Separation of Stem Cells From Differentiated Cells

描述(由申请人提供)：这是一份R21申请的重新提交。我们期望达到的目标是在体外和体内标记可再生的神经干细胞，以确定神经干细胞在大脑中的准确位置及其发育命运，这一知识是开发神经干细胞安全临床应用的先决条件，目的是治疗神经退行性疾病和中枢神经系统损伤。我们建议使用干细胞特异性的Melk基因调控元件来驱动荧光计时器报告。更多的初步数据提供了令人信服的证据，证明Melk基因在两种类型的体细胞干细胞中特异性表达，即神经干细胞和造血细胞。关键的是，Melk编码区上游3.4kb的基因组片段在神经球形成细胞和一些Nestin阳性细胞中选择性地活跃，而在GFAP阳性的星形胶质细胞或TuJ1阳性的年轻神经元中不活跃。对于报告基因，我们将利用荧光计时器蛋白，这是红色荧光蛋白的一种特殊变体，随着成熟，它的荧光颜色会自发地从绿色变成红色。初步研究表明，在巢蛋白增强子控制下表达的荧光计时器在所有神经前体和终末分化的神经细胞中提供了明显的荧光区分。然而，巢蛋白增强子的广泛表达模式使这种组合不适合在体外或体内对神经干细胞进行精确标记。综上所述，我们目前的数据有力地支持了这样的假设，即Melk-Timer组合将允许在体外和体内更精确地标记和分离神经干细胞。我们的具体目标是：1.研究Melk启动子-荧光计时器组合是否标记神经球中的神经干细胞以及在ES细胞体外分化过程中，2.使用Melk-荧光计时器转基因或敲入策略在体内标记神经干细胞。神经干细胞的基因标记将提供模型系统，以允许对干细胞生态位进行明确的研究，干细胞生态位是控制其自我更新和分化的专门微环境。哺乳动物的大脑中可能存在不止一种类型的神经干细胞。基因标记方法将解决这一问题，为基因定义的干细胞群体提供一个明确的标记，然后可以在体外和体内精确地研究干细胞的发育命运。

DESCRIPTION (provided by applicant): This is a resubmission of an R21 application. The goal we expect to reach is to label renewable neural stem cells in vitro and in vivo to identify the precise location of neural stem cells in the brain and their developmental fates, this knowledge is a prerequisite for developing safe clinical applications of neural stem cells for the purpose of treating neurodegenerative disorders and CNS injuries. We propose to use a stem cell specific, MELK gene regulatory element to drive the Fluorescent Timer reporter. Additional preliminary data provide convincing evidence of the specific expression of the MELK gene in two types of somatic stem cells, neural and hematopoietic. Critically, a 3.4kb genomic fragment upstream of the MELK coding region is selectively active in neurosphere-forming cells and in some Nestin positive cells but not in GFAP positive astrocytes or TuJ1 positive young neurons. For the reporter gene, we will take advantage of the Fluorescent Timer protein, a special variant of the red fluorescent protein that spontaneously changes its fluorescence color from green to red with maturation. Preliminary studies have shown that the Fluorescent Timer expressed under the control of a Nestin enhancer provides a clear fluorescent distinction among all neural precursors and terminally differentiated neural cells. The broad expression pattern of the Nestin enhancer, however, makes this combination unsuitable for the precise labeling of neural stem cells in vitro or in vivo. In summary, our current data strongly supports the hypothesis that the MELK-Timer combination will permit more precise labeling and the separation of neural stem cells in vitro and in vivo. Our specific Aims are: 1. To investigate if the MELK promoter - Fluorescent Timer combination labels neural stem cells in neurospheres and during in vitro differentiation of ES cells, and, 2. To genetically label neural stem cells in vivo using the MELK - Fluorescence Timer transgenic or knock-in strategies. The genetic 'labeling' of neural stem cells will provide the model system to permit definitive studies of the stem cell niche, a specialized microenvironment that controls their self-renewal and differentiation. It is likely that more than one type of neural stem cells exist in the mammalian brain. The genetic labeling approach will address this issue providing a clear marker for the genetically defined stem cell population, whose developmental fate could then be precisely addressed both in vitro and in vivo.