Homing of Stem Cells in Stroke Therapeutics

描述（由申请人提供）： 体细胞干细胞对中风治疗的最大影响似乎不是通过字面上的替代细胞，而是通过保护宿主神经细胞和电路免受进行性损伤以及催化内源性宿主再生反应。当这种干细胞介导的作用被调用时，已经看到了行为的改善。神经外胚层来源的干细胞（“神经干细胞[NSC]”）已成为此类治疗的“金标准”，任何替代的干细胞类型都必须与之进行比较。然而，许多实验室已经发现了来自人脐带血衍生干细胞（hUCSC）的血管内施用的功效。假设干细胞可以有效地归巢到损伤区域，那么干细胞的血管内给药允许人们规避更具侵入性的神经外科植入。细胞介导的保护性治疗（与药理学介导的干预相反）的另一个优点是，机会之窗可以宽至24-72小时，使其非常适合中风患者前往医疗机构的现实。虽然UCSC一直很有前途，但将其应用于实际患者的能力具有实际和免疫学限制。特别是，根据动物研究的推断，成年患者需要合并多个脐带血单位，以获得足够数量的干细胞来实现疗效。此外，组合单元强调识别具有不超过1-2个HLA错配的相容单元，以使免疫反应最小化。此外，有效地将细胞输送到最需要它们的地方，而不是输送到不需要它们或甚至可能产生问题的地方，即，归巢-是所有基于细胞的治疗方法成功治疗任何病理状况的关键。如果要最大限度地提高功效和安全性，UCSC必须能够最佳地接触受损的宿主细胞及其环境。当考虑全身施用UCSC或NSC以介导中风后的脑修复时，内皮细胞上的滚动和粘附是归巢级联中的关键第一步，这是将这些细胞靶向损伤的脑所必需的。大多数hUCSC在内皮细胞上表现出滚动和粘附缺陷，严重限制了它们的治疗潜力。NSC也有类似的局限性。最近的研究表明，这种缺陷可以通过用岩藻糖基转移酶-VI预处理hUCSC来纠正，岩藻糖基转移酶-VI使sLeX岩藻糖基化并完成CD 34+细胞上的P-选择素糖蛋白配体（PSGL）。净效应是增加PSGL对内皮细胞上的选择素的识别。然而，这是否对脑血管内皮细胞有效还不清楚。这是否适用于神经干细胞也是未知的-再次，“金标准”-尽管它们也有一个不完整的未岩藻糖基化的选择素配体，这损害了它们的归巢。我们首先提出[目的1]在体外确定岩藻糖基化的最佳参数，从而在生理剪切应力下增强hUCSC和人NSC（hNSC）与发炎的人脑源性内皮细胞的结合（采用专门的流动室）。随后[目的2]，我们将使用这些条件向中风大鼠模型施用岩藻糖基化（相对于对照）hUCSC和hNSC，头对头比较2种干细胞类型（第一次）。在大脑中动脉闭塞后24-72小时，向大鼠静脉内注射干细胞。结果测量将包括行为;梗死体积（通过MRI）;光谱学（通过MRS）;迁移（通过MRI和生物发光成像）;保留宿主神经元及其连接（通过免疫组织化学和束追踪）。这些数据将与组织学相关。这些研究可以帮助提供一种以微创方式增强干细胞向大脑归巢的策略，使用细胞数量和时间框架，这对于在急性/亚急性中风中提供临床益处是实用的，特别是如果干细胞用于保护现存的神经组织和连接;减少炎症，疤痕和二次损伤过程;解毒环境;促进新血管形成。这些研究还可以提供扩展到其他类型的CNS病理学和其他类型的干细胞的见解。 公共卫生相关性： 外周血管内施用人脐带来源的干细胞（hUCSC）或神经干细胞（hNSC）用于成人中的急性中风治疗需要足够数量的具有完全表达的归巢机制的细胞以最佳靶向受损区域。这项提案将评估一项新技术，该技术可以扩大两种干细胞来源中任何一种的使用。通过简单的酶预处理过程将糖基-岩藻糖基-置于干细胞膜上的过程已经在临床前研究中显示出增加hUCSC的归巢，并且可能也将扩展到hNSC。目的1将确定用于制备干细胞的最佳条件，而目的2将在大鼠中风模型中比较评估中风后24小时外周施用后hUCSC与hNSC的功效，特别是在它们的归巢已通过岩藻糖基化优化的条件下。

DESCRIPTION (provided by applicant): Somatic stem cells appear to have their greatest impact on stroke therapeutics not by literally replacing cells but by protecting host neural cells & circuitry from progressive damage as well as catalyzing endogenous host regenerative responses. When such stem cell-mediated actions have been invoked, behavioral improvements have been seen. Neuroectoderm-derived stem cells ("neural stem cells [NSCs]") have been the "gold-standard" for such therapeutics against which any alternative stem cell type must be compared. However, a number of labs have found efficacy from the intravascular administration of human umbilical cord blood-derived stem cells (hUCSCs). The intravascular administration of stem cells allows one to circumvent more invasive neurosurgical implantation, assuming that the cells can efficiently home to the region of injury. An additional advantage of cell-mediated protective therapies (as opposed to pharmacological-mediated interventions) is that the window of opportunity can be as wide as 24-72 hrs, making them well-suited to the realities of when patients with stroke present to health care facilities. Although UCSCs have been promising, the ability to extend their use to actual patients has practical & immunological limitations. In particular, based on extrapolating from animal studies, adult patients would require combining multiple cord blood units in order to obtain sufficient numbers of stem cells to achieve efficacy. Furthermore, combining units places stress on identifying compatible units with no more than 1-2 HLA mismatches to minimize an immune reaction. Also, efficient delivery of cells to where they are most needed & not to areas where they are not required or could even create problems - i.e., homing - is pivotal to the success of all cell-based therapeutics for any pathological condition. UCSCs must have optimal access to injured host cells & their milieu if efficacy & safety are to be maximized. When considering systemic administration of either UCSCs or NSCs for mediating brain repair following stroke, rolling & adhesion on endothelial cells is the critical 1st step in the homing cascade that is necessary for targeting these cells to the injured brain. Most hUCSCs exhibit deficient rolling & adhesion on endothelial cells, severely limiting their therapeutic potential. NSCs have a similar limitation. Recent studies have shown that this deficit can be corrected by pretreating hUCSCs with fucosyltransferase-VI which fucosylates sLeX & completes the P-selectin glycoprotein ligand (PSGL) on the CD34+ cells. The net effect is to increase recognition of the PSGL for selectins on endothelial cells. It is unknown whether this works, however, for brain vascular endothelium. It is also unknown whether this works for NSCs -- again, the "gold standard" -- although they, too, have an incomplete unfucosylated selectin ligand which compromises their homing. We propose first [Aim 1] to determine in vitro the optimal parameters for fucosylating & thereby enhancing the binding of hUCSCs & human NSCs (hNSCs) to inflamed human brain-derived endothelial cells under physiological shear stress (employing a specialized flow chamber). Subsequently [Aim 2], we will use these conditions to administer fucosylated (vs. control) hUCSCs & hNSCs to a rat model of stroke, comparing the 2 stem cell types (for the 1st time) head-to-head. Rats will be injected iv with the stem cells at 24-72 hrs post-middle cerebral artery occlusion. Outcome measures will include behavior; infarct volume (by MRI); spectroscopy (by MRS); migration (by MRI & bioluminescence imaging); sparing of host neurons & their connections (by immunohistochemistry & tract tracing). These data will be correlated with histology. These studies could help provide a strategy for enhancing the homing of stem cells to the brain in a minimally-invasive manner, using cell numbers & a time frame that is practical for providing clinical benefit in acute/subacute stroke, particularly if the stem cells serve to protect extant neural tissue & connections; diminish inflammation, scarring & secondary injury processes; detoxify the milieu; & promote neovascularization. Such studies could also provide insights that extend to other types of CNS pathology & other types of stem cells. PUBLIC HEALTH RELEVANCE: Peripheral intravascular administration of either human umbilical cord-derived stem cells (hUCSCs) or neural stem cells (hNSCs) for acute stroke therapeutics in adults requires a sufficient number of cells with fully expressed homing mechanisms for optimal targeting to the area of compromise. This proposal will assess a new technology that could expand the use of available supplies of either source of stem cells. The process of placing a sugar group - a fucosyl group - on stem cell membranes through a simple enzymatic pre-treatment process has shown in preclinical studies to increase homing of hUCSCs and likely will extend to hNSCs as well. Aim 1 will identify the optimal conditions for preparing the stem cells while Aim 2 will comparatively assess in a rat stroke model the efficacy of hUCSCs vs. hNSCs following peripheral administration 24 hrs post-stroke, particularly under conditions where their homing has been optimized by fucosylation.