Pollard Steven M
Department of Cancer Biology, Samantha Dickson Brain Cancer Unit, UCL Cancer Institute, University College London, London, UK.
Methods Mol Biol. 2013;1059:13-24. doi: 10.1007/978-1-62703-574-3_2.
In vitro studies of neural progenitors isolated from the developing mouse have provided important insights into intrinsic and extrinsic pathways that control their behavior. However, use of primary cultures or neurospheres established from fetal tissues in cell population-based assays can be compromised by cellular heterogeneity. A complementary approach that addresses this issue is the establishment of adherent clonal neural stem (NS) cell lines. Here I describe protocols and troubleshooting advice for establishing adherent NS cell lines from the mouse fetal forebrain. NS cells grow as pure cultures in defined serum-free conditions as adherent monolayers and are therefore amenable to chemical/genetic screens, biochemical studies, and population-based analysis of gene expression or transcriptional regulation (e.g. RNA-Seq and ChIP-Seq). NS cell lines therefore represent a tractable cellular model system to explore the molecular and cellular biology of neural stem cell self-renewal and differentiation. Similar protocols can be extended to rat and human embryos, as well as human brain tumors.
对从小鼠发育大脑中分离出的神经祖细胞进行的体外研究，为控制其行为的内在和外在途径提供了重要见解。然而，在基于细胞群体的分析中，使用从胎儿组织建立的原代培养物或神经球可能会受到细胞异质性的影响。解决这一问题的一种补充方法是建立贴壁克隆神经干细胞（NS）系。在这里，我描述了从小鼠胎儿前脑建立贴壁NS细胞系的方案和故障排除建议。NS细胞在确定的无血清条件下以贴壁单层的形式作为纯培养物生长，因此适用于化学/遗传筛选、生化研究以及基于群体的基因表达或转录调控分析（例如RNA测序和染色质免疫沉淀测序）。因此，NS细胞系代表了一个易于处理的细胞模型系统，用于探索神经干细胞自我更新和分化的分子和细胞生物学。类似的方案可以扩展到大鼠和人类胚胎，以及人类脑肿瘤。
