Application of optogenetics in iPS cell transplantation therapy for ischemic stro

描述（由申请人提供）：中风仍然是人类死亡和残疾的主要原因，而很少有有效的治疗方法可用于中风患者。干细胞移植治疗为缺血性中风后受损脑组织的再生和修复提供了可能。该研究采用全面和前所未有的方法来促进多能干细胞的营养支持和细胞替代潜力，以开发一种高效的缺血性卒中干细胞治疗方法。我们认为，增强移植细胞的存活和再生特性以及改善宿主环境对于成功的干细胞中风治疗至关重要。为了实现这一目标，我们之前和初步的研究已经证明，将缺氧预处理（HP）和其他再生策略（包括光遗传技术）以及外周刺激促进缺血脑中上调的多种营养因子相结合，具有显著的保护作用和增加的功能益处。我们的中心假设是，将hp启动的npc置于光遗传学操作和改善宿主环境的组合策略下，可以使移植细胞和内源性细胞更好地存活，通过外源性和内源性机制增强神经发生/血管生成，并导致中风后最佳的组织修复和功能恢复。在小鼠诱导多能干细胞（iPS）衍生的神经祖细胞（npc）中，我们将表达蓝光敏感通道视紫红质（ChR）通道，并测试蓝光刺激或荧光素酶/ChR蛋白（luminopsis）底物coelenterazine （CTZ）激活ChRs的可能性，这是一种改善和评估神经元分化的可行而有效的方法。移植入缺血性脑后与宿主神经网络的整合和神经元连接。我们将研究促进组织修复的策略，并为独特的小鼠桶状皮层缺血性脑卒中模型中缺血性脑结构的形态和功能恢复提供证据。我们将在体外（特异性目的1）和植入缺血后桶状皮质后（目的2）证明ips - npc中表达/激活ChR通道的可行性和益处。基于明确定义的须-丘脑-脑桶皮层通路，将结合细胞特异性和神经元通路特异性测量，包括光遗传学、电生理和光学成像记录，评估中断的须-脑桶活动的结构和功能恢复（目标3）。该提案来自三个研究实验室，它们在生物物理学、电生理学、细胞/分子生物学和临床神经科学方面具有互补的专业知识。证明ips - npc在特定脑结构中的细胞和组织修复作用对于基于机制的细胞移植治疗的发展至关重要，这些策略将对临床前和临床研究产生重大影响。

DESCRIPTION (provided by applicant): Stroke remains a leading cause of human death and disability while very few effective treatments are available for stroke patients. Stem cell transplantation therapy provides the possibility to regenerate and repair damaged brain tissues after ischemic stroke. The investigation takes a comprehensive and unprecedented approach to promote both trophic supports as well as cell replacement potential of pluripotent stem cells to develop a highly effective stem cell therapy for ischemic stroke. We propose that enhancing the survival and regenerative properties of transplanted cells as well as an improved host environment are critical for a successful stem cell stroke therapy. To reach this goal, our previous and preliminary studies have demonstrated a marked protective effect and increased functional benefits of combining hypoxia preconditioning (HP) and other regenerative strategies including optogenetic techniques and up regulated multiple trophic factors in the ischemic brain promoted by peripheral stimulation. Our central hypothesis is that a combination strategy of HP-primed NPCs subjected to optogenetic manipulations and improved host environment will allow better survival of transplanted as well as endogenous cells, enhance neurogenesis/angiogenesis via both exogenous and endogenous mechanisms, and results in optimal tissue repair and functional recovery after stroke. In neural progenitor cells (NPCs) derived from mouse induced pluripotent stem (iPS) cells, we will express the blue light- sensitive channelrhodopsin (ChR) channels and test the possibility that activation of ChRs by blue light stimuli or by the luciferase/ChR proten (luminopsis) substrate coelenterazine (CTZ) is a feasible and effective method to improve and evaluate neuronal differentiation, integration into host neural networks and neuronal connections after transplantation into the ischemic brain. We will examine the strategies to promote tissue repair and provide evidence for the morphological and functional restoration of ischemic brain structures in the unique barrel cortex ischemic stroke model of mice. We will demonstrate the feasibility and benefits of expression/activation of ChR channels in iPS-NPCs in vitro (Specific Aim 1) and after implantation into the post-ischemic barrel cortex (Aim 2). Based on the well-defined whisker- thalamus-barrel cortex pathway, structural and functional restoration of disrupted whisker-barrel activities will be evaluated usin a combination of cell specific and neuronal pathway specific measurements, including optogenetic, electrophysiological and optical imaging recordings (Aim 3). The proposal is from three research laboratories with complementary expertise in biophysics, electrophysiology, cellular/molecular biology and clinical neurosciences. The demonstration of cellular and tissue repairing benefits of iPS-NPCs in a particular brain structure is critical for the development of mechanism based cell transplantation therapy and the strategies will have great impacts on pre-clinical and clinical studies.