Molecular Regulation of Human Glial Progenitor Cell-Based Remyelination

描述（由申请人提供）：髓鞘疾病包括遗传性白质营养不良症和脑瘫，以及成人血管、创伤和炎症性脱髓鞘综合征。为了解决这一庞大而多样的疾病群体，我们建立了一种细胞治疗方法来治疗中枢髓鞘再生，通过将分离的人类神经胶质祖细胞（GPCs）移植到新生儿脑内，然后让其成熟到成年。当受体是低髓鞘突变体时，如shiverer小鼠，移植细胞大部分成熟为髓鞘少突胶质细胞，并且可以挽救治疗动物的神经表型和寿命。然而，值得注意的是，大量的人类祖细胞整合到受体大脑中，在那里它们有效地竞争了小鼠祖细胞，产生了具有基本人源化白质的小鼠，以及灰质中主要的人类神经胶质祖细胞——最终是星形胶质细胞。由此产生的人类神经胶质嵌合小鼠大脑为我们提供了多种迄今为止无法获得的研究人类神经胶质细胞及其体内祖细胞的机会，包括它们对体外无法充分模拟的损伤和疾病过程的反应。在本实验中，我们将利用这些小鼠在体内评估毒性脱髓鞘对人GPCs的影响，以确定它们在代偿性髓鞘再生过程中对损伤诱导的动员和少突胶质细胞分化的分子反应。特别是，我们将使用表型特异性细胞分选和基因表达分析来确定这些异种移植的人类GPCs对体内脱髓鞘的反应。这些数据是首次从体内脱髓鞘和再髓鞘形成过程中特异性地从人类GPCs中获得的数据，应该为我们提供与再髓鞘形成相关的信号事件及其潜在的调控靶点的基本新见解。为了实现这一目标，我们提出了以下目标：在Aim 1中，我们将用铜酮治疗胶质嵌合小鼠，以便更好地了解人类GPCs对脱髓鞘的反应，评估它们的动员、分化、对重复诱导的反应，以及它们的有丝分裂衰老阈值及其调控。在Aim 2中，我们将研究人类GPC在体内的表达模式，从嵌合性震颤小鼠中分离出基线和脱髓鞘反应时的GPC，从而确定GPC动员和脱髓鞘再生过程中差异调节的基因和途径。在Aim 3中，我们将比较共同居住的人类和小鼠GPCs对铜嘧啶脱髓鞘的反应，从而确定那些可能成为药物开发中高价值靶点的共享途径，以及那些在小鼠中研究可能无法预测人类治疗结果的物种特异性途径。总之，这些实验有望为我们确定治疗脱髓鞘性脑或脊髓损伤的新策略提供信息。此外，在这项工作过程中产生的数据库，作为该领域的免费资源，应该证明促进我们对体内髓鞘再生的理解。

DESCRIPTION (provided by applicant): Disorders of myelin include the hereditary leukodystrophies and cerebral palsies, as well as adult vascular, traumatic and inflammatory demyelination syndromes. To address this large and diverse group of disease, we established a cell-therapeutic approach to central remyelination, by which transplants of isolated human glial progenitor cells (GPCs) are delivered intracerebrally to neonatal recipients, which are then allowed to mature to adulthood. When the recipients are hypomyelinated mutants, such as the shiverer mouse, the transplanted cells mature largely as myelinating oligodendrocytes, and can rescue both the neurological phenotype and lifespan of the treated animals. Remarkably though, large numbers of human progenitors integrate into the recipient brains, wherein they effectively out-compete mouse progenitors, yielding mice with a substantially humanized white matter, and a major contingent of human glial progenitors - and ultimately astrocytes - in the gray matter as well. The resultant human glial-chimeric mouse brains provide us a variety of hitherto unavailable opportunities for studying human glial cells and their progenitors in vivo, including their responses to injury and disease processes that cannot be adequately modeled in vitro. In the proposed experiments, we will use these mice to assess the effects of toxic demyelination on human GPCs in vivo, so as to identify their molecular responses to injury-induced mobilization and oligodendrocytic differentiation during compensatory remyelination. In particular, we will use phenotype-specific cell sorting and gene expression analysis, to define the responses of these xenografted human GPCs to demyelination in vivo. These data, the first ever obtained specifically from human GPCs during demyelination and remyelination in vivo, should afford us fundamental new insights into the signaling events associated with remyelination, and their potentially targetable points of regulatory control. To achieve that end, we propose the following Aims: In Aim 1, we will treat glial-chimeric mice with cuprizone so as to better understand the responses of human GPCs to demyelination, assessing their mobilization, differentiation, responses to repetitive induction, as well as their thresholds for mitotic senescence and the regulatory control thereof. In Aim 2, we will examine the expression patterns of human GPCs in vivo, sorting them from chimeric shiverer mice both at baseline and in response to demyelination, so as to define those genes and pathways differentially regulated during GPC mobilization and remyelination. In Aim 3 we will compare the responses of co-resident human and mouse GPCs to cuprizone demyelination, so as to identify those shared pathways likely to be high-value targets in drug development, as well as those species-specific pathways whose investigation in mice might not predict human therapeutic outcome. Together, these experiments promise to inform our efforts to define new strategies for treating demyelinating brain or spinal cord injury. In addition, the databases to be generated in the course of this work, as freely available resources to the field should prove catalytic in advancing our understanding of remyelination in vivo. PUBLIC HEALTH RELEVANCE: In the course of developing new human cell therapeutics for treating brain disease, we established mice in which a substantial proportion of all CNS glial cells were of human origin, especially in the white matter, the region affected in human myelin disease. In this application, we propose to use perinatal transplants of human glial progenitor cells to establish mice with largely humanized white matter, and to then subject these mice to experimental demyelination, yielding pathology similar to that of disorders such as subcortical stroke and multiple sclerosis. We will then use cell sorting techniques to separate the human glial progenitor cells from the affected mouse brains, followed by gene expression analyses to identify which genes respond to the demyelinating insult, and by what time course. By this means, we expect to define those signaling pathways involved in remyelination by normal human glial progenitor cells in vivo, knowledge that should provide us great insight into how to induce and regulate this process therapeutically.