Targeting chronic neuropathic pain after SCI using human iPS cell transplantation

项目摘要/摘要 基于干细胞/祖细胞移植的星形胶质细胞替代是一种新的和潜在的强大功能 创伤性脊髓损伤(SCI)等中枢神经系统疾病的治疗策略。为了实现这一目标， 我们建议测试人类诱导多能干细胞(IPS)来源的星形胶质细胞(HiPSA)的移植。 用于减轻脊髓损伤引起的神经病理性疼痛。大部分脊髓损伤患者患有神经病理性疼痛， 结果往往造成长期的身体和心理负担。治疗这种衰弱的有效方法 脊髓损伤的预后目前尚不清楚，因此迫切需要新的、强有力的治疗方法。 脊髓损伤后，细胞外谷氨酸稳态的慢性失调在持续 介导痛觉神经传递的浅背角神经元的中枢性超兴奋性 神经性疼痛。星形胶质细胞是表达谷氨酸转运体GLT1的主要细胞类型， 它负责在完整的中枢神经系统中摄取绝大多数谷氨酸，特别是在脊髓中。在……里面 我们的颈部挫伤脊髓损伤的啮齿动物模型导致持续的神经病理性疼痛，我们发现有意义的和 宫颈发育迟缓患者GLT1表达和功能性GLT1介导的谷氨酸摄取的持续减少。 IPS细胞是一种新的和临床相关的来源，用于同种来源的成熟细胞类型的大 移植等应用的数量，可能从最终的自体方式 病人接受者。使用iPS细胞移植来运送星形胶质细胞代表了一种令人兴奋的策略， 到目前为止还没有得到广泛的研究，特别是在靶向关键星形胶质细胞功能方面 例如在机械性水平上的谷氨酸摄取和解决神经病理性疼痛。 重要的是，大多数人类脊髓损伤病例都会影响到颈椎区域，并且是 挫伤/压迫型，迫切需要对其病因和治疗进行评估 神经病理性疼痛在颈椎挫伤模型中的特异性，以提供与患者群体的相关性。 在目标1a中，我们将确定hiPSA移植是否可以逆转几种形式的已建立的 颈部挫伤大鼠的神经病理性疼痛相关行为。重要的是，我们将评估激励因素 以及疼痛的情感成分。在目标1b中，我们将确定HiPSA是否 全细胞膜片钳记录可降低完整的离体痛神经元的超兴奋性 准备工作。在目标2a中，我们将检测hiPSA移植表达GLT1和恢复GLT1的能力- 颈部挫伤后肝细胞谷氨酸摄取的调节水平。我们还将设计HiPSA以过度表达 GLT1以增强其治疗潜力。在目标2b中，我们将研究HiPSA是否可以改变卫生署 小胶质细胞/巨噬细胞反应，在过度兴奋和慢性疼痛中发挥核心作用。我们将评估 HiPSA是否可以将SCI后的这种反应从M1促炎表型转变为 M2修复表型)作为hiPSA移植作用的一种额外的和潜在的强大机制。

Project Summary / Abstract Stem/progenitor cell transplantation-based replacement of astrocytes is a novel and potentially powerful therapeutic strategy for treating CNS diseases such as traumatic spinal cord injury (SCI). Towards this goal, we propose to test transplantation of human induced Pluripotent Stem (iPS) cell-derived astrocytes (hiPSAs) for mitigating SCI-induced neuropathic pain. A major portion of SCI patients suffer from neuropathic pain, resulting in often long-term physical and psychological burdens. Effective treatments for this debilitating outcome of SCI are currently lacking; therefore, novel and robust therapeutic approaches are urgently needed. Following SCI, chronic dysregulation of extracellular glutamate homeostasis plays a key role in persistent central hyperexcitability of superficial dorsal horn (DH) neurons that mediate pain neurotransmission, leading to neuropathic pain. Astrocytes are the principal cell type that expresses the glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in the intact CNS, particularly in spinal cord. In our rodent model of cervical contusion SCI that results in persistent neuropathic pain, we find significant and long-lasting reductions in GLT1 expression and functional GLT1-mediated glutamate uptake in cervical DH. iPS cells are a novel and clinically-relevant source for homogeneous derivation of mature cell types in large quantities for applications such as transplantation, potentially in an autologous fashion from the eventual patient recipient. The use of iPS cell transplantation to deliver astrocytes represents an exciting strategy that has not been extensively studied to date, particularly with respect both to targeting key astrocyte functions such as glutamate uptake at a mechanistic level and to addressing neuropathic pain. Importantly, a majority of human spinal cord trauma cases affect cervical regions and are of the contusion/compression type, urgently calling for the assessment of disease etiology and treatment of neuropathic pain specifically in models of cervical contusion SCI to provide relevance to the patient population. In Aim 1a, we will determine whether hiPSA transplantation can reverse several forms of established neuropathic pain-related behavior in a rat model of cervical contusion. Importantly, we will assess motivational and affective components of pain using an operant paradigm. In Aim 1b, we will determine whether hiPSAs can reduce hyperexcitability of DH pain neurons using whole-cell patch clamp recording in an intact ex vivo preparation. In Aim 2a, we will examine the ability of hiPSA transplants to express GLT1 and to restore GLT1- mediated glutamate uptake levels in DH after cervical contusion. We will also engineer hiPSAs to overexpress GLT1 to enhance their therapeutic potential. In Aim 2b, we will examine whether hiPSAs can alter the DH microglia/macrophage response that plays a central role in hyperexcitability and chronic pain. We will assess whether hiPSAs can shift this response after SCI away from a M1 pro-inflammatory phenotype (and toward a M2 reparative phenotype) as an additional - and potentially powerful - mechanism of hiPSA transplant action.