Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2™ cells using automated high-content image analysis
Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2™ cells using automated high-content image analysis
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DOI：
10.1016/j.neuro.2010.02.003
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发表时间：
2010-06-01
期刊：
影响因子：
3.4
通讯作者：
Mundy, William R.
中科院分区：
文献类型：
--
作者：
Harrill, Joshua A.;Freudenrich, Theresa M.;Mundy, William R.
在整个发育过程中，神经元经历了许多形态学变化，包括从细胞体中长出神经突。暴露于干扰这一过程的神经毒性化学物质可能导致神经系统功能的永久性缺陷。传统上，啮齿动物的原代神经培养物和永生化的人类和非人类克隆细胞系已被用于研究控制神经突生长的分子机制，并检查化学对这一过程的影响。本研究描述了hN 2（TM）人胚胎干细胞（hESC）衍生的神经细胞的分子表型，并使用自动化高内容图像分析来测量体外神经突生长。在接种后24小时，hN 2（TM）细胞表达许多指示神经元表型的蛋白质标志物，包括：巢蛋白。β（III）-微管蛋白、微管相关蛋白2（MAP 2）和磷酸化神经丝。hN 2（TM）细胞中的神经突生长迅速进行，大多数细胞在培养48小时时延伸出一到三个神经突。此外，在用双吲哚基马来酰亚胺I、U 0126、氯化锂、原钒酸钠和布雷菲德菌素A处理hN 2（TM）细胞后，观察到神经突生长和ATP含量的浓度依赖性降低，所有这些都已显示出在原代啮齿动物神经培养物中抑制神经突生长。总体而言，hN 2（TM）细胞的分子表型、神经突生长速率和对神经突生长抑制剂的敏感性与文献中先前描述的其他体外模型相当。hN 2（TM）细胞提供了一种模型，在该模型中研究化学作用对非转化的人源细胞中神经突生长的影响，并提供了使用原代啮齿动物神经培养物或永生化克隆细胞系的替代方法。爱思唯尔公司出版
Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary neural cultures and immortalized human and non-human clonal cell lines have been used to investigate the molecular mechanisms controlling neurite outgrowth and examine chemical effects on this process. The present study characterizes the molecular phenotype of hN2 (TM) human embryonic stem cell (hESC)-derived neural cells and uses automated high-content image analysis to measure neurite outgrowth in vitro. At 24 h post-plating hN2 (TM) cells express a number of protein markers indicative of a neuronal phenotype, including: nestin. beta(III)-tubulin, microtubule-associated protein 2 (MAP2) and phosphorylated neurofilaments. Neurite outgrowth in hN2 (TM) cells proceeded rapidly, with a majority of cells extending one to three neurites by 48 h in culture. In addition, concentration-dependent decreases in neurite outgrowth and ATP-content were observed following treatment of hN2 (TM) cells with either bisindolylmaleimide I, U0126, lithium chloride, sodium orthovanadate and brefeldin A, all of which have previously been shown to inhibit neurite outgrowth in primary rodent neural cultures. Overall, the molecular phenotype, rate of neurite outgrowth and sensitivity of hN2 (TM) cells to neurite outgrowth inhibitors were comparable to other in vitro models previously characterized in the literature. hN2 (TM) cells provide a model in which to investigate chemical effects on neurite outgrowth in a non-transformed human-derived cells and provide an alternative to the use of primary rodent neural cultures or immortalized clonal cell lines. Published by Elsevier Inc.