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间充质干细胞（hMSCs）修复受损的脊髓，使脊髓损伤（SCI）后的美国退伍军人能够增强功能康复。先前在实验室SCI模型中报道了MSC移植后的组织学和分子学改善;然而，迄今为止，很少有研究显示实质性的功能恢复。这在很大程度上归因于观察到的植入细胞的低活力和差定位。我们已经发现，将神经干细胞纳入可生物降解的聚合物支架中可以提高它们在大鼠SCI中的存活率和神经营养作用，从而使功能得到显著改善。因此，我们建议在聚乳酸-羟基乙酸共聚物（PLGA）支架中稳定hMSCs，以测试我们的中心假设，即hMSCs衍生的免疫调节和神经可塑性作用可用于阻止变性，促进修复和神经可塑性，这是SCI康复的生物学必需品。研究设计：目的1旨在确定接种在PLGA中的hMSCs的神经营养和免疫调节作用（1年）。在目标2中，我们将评估在急性SCI（1.5年）中植入支架hMSCs的治疗潜力。最后，目标3研究将确定SCI后70天（1.5年）植入支架hMSC治疗慢性SCI的治疗效果。我们将首先使用我们的体外系统来评估接种在PLGA中的hMSCs的生物学。然后，将进行系统表征的影响，提供PLGA支持，以增加在大鼠模型的节段性半切SCI损伤部位的hMSCs的活力。将在损伤后2或4周分析组织学、免疫细胞化学和分子结果，这将与亚急性和慢性SCI后的功能损害相关。研究将按照随机区组设计进行。实验组的大小由先前报道的功率分析确定。所有结果值均表示为平均值1 SEM。在测试中应用术语“显著”意味着p < 0.05。所有实验程序将由VA Boston Healthcare System的动物护理和使用委员会审查和批准。使用重复测量ANOVA分析行为评分、细胞存活和免疫组织化学结果，然后进行非配对学生t检验。使用Fisher精确检验和配对Student t检验对脊髓反射恢复以及组织细胞学和分子学结果进行统计学评价。方法学：我们将使用良好建立的体外hMSC培养物和器官型系统，以及我们的穿透性SCI大鼠模型（即，T9-T10中线节段性半切）来验证我们的假设。还将部署定量行为电池、脊髓反射电生理学和感觉运动测试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，用于测量在炎症，神经可塑性和再生中起关键作用的分子表达变化的研究。调查结果：我们有坚实的试点和初步数据来支持我们的假设。从这些互锁而又独立的目标中获得的发现将大大增加我们对hMSCs的活力和治疗相互作用的体内问题的理解，以设计基于聚合物和hMSCs的多机制策略来治疗SCI并建立SCI后康复的新治疗窗口。临床关系：到目前为止，仍然没有有效的治疗SCI。该项目中提出的研究专门针对将hMSCs开发为治疗方案，以治疗脊髓损伤后的瘫痪和感觉障碍，如神经性疼痛。由于PLGA和hMSCs已经在其他条件的临床应用中，我们的研究可能会引入一种非常有效的方法，将hMSCs作为SCI，创伤性脑损伤和神经退行性疾病的多模式抗变性和促恢复剂。影响/意义：创伤性脊髓损伤的有效恢复代表了约42，000名美国退伍军人目前未满足的临床需求。我们的初步研究表明，hMSCs（一种原型成体干细胞）在神经系统中发挥多机制治疗作用。这些hMSC衍生的免疫调节和神经可塑性作用可用于阻止变性以及促进恢复和神经可塑性，这为增加SCI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.