Targeting chronic neuropathic pain after SCI using human iPS cell transplantation

项目总结/摘要 基于干/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的 用于治疗CNS疾病如创伤性脊髓损伤（SCI）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）衍生的星形胶质细胞（hiPSAs）的移植， 减轻SCI引起的神经性疼痛大部分SCI患者患有神经性疼痛， 导致长期的身体和心理负担。有效的治疗方法 SCI的结果是目前缺乏的;因此，新的和强大的治疗方法是迫切需要的。 脊髓损伤后，细胞外谷氨酸稳态的慢性失调在持续性神经损伤中起着关键作用。 介导疼痛神经传递的浅表背角（DH）神经元的中枢过度兴奋，导致 神经性疼痛星形胶质细胞是表达谷氨酸转运蛋白GLT 1的主要细胞类型， 其负责在完整CNS中，特别是在脊髓中的绝大多数谷氨酸摄取。在 我们的啮齿动物模型的颈部挫伤脊髓损伤，导致持续的神经病理性疼痛，我们发现显着， 宫颈DH中GLT 1表达和功能性GLT 1介导的谷氨酸摄取的持久降低。 iPS细胞是一种新的和临床相关的来源，用于大规模的成熟细胞类型的同质衍生。 用于诸如移植的应用的量，可能以自体方式从最终的移植物中去除。 患者接受者。使用iPS细胞移植来递送星形胶质细胞代表了一种令人兴奋的策略， 迄今为止尚未进行广泛的研究，特别是在靶向关键星形胶质细胞功能方面， 例如在机械水平上的谷氨酸摄取，以及解决神经性疼痛。 重要的是，大多数人类脊髓创伤病例影响颈部区域，并且是最常见的脊髓损伤。 挫伤/压迫型，迫切需要评估疾病病因和治疗， 神经性疼痛，特别是在颈部挫伤SCI模型中，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 神经病理性疼痛相关的行为在大鼠模型的颈部挫伤。重要的是，我们将评估动机 和疼痛的情感成分。在目标1b中，我们将确定hiPSA是否 在完整的离体条件下，使用全细胞膜片钳记录， 准备.在目标2a中，我们将检查hiPSA移植物表达GLT 1和恢复GLT 1的能力。 介导的谷氨酸摄取水平在DH后颈部挫伤。我们还将设计hiPSAs， GLT 1，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DH 小胶质细胞/巨噬细胞反应，在过度兴奋和慢性疼痛中起核心作用。我们将评估 hiPSAs是否可以使SCI后的这种反应从M1促炎表型（并朝向M2促炎表型）转变， M2修复表型）作为hiPSA移植作用的额外且潜在强大的机制。

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.