Lumbar Puncture Delivery of MSC & Function in Spinal Cord Injury

描述（由申请人提供）：实验数据表明，间充质干细胞（MSC）可调节炎症反应、瘢痕组织形成和对驻留细胞的旁分泌作用，支持间充质干细胞（MSC）用于包括脊髓损伤（SCI）在内的临床相关疾病治疗。这些研究的一个主要目的是确定间充质干细胞的抗炎作用是否需要它们存在于损伤部位，还是可以通过植入更容易到达的位置的微型生物反应器（即脊髓的腰椎扩大）并通过分泌因子传递到脑脊液中来实现。尽管有证据表明间充质干细胞可以调节受损脊髓的环境以限制组织损伤，但可能需要提供更多的因素来实现更广泛的功能恢复，包括轴突再生。因此，本研究的目的是研究包膜间充质干细胞单独和联合神经干/前体细胞植入在减轻炎症、增加轴突延伸和功能恢复方面的作用，作为一种潜在的（亚）急性脊髓损伤治疗方法。为了实现这些目标，我们将追求三个目标。1. 目的：建立一种高效的MSC包封方法。我们将利用海藻酸-聚l -赖氨酸-海藻酸盐间充质干细胞微胶囊来促进细胞活力和抗炎药物的分泌。我们期望这些研究将产生一种新的MSC包封技术，该技术将维持MSC在体外的活力和功能，并可用于制造用于SCI移植实验的可植入MSC生物反应器。2. 分析间充质干细胞的免疫调节作用。将自由迁移和封装的MSC注入腰椎肿大，以进入CSF，并随时间分析其分布和生存能力。在组织提取物中分析损伤部位及其周围mRNA和蛋白表达的变化。3. 将间充质干细胞在脊髓损伤中的免疫调节作用与神经干/前体细胞相结合。我们将探索腰椎穿刺给药作为MSC与其他细胞联合治疗的临床相关方法。组织学上分析MSC对内源性神经干/前体细胞的影响。我们还将神经干细胞/前体细胞与MSC联合移植到脊髓损伤中，分析损伤脊髓环境的调节是否能促进其他细胞的作用。根据上述研究，我们期望：i)开发可植入的MSC生物反应器，ii)比较包被和迁移的MSC对脊髓损伤后组织恢复的作用，iii)比较人和大鼠MSC在脊髓损伤中的作用，iv)在广泛使用的大鼠脊髓损伤模型中评估MSC和神经干/前体细胞输注的潜在协同或互补作用，最终将有助于在大型动物模型和人体中进行测试。本文描述的研究将使用一种与临床相关的方法将MSC输送到脊髓，这将更好地了解MSC作为抗炎剂单独或与其他细胞联合治疗脊髓损伤的能力。

DESCRIPTION (provided by applicant): The use of mesenchymal stem cells (MSC) for clinically-relevant treatments of disorders including spinal cord injury (SCI) is supported by experimental data indicating that MSC modulate inflammatory reactions, scar tissue formation, and paracrine actions on resident cells. A main objective of these studies is to determine whether the anti-inflammatory benefit of MSC requires their presence in the injury site or can be achieved through microscopic bioreactors implanted in a more accessible location (i.e. lumbar enlargement of the spinal cord) and mediated by secreted factors delivered into the CSF. Although evidence suggests that MSC can modulate the environment in the injured spinal cord to limit tissue damage, it is likely that additional factors need to be provided for more extensive functional recovery, including axonal regeneration. Therefore, an objective of this study is to investigate the effects of encapsulated MSC alone and in combination with neural stem/precursor cell implantation in reducing inflammation, and increasing axonal extension and functional recovery, as a potential (sub)-acute SCI treatment. Toward these goals, we will pursue three aims. 1. To develop an efficient method of MSC encapsulation. We will utilize alginate-poly L-lysine-alginate MSC micro-encapsulation to promote cell viability and secretion of anti-inflammatory agents. We expect that these studies will yield a novel MSC encapsulation technique that will sustain MSC viability and function in vitro, and which can be used to generate implantable MSC bioreactors for transplantation experiments in SCI. 2. To analyze immunomodulatory effects of MSC. Freely migrating and encapsulated MSC will be injected into the lumbar enlargement to gain access to the CSF, and their distribution and viability will be analyzed over time. Changes in mRNA and protein expression in and around the injury site will be analyzed in tissue extracts. 3. To combine the immunomodulatory effects of MSC in SCI with neural stem/precursor cells. We will explore lumbar puncture administration as a clinically-relevant approach for combination treatment of MSC with other cells. The effects of MSC on endogenous neural stem/precursor cells will be analyzed histologically. We will also transplant neural stem/precursor cells into SCI in combinations with MSC to analyze whether modulation of the injured spinal cord environment can facilitate the action of other cells. As a result of the studies described above, we expect to: i) develop an implantable MSC bioreactor, ii) compare the effect of encapsulated and migrating MSCs in promoting tissue recovery after SCI, iii) compare the effect of human and rat MSC in SCI, and iv) evaluate potential synergistic or complementary effects of MSC and neural stem/precursor cell infusion in a widely used rat SCI model, which eventually will facilitate testing in large animal models and humans. The studies described here will use a clinically-relevant method of delivering MSC to the spinal cord that will create a better understanding of the capabilities of MSC as anti-inflammatory agents for SCI treatment alone and in combination with other cells. PUBLIC HEALTH RELEVANCE: We will investigate the anti-inflammatory effects of human and rat bone marrow mesenchymal stem cells (MSC) for clinically-relevant treatments of acute spinal cord injury (SCI) by introducing these cells through less invasive procedures that do not require surgery but resemble a spinal tap. We will also test enclosing the cells in alginate capsules that prevent them from getting rejected. The combined approach may open widely applicable and less invasive treatments for early intervention after spinal cord injury with partially matched MSC that can be stored in frozen cell banks.