Paracrine and synaptic mechanisms underlying recovery using human stem cell thera

描述(申请人提供)：干细胞疗法在寻找有效治疗中风方面提供了巨大的希望，中风是美国成人严重残疾的主要原因，每年困扰着近80万美国人，社会成本高达数十亿美元。临床前 数据表明，干细胞疗法通过针对大脑可塑性等大脑修复过程， 有几周到几个月的治疗窗口，这意味着它可以使大多数中风患者受益。然而，调节干细胞介导的恢复的机制却知之甚少。这将是重要的剖析，因为阐明干细胞的作用机制将开始描绘脑修复的分子途径，并可能导致干细胞治疗的有效性和安全性的提高，从而在其转化到临床时取得成功。我们提出的使用尖端工具--陷阱、阵列断层扫描和电生理学--的研究将克服现有的技术障碍，使我们能够解决我们对干细胞机制理解中的两个重大差距：1)移植的干细胞在体内表达什么，促进大脑的可塑性和恢复？基于我们和其他人之前的工作，我们假设人类神经干细胞(HNPC)通过分泌旁分泌因子来调节大脑的可塑性，从而诱导恢复。然而，由于很难分离hNPC和宿主的表达谱，在体内识别这些旁分泌因子一直是一个挑战。我们克服了这一障碍，使用新的陷阱技术将移植的hNPC mRNA从宿主脑中分离出来。在目标1中，我们使用TRAP+微阵列来生成第一个移植到卒中脑内的人神经干细胞的体内转录图谱，并寻找候选的可塑性调节因子。然后，我们将在hNPC中对这些候选基因进行shRNA敲除，并通过补充体外和体内实验来评估它们对功能恢复和轴突可塑性的影响。2)促进康复的干细胞引发了哪些突触水平的大脑变化？我们报道了hNPC通过促进轴突和树突的萌发来增强卒中后的可塑性。目前尚不清楚中风和hNPC如何影响突触的形成和功能，而突触最终必须发生可塑性变化才能促进恢复。由于突触的复杂性和细枝末节，这些细节以前是无法获得的。新的阵列断层扫描技术克服了这一障碍，能够以皮质层特异性对单个突触进行详细的结构分析。使用这种方法，我们已经发现卒中后抑制性GABA能突触增加，hNPC依赖的兴奋性谷氨酸能突触增加。这导致了一种假说，即hNPC通过将兴奋性/抑制性突触的平衡转向兴奋来促进恢复。在目标2中，我们将使用阵列断层扫描和电生理学，以及我们从目标1中敲除的hNPC种群，来确定hNPC如何在体外和体内影响这种平衡，以及由此对卒中后功能恢复的影响。

DESCRIPTION (provided by applicant): Stem cell therapy offers great promise in the search for an effective treatment for stroke, a leading cause of serious adult disability in the US afflicting nearly 800,000 Americans annually at a societal cost of billions of dollars. Preclinical data indicate that stem cell therapy, by targeting brain repair processes such as brain plasticity, has a therapeutic window of weeks to months implying it could benefit the majority of stroke patients. The mechanisms mediating stem cell-mediated recovery are, however, poorly understood. This will be important to dissect, as elucidating stem cell mechanisms of action will begin to delineate the molecular pathways of brain repair, and could also lead to improved efficacy and safety, and thus success, of stem cell therapy as it translates to the clinic. Our proposed study using cutting edge tools-TRAP, array tomography, and electrophysiology-will overcome existing technical barriers and enable us to address two significant gaps in our understanding of stem cell mechanisms: 1) What do transplanted stem cells express in vivo that promote brain plasticity and recovery? Based on previous work from us and others we hypothesize that human neural stem cells (hNPCs) elicit recovery by secreting paracrine factors that modulate brain plasticity. However, identifying these paracrine factors in vivo has been a challenge due to difficulties separating the hNPC and host expression profiles. We overcome this hurdle using the novel TRAP technique to separate transplanted hNPC mRNA from host brain mRNA. In Aim 1 we use TRAP plus microarray to generate the first in vivo transcriptional profile of hNPCs transplanted into the stroke brain and to identify candidate plasticity-modulating factors. We will then perform shRNA knockdown of these candidates in hNPCs and evaluate their effects on functional recovery and neurite plasticity using complementary in vitro and in vivo assays. 2) What synapse-level brain changes are elicited by stem cells that promote recovery? We reported that hNPCs enhance post-stroke plasticity by promoting axonal and dendritic sprouting. What is not known is how stroke and hNPCs affect synapse formation and function, which is ultimately where plasticity changes must occur to promote recovery. Such details were previously unattainable due to the complexity and minutia of synapses. The novel array tomography technique overcomes this hurdle enabling detailed structural analysis of individual synapses with cortical-layer specificity. Using this approach, we have identified a post- stroke increase in inhibitory GABAergic synapses and an hNPC-dependent increase in excitatory glutamatergic synapses. This has led to the hypothesis that hNPCs promote recovery by shifting the excitatory/inhibitory synaptic balance towards excitation. In Aim 2 we will use array tomography and electrophysiology, and our knockdown hNPC populations from Aim 1, to determine how hNPCs affect this balance, both in vitro and in vivo, and the resulting impact on post-stroke functional recovery.