Paracrine and synaptic mechanisms underlying recovery using human stem cell thera

使用人类干细胞疗法进行恢复的旁分泌和突触机制

• 批准号: 8692028
• 负责人: GARY K STEINBERG
• 金额: $ 45.55万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8692028
• 关键词: Address; Adult; Adverse effects; Affect; Affinity Chromatography; American; Animals; Apolipoprotein E; Biological Assay; Brain; Cell Line; Cell Transplants; Cells; Clinic; Clinical; Clinical Trials; Collaborations; Critical Pathways; Data; Electrophysiology (science); Equilibrium; Excitatory Synapse; Family; Figs - dietary; Future; Gene Expression Profile; Glutamates; Human; Image; Immunohistochemistry; In Vitro; Individual; Inhibitory Synapse; Injury; Kinetics; Lead; Life; Mediating; Messenger RNA; Microarray Analysis; Molecular; Molecular Profiling; Neurites; Neurologic; Neurons; Pathology; Pathway interactions; Patients; Pharmacologic Substance; Platelet Factor 4; Play; Population; Positioning Attribute; Process; Recovery; Recovery of Function; Reporting; Ribosomes; Role; Safety; Specificity; Stem cell transplant; Stem cells; Stroke; Synapses; System; Techniques; Testing; Therapeutic; Therapeutic Effect; Thrombospondin 1; Thrombospondins; Translating; Translations; Transplantation; Work; base; brain repair; cost; disability; effective therapy; expectation; gamma-Aminobutyric Acid; human stem cells; improved; in vivo; knock-down; nerve stem cell; novel; paracrine; patient population; post stroke; pre-clinical; prevent; public health relevance; repaired; small hairpin RNA; stem cell therapy; stroke recovery; success; synaptic function; synaptogenesis; therapeutic target; tomography; tool

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.

描述（由申请人提供）：干细胞疗法在寻求有效治疗中风方面提供了巨大的希望，中风是美国严重成人残疾的主要原因，每年折磨近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，通过靶向脑修复过程，如脑可塑性， 有一个数周至数月的治疗窗口，这意味着它可以使大多数中风患者受益。然而，对干细胞介导的恢复机制知之甚少。这将是重要的解剖，阐明干细胞的作用机制将开始描绘脑修复的分子途径，也可能导致提高疗效和安全性，从而成功，干细胞治疗，因为它转化为临床。我们使用尖端工具TRAP，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的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？基于我们和其他人以前的工作，我们假设人神经干细胞（hNPC）通过分泌调节脑可塑性的旁分泌因子引起恢复。然而，由于难以分离hNPC和宿主表达谱，在体内鉴定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TRAP技术，从宿主脑mRNA中分离移植的hNPC mRNA。在目标1中，我们使用TRAP加微阵列生成的第一个在体内的hNPC移植到中风脑的转录谱，并确定候选可塑性调节因子。然后，我们将在hNPC中进行这些候选物的shRNA敲除，并使用互补的体外和体内测定来评估它们对功能恢复和神经突可塑性的影响。2)促进恢复的干细胞引起了哪些突触水平的大脑变化？我们报道了hNPCs通过促进轴突和树突发芽增强卒中后可塑性。目前尚不清楚中风和hNPC如何影响突触的形成和功能，这最终是可塑性变化必须发生以促进恢复的地方。由于突触的复杂性和细节，这些细节在以前是无法获得的。新的阵列断层扫描技术克服了这一障碍，使详细的结构分析个别突触与皮质层特异性。使用这种方法，我们已经确定了中风后抑制性GABA能突触的增加和兴奋性GABA能突触的hNPC依赖性增加。这导致了这样的假设：hNPC通过将兴奋性/抑制性突触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中敲低的hNPC群体，以确定hNPC如何影响这种平衡，在体外和体内，以及对中风后功能恢复的影响。