng ...
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