Genes, Environment & Neural Stem Cell Transplantation in the Gut

描述（由申请人提供）：神经干细胞（NSC）移植是一种新兴技术，具有治疗外周（PNS）和中枢神经系统（CNS）疾病的巨大前景。最近，我们的实验室已经开始研究使用NSC作为胃肠动力障碍的可行治疗策略。使用在具有神经元型一氧化氮合酶（nNOS）酶的基因缺失的小鼠中移植CNS衍生的NSC（CNS-NSC）的策略，我们已经表明CNS-NSC可以成功地移植并分化成表达nNOS的神经元，可以恢复这些小鼠中的氮能松弛和“拯救”胃功能（排空）。我们还表明，内在的凋亡信号是更重要的决定移植的神经干细胞的生存比宿主的免疫反应。这些实验的结果产生了相当大的信心，这种策略作为一种潜在的治疗的可行性。进一步验证这种方法将需要更多的知识，负责分化和生存的NSC在肠道中的机制。在这方面，我们最初集中在负责肠神经系统的正常发育，胶质源性神经营养因子或GDNF，及其同源受体，RET的最好的表征途径。我们的初步数据，使用CNS-NSC转染与组成型活性RET突变体（RET/PTC 2）表明，激活这一途径的结果在更大的增殖，生存和神经元分化的NSC。我们的总体假设是，RET的激活足以改善移植到肠道后的CNS-NSC的存活和分化，并且这可以是替代肠神经系统功能的有效治疗策略。为了验证这一假设，我们将使用从CNS（CNS-NSC）和肠道（ENS-NSC）分离的NSC追求以下具体目标：具体目标1：使用可诱导的组成型活性RET/PTC 2基因构建体来确定移植后最佳结果所需的RET激活的持续时间。具体目标2：确定RET激活是否可以在CNS或ENS衍生的NSC中产生相似的表型。具体目标3：确定RET激活的CNS和ENS衍生的NSC是否能有效恢复临床相关疾病模型（如先天性巨结肠和炎性神经病）中的肠神经元功能。我们的长期目标是发展神经元移植作为治疗人类肠神经系统疾病的一种方法。建立干细胞的最佳来源（成人与胚胎; CNS与ENS等）目前仍然是一个难以实现的目标，需要世界各地许多实验室再进行几年的工作。通过建立一个允许的和必要的作用，RET，拟议的研究将提供关键的基础，从各种来源的神经元前体的治疗使用。公共卫生相关性：胃肠动力障碍很常见，给我们的社会带来了重大的疾病负担。这些疾病是由于负责消化道内有序运动的神经和肌肉功能紊乱引起的，并导致症状，如严重便秘、肠梗阻、胃排空延迟或吞咽困难引起的恶心和呕吐。这些病症中有几种是由胃肠神经的丧失或功能异常引起的，单独使用药物不能令人满意地治疗。治疗这些疾病的理想策略是通过移植神经干细胞来恢复神经功能。本提案的目的是通过实验研究这种方法，并确定可以优化神经干细胞治疗和治愈这些疾病的生物学因素。

DESCRIPTION (provided by applicant): Neural stem cell (NSC) transplantation is an emerging technique with immense promise for the treatment of disorders of the peripheral (PNS) and central nervous system (CNS). More recently, our laboratory has begun investigating the use of NSC as a viable therapeutic strategy for disorders of gastrointestinal motility. Using a strategy of transplanting CNS-derived NSC (CNS-NSC) in mice with a genetic deletion of the neuronal nitric oxide synthase (nNOS) enzyme, we have shown that CNS-NSC can successfully engraft and differentiate into nNOS expressing neurons, can restore nitrergic relaxation and "rescue" gastric function (emptying) in these mice. We have also shown that intrinsic apoptotic signaling is more important in determining the survival of transplanted NSC than the host immune response. The results of these experiments have generated considerable confidence in the viability of this strategy as a potential therapy. Further validation of this approach will require a greater knowledge of the mechanisms responsible for differentiation and survival of NSC in the gut. In this regard, we have initially focused on the most well characterized pathway responsible for the normal development of the enteric nervous system, glial derived neurotrophic factor or GDNF, and its cognate receptor, RET. Our preliminary data, using CNS-NSC transfected with a constitutively active RET mutant (RET/PTC2) suggest that activation of this pathway results in greater proliferation, survival and neuronal differentiation of NSC. Our overall hypothesis is that activation of RET is sufficient to improve the survival and differentiation of CNS-NSC after transplantation into the gut and that this can be an effective therapeutic strategy to replace enteric nervous system function. To test this hypothesis, we will pursue the following specific aims using NSC isolated from the CNS (CNS-NSC) and the gut (ENS-NSC): Specific Aim 1: To use an inducible, constitutively active RET/PTC2 gene construct to determine the duration of RET activation required for an optimal outcome after transplantation. Specific Aim 2: To determine if RET-activation can produce a similar phenotype in either CNS or ENS derived NSC. Specific Aim 3: To determine if RET-activated CNS and ENS-derived NSC can effectively restore enteric neuronal function in clinically relevant models of disease such as Hirschsprung's disease and inflammatory neuropathies. Our long-term objective is to develop neuronal transplantation as a treatment for human diseases of the enteric nervous system. To establish the optimal source of stem cells (adults versus embryonic; CNS versus ENS etc.) currently remains an elusive goal and will need several more years of work by many laboratories across the world. By establishing a permissive and necessary role of RET, the proposed studies will provide the critical groundwork for the therapeutic use of neuronal precursors from a variety of sources. PUBLIC HEALTH RELEVANCE: Disorders of gastrointestinal motility are common and pose a significant burden of disease on our society. These disorders results from disturbances in the function of the nerves and muscle responsible for orderly movement within the digestive tract and result in symptoms such as severe constipation, intestinal obstruction, nausea and vomiting from delayed gastric emptying or difficulty in swallowing. Several of these conditions are caused by loss or abnormal function of gastrointestinal nerves and cannot be treated satisfactorily by drugs alone. An ideal strategy for these disorders is to restore nerve function by transplanting neural stem cells. It is the purpose of this proposal to study this approach experimentally and determine the biological factors that can optimize the use of neural stem cells for the treatment and perhaps cure of these diseases.