Lumbar Puncture Delivery of MSC & Function in Spinal Cord Injury

描述（由申请人提供）：间充质干细胞（MSC）用于包括脊髓损伤（SCI）在内的疾病的临床相关治疗的用途得到了实验数据的支持，这些数据表明MSC调节炎症反应、瘢痕组织形成和对驻留细胞的旁分泌作用。这些研究的主要目的是确定MSC的抗炎益处是否需要它们存在于损伤部位，或者可以通过植入更容易接近的位置（即脊髓的腰椎扩大）的显微生物反应器并通过递送到CSF中的分泌因子介导来实现。尽管有证据表明MSC可以调节受损脊髓中的环境以限制组织损伤，但可能需要提供额外的因素以实现更广泛的功能恢复，包括轴突再生。因此，本研究的一个目的是研究包封MSC单独和与神经干细胞/前体细胞植入组合在减少炎症、增加轴突延伸和功能恢复方面的作用，作为潜在的（亚）急性SCI治疗。为了实现这些目标，我们将追求三个目标。1.提出一种高效的MSC封装方法。我们将利用藻酸盐-聚L-赖氨酸-藻酸盐MSC微囊化来促进细胞活力和抗炎剂的分泌。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，并且可用于产生用于SCI移植实验的可植入MSC生物反应器。2.分析MSC的免疫调节作用。将自由迁移和包封的MSC注射到腰椎膨大处，以获得CSF，并分析其随时间的分布和活力。将在组织提取物中分析损伤部位内和周围的mRNA和蛋白质表达的变化。3.联合收割机将脊髓间充质干细胞的免疫调节作用与神经干/前体细胞结合起来。我们将探讨腰椎穿刺管理作为临床相关的方法与其他细胞的MSC的联合治疗。MSC对内源性神经干细胞/前体细胞的影响将进行组织学分析。我们还将神经干细胞/前体细胞与MSC联合移植到SCI中，以分析受损脊髓环境的调节是否可以促进其他细胞的作用。 通过上述研究，我们希望：i）开发可植入的MSC生物反应器，ii）比较包封的和迁移的MSC在SCI后促进组织恢复的作用，iii）比较人和大鼠MSC在SCI中的作用，以及iv）评估MSC和神经干细胞/前体细胞输注在广泛使用的大鼠SCI模型中的潜在协同或互补作用，这最终将有助于在大型动物模型和人类中进行测试。这里描述的研究将使用一种临床相关的方法将MSC输送到脊髓，这将更好地理解MSC作为单独和与其他细胞联合治疗SCI的抗炎剂的能力。 公共卫生相关性：我们将研究人类和大鼠骨髓间充质干细胞（MSC）的抗炎作用，用于急性脊髓损伤（SCI）的临床相关治疗，通过不需要手术但类似于脊髓穿刺的微创手术引入这些细胞。我们还将测试将细胞封闭在藻酸盐胶囊中，以防止它们被排斥。这种联合方法可能会为脊髓损伤后的早期干预提供广泛适用且侵入性较小的治疗方法，部分匹配的MSC可以储存在冷冻细胞库中。

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.