Stem Cell-Derived Developmental Human Cortical Interneurons to Treat Intractable Epilepsy

摘要 癫痫是一种严重的神经系统疾病，影响着全球6500万人，其特点是 通过不可预测的异常放电导致反复发作。约三分之一的患者患有 癫痫是指对抗癫痫药物(AEDs)无效的顽固性癫痫发作。神经外科 介入和神经刺激器设备只是一小部分药物耐药患者的有用选择 癫痫发作，强调迫切需要开发新的治疗方法。一种前景看好的战略是 植入新的神经元，以活动依赖的方式提供增强的GABA能抑制。但是，请使用 将胎儿神经元用于细胞治疗涉及到实际和伦理问题。因此，要克服这样的问题 障碍，在我们之前的研究中，我们开创了人类多能干细胞(HPSCs)移植的先河- 内侧神经节隆起(MGE)型人发育皮质中间神经元(CIN)分化为 癫痫小鼠的大脑，并展示了它们整合到功能失调的电路中，伴随着 抑制癫痫发作和共病的行为异常。此外，我们还确定了 人类CIN分化的最佳阶段，以确保最大限度地整合到宿主电路中并确保安全 没有肿瘤形成的风险，并开发了一种有效地产生这些安全和高度迁移的方法 大量来自hPSCs的CIN群体，使癫痫的细胞治疗更接近现实。 然而，在这种有希望的恢复剂临床翻译之前，仍然有一些重要的问题需要解决 治疗：1)人类发育期CIN在成人癫痫环路中的突触连接特异性是什么？ 2)人类CIN移植物抑制癫痫宿主回路的安全和最佳浓度是多少？3)人类 发育性CIN移植在癫痫脑中保持长期疗效和安全性？为了解决这些问题，我们 将检验我们的假设，即人类IPSC来源的发育颈动脉具有最佳的移植密度 优先支配宿主兴奋性神经元，改善癫痫发作活动，具有长期疗效和 安全。我们将移行性人类CIN移植到NOD SCID Gamma(NSG)小鼠的海马区 红藻氨酸诱导的颞叶癫痫(KA-TLE)是最常见的人类海马区硬化模型 耐药癫痫的常见病因，并分析移植CINS的突触整合特异性和宿主 癫痫患者大脑中的抑制作用。抗癫痫疗效的长期维持将得到广泛的重视 分别于移植后3个月、6个月、9个月进行全天候视频脑电分析。我们会 对移植物进行免疫组织化学分析，以确定细胞存活、成熟、整合和 最重要的是，将细胞增殖作为移植安全性的衡量标准，而不存在不受控制的生长风险。完成 这些研究对于将这种实验性疗法转化为可行的治疗策略至关重要 顽固性癫痫。

Abstract Epilepsy is a severe neurological disease affecting more than 65 million people worldwide and is characterized by unpredictable abnormal electrical discharges resulting in recurrent seizures. About one third of patients with epilepsy suffer from intractable seizures that do not respond to antiepileptic drugs (AEDs). Neurosurgical interventions and neurostimulator devices are useful options for only a fraction of patients with drug-refractory seizures, underscoring the urgent need to develop new therapies. One strategy with considerable promise is to engraft new neurons to provide enhanced GABAergic inhibition in an activity-dependent manner. However, use of fetal neurons for cell therapy is associated with practical and ethical issues. Therefore, to overcome such hurdles, in our previous studies, we pioneered the transplantation of human pluripotent stem cells (hPSCs)- derived medial ganglionic eminence (MGE)-type human developmental cortical interneurons (cINs) into epileptic mouse brains and demonstrated their integration into dysfunctional circuitry, accompanied by the suppression of seizures and comorbid behavioral abnormalities. Furthermore, we have also determined the optimal stage of human cIN differentiation to ensure maximal integration into host circuitry as well as safety without risk of tumor formation, and developed a method to efficiently generate these safe and highly migratory populations of cINs from hPSCs in large quantities, bringing cell therapy for epilepsy one step closer to reality. However, there are still important issues to address prior to the clinical translation of this promising restorative therapy; 1) what is the synaptic connection specificity of human developmental cINs in adult epileptic circuitry? 2) what are safe and optimal densities of human cIN grafts for inhibition of epileptic host circuitry? 3) do human developmental cIN grafts maintain long-term efficacy and safety in epileptic brains? To tackle these issues, we will test our hypothesis that human iPSC-derived developmental cINs with optimal grafting densities preferentially innervate host excitatory neurons and ameliorate seizure activity with long-term efficacy and safety. We will transplant migratory human cINs into Nod Scid gamma (NSG) mice with intrahippocampal kainic acid-induced temporal lobe epilepsy (KA-TLE), a model of human hippocampal sclerosis, the most common cause of drug-resistant epilepsy, and analyze grafted cINs’ synaptic integration specificity and host inhibition in the epileptic brains. The long-term maintenance of anti-epileptic efficacy will be extensively analyzed by 24/7 video-EEG recordings 3 months, 6 months and 9 months after transplantation. We will analyze the grafts immunohistochemically to determine the extent of cell survival, maturation, integration, and most importantly, cell proliferation as a measure of graft safety without risk of uncontrolled growth. Completion of these studies is pivotal for translating this experimental therapy into a viable therapeutic strategy for intractable epilepsy.