Neuro-Vascular Regeneration

描述（由申请人提供）：本申请的目的是在体外设计神经血管再生单元，为与神经和血管系统进行性变性相关的疾病（如中风）提供优化的细胞疗法。我们的方法将产生能够在可移植血管微环境（或生态位）中体外培养人类神经干细胞（NSC）的系统，这将使NSC在体内长期存活、繁殖和分化。因此，我们提出的研究的首要假设是，有可能产生一个离体神经血管单元，可以植入中风患者的中枢神经系统，并提供神经血管细胞的来源，这些细胞将在植入物内继续发育，并与现有组织结合，以防止进行性损失和恢复功能。为了验证这一假设，我们进行了免疫组织化学分析、生物成像和NSC龛的定量细胞结构制图，包括成年小鼠大脑的室下区和嗅球，以及新生小鼠的室下区，以确定这些龛内的细胞结构和物理相互作用。我们将类似地绘制皮层的神经血管结构，并确定它如何在中风损伤的反应中改变。我们还建立了体外共培养系统，并进行了研究，以检查NSC和从这些组织中分离的血管内皮细胞之间的旁分泌信号，以确定利基特异性流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。我们已经设计和制造了生物材料和生物反应器，使干细胞能够存活和繁殖，并制造灌注微血管网络。我们还建立了啮齿动物中风性损伤模型，设计了用NSC治疗中风的方法，并开发了mri辅助方法来跟踪植入细胞和监测受损神经组织的功能恢复。因此，这些拟议的研究将整合我们在初步实验中产生的知识和工具。通过体外重现NSC生态位微环境来制造可移植的神经血管再生单元，在后续的研究阶段，我们将能够使用这些单元来替换和修复中风损伤的组织。这些研究将只代表这种新方法用于纠正退行性神经疾病的初步应用。一些拟议的研究将使用美国国立卫生研究院批准的人类胚胎干细胞系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.