Lumbar Puncture Delivery of MSC & Function in Spinal Cord Injury

描述(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得到了实验数据的支持，实验数据表明，MSC调节炎症反应、疤痕组织形成和对驻留细胞的旁分泌作用。这些研究的一个主要目的是确定MSC的抗炎益处是需要它们出现在损伤部位，还是可以通过在更容易接近的位置植入微型生物反应器(即腰椎增大脊髓)并通过分泌因子进入脑脊液来实现。尽管有证据表明，MSC可以调节受损脊髓的环境以限制组织损伤，但很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，本研究的一个目的是探讨微囊化MSC单独和联合神经干细胞/前体细胞移植在减轻炎症、促进轴突伸展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。朝着这些目标，我们将追求三个目标。1.开发一种高效的MSC封装方法。我们将利用海藻酸盐-聚L-赖氨酸-海藻酸盐间充质干细胞微胶囊来促进细胞存活和抗炎药的分泌。我们希望这些研究将产生一种新的MSC包裹技术，该技术将在体外维持MSC的活性和功能，并可用于制造可植入的MSC生物反应器，用于脊髓损伤的移植实验。2.分析MSC的免疫调节作用。自由迁移和包裹的MSC将被注入腰椎膨大区以获得脑脊液，并将随着时间的推移分析它们的分布和活性。损伤部位及其周围的mRNA和蛋白质表达的变化将在组织提取液中进行分析。3.将MSC对脊髓损伤的免疫调节作用与神经干细胞/前体细胞相结合。我们将探索腰椎穿刺给药作为一种与临床相关的方法，用于联合治疗MSC和其他细胞。组织学将分析MSC对内源性神经干细胞/前体细胞的影响。我们还将联合MSC将神经干细胞/前体细胞移植到脊髓损伤中，以分析损伤脊髓环境的调制是否可以促进其他细胞的活动。由于上述研究的结果，我们期望：i)开发可植入的MSC生物反应器，ii)比较包裹和迁移MSCs在促进脊髓损伤后组织恢复方面的效果，iii)比较人和大鼠MSC在脊髓损伤中的作用，以及iv)在广泛使用的大鼠脊髓损伤模型中评估MSC和神经干细胞/前体细胞输注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，这将使人们更好地了解MSC作为抗炎剂单独和与其他细胞联合治疗脊髓损伤的能力。 公共卫生相关性：我们将研究人和大鼠骨髓间充质干细胞(MSC)在急性脊髓损伤(SCI)临床相关治疗中的抗炎作用，方法是通过不需要手术但类似于脊椎抽液的侵入性较小的程序引入这些细胞。我们还将测试将细胞包裹在藻酸盐胶囊中，以防止它们被排斥。这种联合方法可能会为脊髓损伤后早期干预开辟广泛适用且侵入性较小的治疗方法，使部分匹配的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.