Neuro-Vascular Regeneration

描述（由申请人提供）：本申请的目的是离体设计神经血管再生单元，以提供用于与神经和血管系统的进行性变性相关的病症（例如中风）的优化细胞疗法。我们的方法将产生可以在可移植的血管微环境（或小生境）中离体培养人神经干细胞（NSC）的系统，这将使NSC在体内的后续长期存活、繁殖和分化成为可能。因此，我们提出的研究的总体假设是，将有可能离体产生神经血管单元，该神经血管单元可以植入中风患者的CNS中，并提供神经血管细胞的来源，该神经血管细胞将在植入物内继续发育并与现有组织整合，以防止进行性丧失并恢复功能。为了开始验证这一假设，我们对NSC小生境进行了免疫组织化学分析、生物成像和定量细胞结构绘图，包括成年小鼠脑的脑室下区和嗅球以及新生小鼠的脑室下区，以确定这些小生境内的细胞结构和物理相互作用。我们将同样绘制大脑皮层的神经血管结构图，并确定它是如何在中风损伤后发生变化的。我们还建立了体外共培养系统，并进行了研究以检查NSC和从这些组织分离的血管内皮细胞之间的旁分泌信号传导，以确定生态位特异性流动力如何影响这种相互作用，以及这如何影响NSC的生长和分化。我们已经设计和生产的生物材料和生物反应器，将使干细胞的生存和繁殖和灌注微血管网络的制造。我们还建立了中风诱导损伤的啮齿动物模型，设计了用NSC治疗中风的方法，并开发了用于跟踪植入细胞和监测受损神经组织功能恢复的MRI辅助方法。因此，这些拟议的研究将整合我们初步实验中产生的知识和工具。通过体外重现NSC生态位微环境以制造可移植的神经血管再生单元，在随后的研究阶段中，我们将能够使用这些单元来替换和修复中风损伤的组织。这些研究将仅代表这种新方法用于矫正退行性神经疾病的初步应用。一些拟议的研究将利用NIH批准的人ES细胞系WA01和WA09。

DESCRIPTION (provided by applicant): The aim of this application is to engineer neuro-vascular regenerative units ex vivo to provide optimized cellular therapies for disorders associated with progressive degeneration of the neurological and vascular systems, such as stroke. Our approach will produce systems that can grow human neural stem cells (NSC) ex vivo in a transplantable vascular microenvironment (or niche) that will enable the subsequent long-term survival, propagation and differentiation of NSC in vivo. Hence the overarching hypothesis of our proposed studies is that it will be possible to generate a neuro-vascular unit ex vivo that can be implanted into the CNS of stroke patients and provide a source of neuro-vascular cells that will continue to develop within the implant and integrate with existing tissue to prevent progressive loss and restore function. To begin testing this hypothesis, we have conducted immunohistochemical analyses, bioimaging and quantitative cytoarchitectural mapping of NSC niches including the sub-ventricular zone and olfactory bulb of the adult mouse brain and the sub-ventricular zone of the neonatal mouse to define the cellular architecture and physical interactions within these niches. We will similarly map the neurovascular architecture in the cortex, and determine how it is altered in response to stroke injury. We have also established in vitro co- culture systems and conducted studies to examine paracrine signaling between NSC and vascular endothelial cells isolated from these tissues, to determine how niche-specific flow forces impact such interactions, and how this affects the growth and differentiation of NSC. We have designed and generated biomaterials and bioreactors that will enable stem cell survival and propagation and fabrication of perfused microvascular networks. We have also established a rodent model of stroke-induced injury, devised methods to treat stroke with NSC, and developed MRI-assisted methods for tracking implanted cells and monitoring functional recovery of injured neural tissues. These proposed studies will therefore integrate knowledge and tools generated in our preliminary experiments. By recapitulating the NSC niche microenvironment ex vivo to fabricate transplantable neuro- vascular regenerative units, in subsequent phases of study we will be able to use these units to replace and repair stroke damaged tissue. These studies will represent only the initial application of this new approach for the correction of degenerative neural disorders. Some proposed studies will utilize NIH-approved human ES cell lines WA01 and WA09.