Homing of Stem Cells in Stroke Therapeutics

描述(由申请人提供)： 躯体干细胞似乎对中风治疗有最大的影响，不是通过字面上的取代细胞，而是通过保护宿主神经细胞和电路免受进行性损害，以及催化内源性宿主再生反应。当这种干细胞介导的行为被调用时，行为得到了改善。神经外胚层来源的干细胞(“神经干细胞[NSCs]”)一直是此类疗法的“黄金标准”，任何替代干细胞类型都必须与之进行比较。然而，一些实验室发现血管内注射人脐血源干细胞(HUCSCs)是有效的。假设干细胞可以有效地定位于损伤区域，那么血管内注射干细胞可以让人避开更具侵入性的神经外科植入。细胞介导的保护性治疗(与药物介导的干预相反)的另一个优势是，机会窗口可以长达24-72小时，使它们非常适合中风患者何时出现在卫生保健机构的现实。尽管UCSCs前景看好，但将其推广到实际患者的能力存在实际和免疫学方面的限制。特别是，根据动物研究的推断，成年患者需要结合多个脐带血单位才能获得足够数量的干细胞才能达到疗效。此外，合并单元强调识别不超过1-2个HLA错配的兼容单元，以将免疫反应降至最低。此外，有效地将细胞运送到最需要它们的地方，而不是送到不需要它们或甚至可能产生问题的地方--即归巢--是所有基于细胞的疗法成功治疗任何病理疾病的关键。如果要最大限度地提高疗效和安全性，UCSCs必须具有接触受损宿主细胞及其环境的最佳途径。当考虑全身应用UCSCs或NSCs来介导中风后的脑修复时，在归巢过程中，内皮细胞的滚动和黏附是关键的第一步，这是将这些细胞靶向受损大脑所必需的。大多数人脐静脉干细胞在内皮细胞上的滚动和黏附能力不足，严重限制了其治疗潜力。神经干细胞也有类似的限制。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hUCSCs来纠正，岩藻糖基转移酶-VI作用于SLeX，完成CD34+细胞上的P-选择素糖蛋白配体(PSGL)。最终的效果是增加了PSGL对内皮细胞上选择素的识别。然而，目前还不清楚这是否对脑血管内皮细胞起作用。目前还不清楚这是否适用于神经干细胞--同样是“黄金标准”--尽管它们也有一个不完整的未岩藻糖基化的选择素配体，这损害了它们的归宿。我们建议首先在体外确定岩藻糖化的最佳参数，从而增强hUCSCs和人NSCs(HNSCs)在生理剪应力下(使用专门的流动室)与炎症的人脑来源内皮细胞的结合。随后[目标2]，我们将利用这些条件将岩藻糖化(与对照)hUCSCs和hNSCs应用于卒中大鼠模型，(首次)将这两种干细胞类型进行头对头的比较。大鼠在大脑中动脉闭塞后24-72小时静脉注射干细胞。结果指标将包括行为；梗塞体积(通过MRI)；光谱(通过MRS)；迁移(通过MRI和生物发光成像)；宿主神经元及其连接的保留(通过免疫组织化学和轨迹追踪)。这些数据将与组织学相关。这些研究有助于提供一种策略，利用细胞数量以微创的方式加强干细胞向大脑的归巢，这一时间框架对于在急性/亚急性卒中中提供临床益处是可行的，尤其是如果干细胞能够保护现有的神经组织和连接；减少炎症、疤痕形成和二次损伤过程；解除环境毒素；促进新生血管。这样的研究也可以提供延伸到其他类型的中枢神经系统病理&其他类型的干细胞的洞察力。 公共卫生相关性： 外周血管内注射人脐血源性干细胞(HUCSCs)或神经干细胞(HNSCs)用于成人急性卒中治疗需要足够数量的细胞，具有充分表达的归巢机制，以便最佳地靶向折衷区域。这项提案将评估一项新技术，该技术可以扩大任何一种干细胞来源的可用供应的使用。临床前研究表明，通过简单的酶前处理将糖基-岩藻糖基-放置在干细胞膜上的过程可以增加hUCSCs的归巢，而且很可能也将延伸到hNSCs。AIM 1将确定制备干细胞的最佳条件，而AIM 2将在大鼠中风模型中比较评估卒中后24小时外周给药后hUCSCs与hNSCs的疗效，特别是在其归巢已通过岩藻糖化进行优化的条件下。

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.