Polymer-Stabilized Mesenchymal Stem Cells for Spinal Cord Repair

描述(由申请人提供)： 目的：我们的最终目标是应用自体人骨髓间充质干细胞(HMSCs)修复损伤的脊髓，从而增强美国退伍军人脊髓损伤(SCI)后的功能康复。在实验室脊髓损伤模型中，MSC移植后组织学和分子水平的改善已有报道；然而，到目前为止，很少有研究显示有实质性的功能恢复。这在很大程度上归因于观察到的植入细胞的低活性和糟糕的定位。我们发现，将神经干细胞整合到可生物降解的聚合物支架中可以提高它们在大鼠脊髓损伤中的存活率和神经营养作用，从而允许显著的功能改善。因此，我们建议在聚乳酸-乙醇酸(PLGA)支架中稳定hMSCs，以验证我们的中心假设，即hMSC来源的免疫调节和神经可塑性可以用于阻止变性，促进修复和神经可塑性，这是脊髓损伤康复的生物必需品。研究设计：目的1旨在确定人骨髓间充质干细胞在PLGA中的神经营养和免疫调节作用(1年)。在目标2中，我们将评估支架hMSCs在急性脊髓损伤(1.5年)中的治疗潜力。最后，目的3项研究将确定支架hMSCs在脊髓损伤后70天(1.5年)植入治疗慢性脊髓损伤的疗效。我们将首先使用我们的体外系统来评估种植在PLGA中的hMSCs的生物学特性。然后将对提供PLGA支持以提高节段性脊髓损伤大鼠损伤部位hMSCs活性的影响进行系统的表征。组织学、免疫细胞化学和分子结果将在损伤后2周或4周进行分析，这将与亚急性和慢性脊髓损伤后的功能损害相关。研究将按照随机区组设计进行。试验组的规模由先前报道的功率分析确定。所有的结果值以平均1个扫描电子显微镜表示。在测试中使用“显著”一词意味着p&lt；0.05。所有实验程序都将由退伍军人管理局波士顿医疗系统的动物护理和使用委员会审查和批准。行为评分、细胞存活率和免疫组织化学结果分析采用重复测量ANOVA，然后进行非配对学生t检验。脊髓反射恢复及组织细胞学和分子结果的统计评价采用Fisher‘s精确检验和配对学生t检验。方法：我们将使用成熟的体外hMSC培养和器官分型系统，以及我们的大鼠穿透性脊髓损伤模型(即T9-T10中线节段半横断)来验证我们的假设。还将部署定量行为电池、脊髓反射和感觉运动测试的电生理学以及组织病理学分析。最后，我们将进行半定量Western Blot和定量RT-PCR分析，以测量在炎症、神经可塑性和再生中发挥关键作用的分子的表达变化。研究结果：我们有可靠的试点和初步数据来支持我们的假设。这些相互关联但又相互独立的目标的研究结果将大大增加我们对体内hMSCs活性和治疗相互作用的理解，以设计聚合物和基于hMSCs的多机制策略来治疗脊髓损伤，并建立新的脊髓损伤后康复治疗窗口。临床关系：到目前为止，脊髓损伤仍没有有效的治疗方法。本项目中提出的研究特别旨在将hMSCs发展为治疗方案，用于治疗脊髓损伤后的瘫痪和感觉障碍，如神经病理性疼痛。由于PLGA和hMSCs已经在其他条件下应用于临床，我们的研究可能会引入一种高效的方法来将hMSCs作为多模式抗变性和促进修复的药物用于脊髓损伤、创伤性脑损伤和神经退行性疾病。影响/意义：创伤性脊髓损伤的有效恢复是目前约42,000名美国退伍军人尚未满足的临床需求。我们的初步研究表明，hMSCs(一种成体干细胞的原型)在神经系统中发挥多机制治疗作用。这些hMSC产生的免疫调节和神经可塑性作用可用于阻止退行性变，促进恢复和神经可塑性，为提高脊髓损伤康复的治疗效果提供了生物学基础。

DESCRIPTION (provided by applicant): OBJECTIVES: Our ultimate goal is to apply autologous human mesenchymal stromal stem cells (hMSCs) to repair the injured spinal cord, enabling enhanced functional rehabilitation for America's veterans after spinal cord injury (SCI). Histological and molecular improvements were previously reported in lab SCI models after MSC transplantation; however, to date, few studies showed substantial functional recovery. This attributes largely to the observed low viability and poor localization of implanted cells. We have uncovered that incorporation of neural stem cells into biodegradable polymer scaffolds boosted their survival and neurotrophic impacts in rat SCI, thereby permitting marked functional improvement. Thus, we propose to stabilize hMSCs in poly(lactic-co-glycolic acid) (PLGA) scaffolds in order to test our central hypothesis that hMSC-derived immunomodulatory and neuroplastic effects could be used to impede degeneration and promote repair and neuroplasticity, a bionecessity for SCI rehabilitation. RESEARCH DESIGN: Aim 1 is designed for determining the neurotrophic and immunomodulatory effects of hMSCs seeded in PLGA (1 Year). In Aim 2, we will assess the therapeutic potential of implanting scaffolded hMSCs in acute SCI (1.5 Years). Lastly, Aim 3 studies will determine the therapeutic efficacy of treating chronic SCI with scaffolded hMSCs implanted 70 d post SCI; (1.5 Years). We will first use our in vitro systems to evaluate the biology of hMSCs seeded in PLGA. Systematical characterization will then be done on the impact of providing PLGA support to augment viability of hMSCs at the injury site of a rat model of segmental hemisection SCI. Histological, immunocytochemical and molecular outcomes will be analyzed at 2 or 4 weeks post injury, which will be correlated with functional impairment after subacute and chronic SCI. The studies will be conducted according to a randomized block design. The size of the experimental groups is determined by power analyses previously reported. All outcome values are presented as mean 1 SEM. Application of the term "significant" in the tests implies p < 0.05. All experimental procedures will be reviewed and approved by the Animal Care and Use Committee of the VA Boston Healthcare System. Behavioral scores, cell survival and immunohistochemical results are analyzed using repeated measures ANOVA, followed by unpaired Student's t tests. Fisher's exact test and paired Student's t test are used for the statistical evaluation of spinal reflex recovery and histocytological and molecular outcomes. METHODOLOGY: We will use well established in vitro hMSC cultures and organotypic system, as well as our rat model of penetrating SCI (i.e., T9-T10 midline segmental hemisection) to test our hypotheses. Quantitative behavioral batteries, electrophysiology of spinal reflex and sensorimotor tests, and histopathologic assays will be also deployed. Finally, we will perform semi-quantitative Western Blot and quantitative rt-PCR assays for studies that measure expression changes of molecules that play pivotal roles in inflammation, neural plasticity and regeneration. FINDINGS: We have solid pilot and preliminary data to support our hypotheses. Findings obtained from these interlocked and yet independent aims will substantially increase our understanding on in vivo issues of viability and therapeutic interaction of hMSCs for devising polymer and hMSCs based multimechanistic strategies to treat SCI and establish novel therapeutic windows of post-SCI rehabilitation. CLINICAL RELATIONSHIPS: To date, there is still no effective treatment for SCI. Studies proposed in this project are specifically aimed at developing hMSCs into therapeutic regimens to treat paralysis and sensory disorders such as neuropathic pain following SCI. Since PLGA and hMSCs are already in clinical applications for other conditions, our study may introduce a highly effective approach for delivering hMSCs as multimodal anti-degenerative and pro-restorative agents for SCI, traumatic brain injury, and neurodegenerative diseases. IMPACT/SIGNIFICANCE: Effective recovery from traumatic spinal cord injury represents a currently unmet clinical need for about 42,000 US veterans. Our pilot studies demonstrated that hMSCs (a prototype adult stem cell) exert multimechanistic therapeutic effects in neurological systems. These hMSC-derived immunomodulatory and neuroplastic effects could be used to impede degeneration as well as promote restoration and neuroplasticity, which provides a biological foundation for augmenting therapeutic outcomes of SCI rehabilitation.