Daynac Mathieu, Morizur Lise, Kortulewski Thierry, Gauthier Laurent R, Ruat Martial, Mouthon Marc-André, Boussin François D
CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967; INSERM, UMR 967; Université Paris Diderot, Sorbonne Paris Cité, UMR 967; Université Paris Sud, UMR 967; CNRS, Université Paris Sud, UMR 9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department;
CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967; INSERM, UMR 967; Université Paris Diderot, Sorbonne Paris Cité, UMR 967; Université Paris Sud, UMR 967;
J Vis Exp. 2015 Sep 14(103):53247. doi: 10.3791/53247.
Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.
侧脑室室下区（SVZ）的神经干细胞（NSCs）在哺乳动物大脑中终生维持嗅觉神经发生。它们依次产生过渡放大细胞（TACs）和成神经细胞，这些成神经细胞一旦整合到嗅球中就会分化为神经元。新兴的荧光激活细胞分选（FACS）技术已能够分离神经干细胞及其后代，并开始揭示成年神经发生微环境中的基因调控网络。我们在此报告一种细胞分选技术，该技术可通过LeX/EGFR/CD24三重染色追踪和区分成年SVZ中上述细胞群体的细胞周期动态。然后将分离的细胞接种为贴壁细胞，通过延时视频显微镜详细探究其细胞周期进程。为此，我们使用转基因荧光泛素化细胞周期指示剂（FUCCI）小鼠，其中由于G1特异性红色Cdt1报告基因，细胞在G1期呈红色荧光。该方法最近揭示，增殖的神经干细胞在衰老过程中其G1期逐渐延长，导致神经发生受损。该方法可轻松应用于其他系统，对于研究脑部疾病背景下脑细胞的细胞周期动态可能具有重要意义。
