Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics.
Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics.
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DOI：
10.3791/53247
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发表时间：
2015-09-14
期刊：
影响因子：
--
通讯作者：
Boussin FD
中科院分区：
文献类型：
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作者：
Daynac M;Morizur L;Kortulewski T;Gauthier LR;Ruat M;Mouthon MA;Boussin FD
位于侧脑室脑室下区的神经干细胞（Neural stem cells，NSCs）在哺乳动物的大脑中维持着嗅觉神经的发生。它们相继产生转运放大细胞（TAC）和成神经细胞，一旦它们整合嗅球，这些细胞就分化成神经元。新兴的荧光激活细胞分选（FACS）技术已经允许分离神经干细胞以及它们的后代，并已开始阐明成人神经原性龛中的基因调控网络。我们在这里报告的细胞分选技术，允许以下和区分上述细胞群体的细胞周期动力学与成人SVZ与LeX/EGFR/CD 24三重染色。然后将分离的细胞作为贴壁细胞铺板，以通过延时视频显微镜详细探索其细胞周期进程。为此，我们使用转基因荧光泛素化细胞周期指示剂（FUCCI）小鼠，其中细胞在G1期期间由于G1特异性red-Cdt 1报告基因而发出红色荧光。这种方法最近发现，增殖的神经干细胞在衰老过程中逐渐延长其G1期，导致神经发生障碍。这种方法很容易移植到其他系统，并可能是非常感兴趣的脑细胞的细胞周期动力学研究的背景下，脑病理学。
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.