Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection
Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection
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DOI：
10.3791/4093
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发表时间：
2012-10-01
影响因子：
1.2
通讯作者：
Calegari, Federico
中科院分区：
文献类型：
--
作者：
Artegiani, Benedetta;Lange, Christian;Calegari, Federico
成体干细胞可以分裂产生额外的干细胞（扩增）或更多分化的细胞类型（分化），这对于胚胎发育期间的组织形成和成年期间的组织稳态至关重要 (1)。目前，人们投入了大量精力来控制体干细胞从扩增到分化的转变，因为这被认为是开发再生医学新策略的基础 (1,2)。然而，研究和使用成体干细胞的一个主要挑战是它们的扩增已被证明非常难以控制。在这里，我们描述了一种系统，该系统可以通过操纵cdk4/cyclinD1复合物的表达来控制小鼠胚胎皮层或成年海马中的神经干/祖细胞（统称为NSC）的扩增，cdk4/cyclinD1复合物是细胞周期G1期和体干细胞分化的主要调节因子 (3,4)。具体来说，描述了两种不同的方法，通过这两种方法在体内 NSC 中过表达 cdk4/cyclinD1 复合物。通过第一种方法，通过将编码 cdk4/cyclinD1 的质粒注射到小鼠端脑的管腔中，然后通过子宫内电穿孔将其递送至外侧皮层的 NSC，从而触发转基因的附加型表达 (5-8)，从而获得细胞周期调节因子的过度表达 (5-8)。通过第二种方法，将高度浓缩的 HIV 衍生病毒立体定位注射到成年小鼠海马的齿状回中，从而在病毒构建体整合到受感染细胞的基因组中后触发细胞周期调节因子的组成型表达 (9)。这两种方法的基本原理已在其他视频协议 (10-14) 中进行了描述，在此进行了优化，以 i) 减少组织损伤，ii) 针对非常特定的大脑区域的广泛部分，iii) 在每个区域内获得大量被操纵的细胞，以及 iv) 触发每个细胞内转基因的高表达水平。使用这两种方法的转基因的瞬时过表达是通过不同的方式获得的。 e.通过细胞分裂或在表达 Cre 的 NSC 中施用他莫昔芬来自然稀释电穿孔质粒，这些 NSC 感染了两侧分别为 loxP 位点的 cdk4/cyclinD1 病毒 (9,15)。这些方法提供了一个非常强大的平台，可以快速且组织特异性地操纵小鼠大脑中任何基因的表达。特别是，通过操纵 cdk4/cyclinD1 复合物的表达，我们的系统可以暂时控制 NSC 扩张及其向分化的转变，从而最终增加哺乳动物大脑中生成的神经元数量。我们的方法对于基础研究和使用体干细胞治疗哺乳动物中枢神经系统可能至关重要，同时可以更好地理解i）干细胞对发育过程中组织形成的贡献，ii）成年期间的组织稳态，iii）成体神经发生在认知功能中的作用，也许，iv）更好地在神经退行性疾病模型中使用体干细胞。
Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood (1). Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine (1,2). However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation (3,4). Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes (5-8). By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells (9). Both approaches, whose basic principles were already described by other video protocols (10-14), were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i. e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively (9,15).These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.