Forced Differentiation of CNS Neural Precursors in vitro and in vivo

描述（由申请人提供）：齿状回和脑室下区是哺乳动物大脑中神经发生的允许区域，而大多数其他区域不是。许多重点放在寻找这些环境中不同的许可和非许可设置之间的线索集。随着这些因素的阐明，越来越清楚的是，许多在神经源性区域中活跃的信号分子也存在于非允许区域中，特别是在损伤后。那么，为什么神经前体细胞在暴露于某些区域时会产生神经元，而在其他区域则不会？虽然有许多细胞内的途径，是已知的调节神经前体产生神经元的效率和能力，有很少的考虑是否操纵任何这些途径能够克服在体外或体内的非容许环境。在这个建议中，我们将解决的假设，神经前体细胞可以克服非许可的环境，通过操纵的基本途径，控制细胞的命运规范。具体目标1：定义在体外干细胞生长条件下神经前体细胞定向分化为神经元或少突胶质细胞的机制。神经生成素（Ngn）或Mash 1的强制表达驱动神经元分化，Sox 10在24小时内驱动少突胶质细胞分化，而生长条件没有任何变化。我们的初步数据支持这一假设，即这些操作驱动多潜能NPC成为命运限制的前体。在这个目标中，我们将通过几组实验来测试这个过程的机制基础：1）当感染的细胞暴露于分化条件时，用转录因子定向分化是否改变分化的速率或程度？2)Ngn、Mash1或Sox10的表达是否指示向适当的命运分化（Ngn和Mash1的神经元和Sox10的少突胶质细胞），并限制分化为其他命运？3)这些因子表达的影响是可逆的吗？4)神经源性因子与Pax6之间是否存在协同作用？Sox10和Oligs之间的关系？5)与Ngn相比，Mash1强制表达产生的神经元中观察到的形态学差异的基础是什么？这些实验旨在进一步了解神经前体细胞强制分化的机制。具体目标2：确定定向分化是否克服了体内非允许条件。我们将设计前体来表达Ngn 1，Ngn 2，Mash 1或Sox 10，并将它们移植到两种条件下。首先，我们将它们注射到胚胎晚期的小鼠大脑中，以测试这些细胞在正常允许的环境中的行为。第二，我们将细胞注射到成年小鼠纹状体中，以测试这些细胞在神经元或少突胶质细胞分化的不太允许的条件下的行为。存在于已经形成的大脑中的神经前体细胞提供了一个令人兴奋的机会，通过利用内源性前体的潜力来扩增和产生神经元或少突胶质细胞，或者通过移植分裂的前体，然后对受损的环境做出反应以实现修复，来促进神经系统的再生修复。如果我们能够利用这些细胞的再生能力，就有可能考虑以干细胞为基础的治疗多发性硬化症，神经创伤或神经退行性疾病。

DESCRIPTION (provided by applicant): The dentate gyrus and subventricular zone are permissive areas for neurogenesis in the mammalian brain, while most other areas are not. Much emphasis is being placed on finding the set of cues in these environments that differ between permissive and non-permissive settings. As these factors are elucidated it is becoming clear that many signaling molecules active in neurogenic zones are also present in non-permissive areas, particularly after injury. Why then do neural precursor cells respond by making neurons when exposed to some regions while not others? While there are a number of intracellular pathways that are known to modulate the efficiency and ability of neural precursors to produce neurons, there has been little consideration as to whether manipulation of any of these pathways are able to overcome nonpermissive environments in vitro or in vivo. In this proposal we will address the hypothesis that neural precursor cells can overcome non- permissive environments by manipulation of the basic pathways that control cellular fate specification. Specific Aim 1: Define the mechanism(s) underlying directed differentiation of neural precursor cells into neurons or oligodendrocytes in stem cell growth conditions in vitro. Forced expression of Neurogenins (Ngn) or Mash1 drive neuronal differentiation and Sox10 drives oligodendrocyte differentiation within 24 hours without any change in growth conditions. Our preliminary data supports the hypothesis that these manipulations drive multipotential NPC to become fate-restricted precursors. In this aim we will test the mechanistic basis of this process through several sets of experiments: 1) Does directed differentiation with transcription factors change the rate or extent of differentiation when infected cells are exposed to differentiating conditions? 2) Does expression of Ngn, Mash1 or Sox10 instruct differentiation toward the appropriate fate (neurons for Ngn and Mash1 and oligodendrocytes for Sox10) and restrict differentiation into alternative fates? 3) Are the effects of expression of these factors reversible? 4) Is there synergism between neurogenic factors and Pax6? Between Sox10 and Oligs? 5) What is the basis for morphologic differences seen in neurons produced by forced expression of Mash1 compared to Ngn? These experiments are designed to acquire a further understanding behind the mechanism of forced differentiation of neural precursor cells. Specific Aim 2: Determine if directed differentiation overcomes non-permissive conditions in vivo. We will engineer precursors to express Ngn1, Ngn2, Mash1 or Sox10 and transplant them into two conditions. First, we will inject them intraventricularly into the late embryonic mouse brain to test the behavior of these cells in a normally permissive environment. Second, we will inject the cells into the adult mouse striatum to test the behavior of these cells in less permissive conditions for neuronal or oligodendrocyte differentiation. Neural precursor cells present in the already formed brain present an exciting opportunity to promote regenerative repair of the nervous system either by harnessing the potential of endogenous precursors to expand and produce neurons or oligodendrocytes or by transplantation of dividing precursors that then respond to the injured environment to effect repair. If we are able to harness the regenerative capacity of these cells it will be possible to consider stem cell based treatments for multiple sclerosis, neurotrauma or neurodegenerative diseases.