Forced Differentiation of CNS Neural Precursors in vitro and in vivo

描述(申请人提供)：齿状回和脑室下区是哺乳动物大脑中允许神经发生的区域，而大多数其他区域不是。重点是在这些环境中寻找在许可和非许可设置之间存在差异的线索集。随着这些因素的阐明，越来越明显的是，许多活跃在神经源性区域的信号分子也存在于非许可区域，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通过制造神经元来做出反应，而在其他区域则不会呢？虽然已知有许多细胞内通路调节神经前体产生神经元的效率和能力，但很少有人考虑操纵这些通路中的任何一条是否能够在体外或体内克服不允许的环境。在这个提案中，我们将解决这样的假设，即神经前体细胞可以通过操纵控制细胞命运指定的基本途径来克服不允许的环境。具体目的1：明确神经前体细胞在干细胞体外生长条件下定向分化为神经元或少突胶质细胞的机制(S)。神经原蛋白(NGN)或Mash1的强制表达促进神经元分化，Sox10在24小时内促进少突胶质细胞分化，而生长条件没有任何变化。我们的初步数据支持这样的假设，即这些操纵促使多个潜在的NPC成为命运受限的前体。为此，我们将通过几组实验来测试这一过程的机制基础：1)当感染细胞暴露在分化条件下时，转录因子的定向分化是否会改变分化的速度或程度？2)NGN、Mash1或Sox10的表达是否会引导分化走向合适的命运(NGN和Mash1的神经元，Sox10的少突胶质细胞)，并限制分化为另一种命运？3)这些因子的表达影响可逆吗？4)神经源性因子和Pax6之间是否存在协同作用？5)与NGN相比，强制表达Mash1产生的神经元形态差异的基础是什么？这些实验旨在进一步了解神经前体细胞强迫分化的机制。具体目标2：确定体内定向分化是否克服了不允许的条件。我们将设计表达Ngn1、Ngn2、Mash1或Sox10的前体，并将它们移植到两种条件下。首先，我们将把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。其次，我们将把这些细胞注射到成年小鼠的纹状体中，以测试这些细胞在不允许神经元或少突胶质细胞分化的条件下的行为。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来促进神经系统的再生修复，方法是利用内源性前体的潜力来扩大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来实现修复。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.