CHARACTERIZATION OF CNS STEM CELLS USING SOMATIC MOSAICS

在成人大脑的主要有丝分裂后细胞环境中存在多潜能干细胞，这引发了对假定细胞类型的鉴定(S)和潜在功能的深入研究。干细胞特性的阐明可能会对其在神经退行性疾病或年龄相关退行性疾病的治疗中的潜在用途产生立竿见影的影响。关于中枢神经系统干细胞的身份及其从干细胞到有丝分裂后神经元和/或神经胶质细胞的循序渐进过程一直存在争议。最近关于位于脑室下区(SVZ)的干细胞的提示研究表明，在SVZ中发现的星形胶质细胞样细胞具有自我更新和/或在吻端迁移路径下游产生细胞的潜力。本申请描述了一种新的体细胞镶嵌方法的发展，以明确评估SVZ星形胶质细胞作为干细胞的作用，其中总体假设如下：选择性移除GFAP阳性的SVZ星形胶质细胞，据称是多能干细胞，将导致伴随而来的神经母细胞和正常情况下来自SVZ的成熟神经元的丧失。具体地说，将使用生殖系传播的转基因构建物，其携带顺式重组元件loxP，位于强转录终止序列的两侧。当通过缺陷的疱疹病毒扩增载体通过体细胞递送cre重组酶激活时，转基因结构的永久性改变将被特异性地催化在SVZ星形胶质细胞的基因组中，从而影响前药物酶的局部表达，使这些细胞对促进细胞死亡的前药物敏感。重组诱导转基因表达的细胞也将通过组织化学标记物的共同诱导来标记，从而在没有前药物的情况下确定细胞的命运。本提案中描述的实验构成了对体细胞镶嵌系统的特性的分析，预计将允许对未来的实验进行适当的设计、执行和解释，其中该系统将被应用于成年啮齿动物中SVZ星形胶质细胞的功能。

The existence of multipotent stem cells in the primarily post-mitotic cellular environment of the adult brain has sparked intense research into identification of putative cell type(s) and potential functions. Elucidation of stem cell identity could have an immediate impact regarding its potential use in therapies for neurodegenerative diseases or age-related degeneration. Controversy has arisen regarding the identity of the CNS stem cell and its step-wise lineage progression from stem cell to post- mitotic neuron and/or glia. Recent suggestive studies concerning stem cells located in the subventricular zone (SVZ) have shown that astrocyte- like cells found in the SVZ have the potential to self-renew and/or give rise to cells downstream in the rostral migratory pathway. The development of a novel somatic mosaic approach is described in this application to definitively assess the role of SVZ astrocytes as stem cells, where the overall hypothesis is as follows: Selective removal of GFAP- positive SVZ astrocytes, purported to be multipotent stem cells, will result in the concomitant loss of neuroblastic cells and mature neurons that normally arise from the SVZ. Specifically, a germline-transmitted transgene construct will be used that carries cis recombination elements, loxP, flanking strong transcriptional termination sequences. When activated by the somatic delivery of cre recombinase via a defective herpes amplicon vector a permanent alteration in transgene structure will be catalyzed specifically in the genome of SVZ astrocytes, thereby effecting focal expression of a pro-drug enzyme that sensitizes these cells to a cell death-promoting pro-drug. Cells in which recombination induces transgene expression will also be tagged via co-induction of a histochemical marker, allowing for determination of cell fate in the absence of pro-drug. Experiments described in this proposal constitute the analysis of the properties of the somatic mosaic system that are anticipated to allow for appropriate design, execution, and interpretation of future experiments where the system will be applied to address the function of SVZ astrocytes in the adult rodent.