Bioengineering a cortical microtissue model to study human microglia in Alzheimer's disease

项目总结/摘要 小胶质细胞对来自其微环境的外部线索高度敏感， 通过内在的转录程序围绕不同的细胞状态快速改变。特定的小胶质细胞状态 导致阿尔茨海默病（AD）病理学的因素仍然没有明确的定义，主要是由于长期以来的关键因素， 需要一个可靠和强大的建模工具。鉴于人类和老鼠之间的实质性差异 小胶质细胞，iPSC衍生的人类小胶质细胞（iMG）已经成为研究小胶质细胞的严格平台。 神经炎症和AD连锁遗传学。最近从2D到3D培养系统的转变进一步增强了 iMG在了解AD发病机制中的效用，但技术障碍仍然存在。一个改进，以实现 在可扩展的3D神经环境中更好的细胞多样性和功能性将成为一种期望的工具， 在严格控制的类脑环境中精确监测和操纵iMG。我们的长期目标 是了解神经炎症的致病决定因素，为AD的未来治疗提供重要信息， 干细胞和工程创新。为此，我们建议在这里引入一个原创的3D文化平台， 根据患者的情况，检查健康和AD中小胶质细胞激活的外在和内在因素， 衍生的iMG在工程化的原代大鼠皮质微组织中生长。利用经验证的AD突变iPSC和 转基因AD大鼠，我们的总体目标是1）确认我们的杂交模型的能力， 人类特异性的AD相关小胶质细胞特征和标记，以及2）表征“基态”， 在生理或病理微环境下对照和AD iMG的神经炎症反应 (i.e.野生型对AD转基因微组织）。根据我们的初步研究，我们的中心假设是， iMG-微组织生长系统支持人小胶质细胞的成熟和操作 以检测可能导致AD发病机制的小胶质细胞行为的细微差别。我们的具体目标是 测试我们的工作假设：1）iMG在皮质微组织中的分布模拟了稳态的人类小胶质细胞 在体内，并且可以针对由基因型贡献的小胶质细胞内在表现进行表型分型，以及2）iMG 维持在AD突变体微组织中的细胞受到促进AD的外部线索的影响，并显示疾病- 相关特征。我们的预期结果是：1）建立新一代的体外人类小胶质细胞 模型系统，独特地允许解剖AD连锁遗传学的交织影响， 2）获得一个独特的数据集，为小胶质细胞功能提供一个框架， 找出AD的关键小胶质细胞特征我们相信这项研究的扩展超出了这个奖项 深入描述此处发现的小胶质细胞研究结果的一段时间将最终带来宝贵的机会 制定急需的AD干预和预防策略。

PROJECT SUMMARY/ABSTRACT Microglia are highly sensitive to the extrinsic cues from their microenvironment in a context-specific manner and rapidly change around diverse cellular states via intrinsic transcriptional programs. The specific microglial state that contributes to Alzheimer’s Disease (AD) pathology is still illy defined, mostly owing to the longstanding critical need for a reliable and robust modeling tool. In light of the substantial difference between human and mouse microglia, the iPSC-derived human microglia (iMG) have emerged as a rigorous platform to investigate neuroinflammation and AD-linked genetics. A recent transition from 2D to 3D culture systems further enhances the utility of iMG in understanding AD pathogenesis, but technical barriers remain. An improvement to achieve better cellular diversity and functionality in a scalable 3D neural setting will make a desired tool that enables precise monitoring and manipulations of iMG inside a stringently controlled brain-like milieu. Our long-term goal is to understand the pathogenic determinants of neuroinflammation to inform crucial future treatment for AD, via stem cell and engineering innovations. To this end, we propose here to introduce an original 3D culture platform to examine the extrinsic and intrinsic elements of microglial activation in health and in AD, based on patient- derived iMG grown in engineered primary rat cortical microtissues. Leveraging validated AD mutant iPSCs and transgenic AD rats, our overall objectives are to 1) confirm the capability of our hybrid model to recapitulate human-specific, AD-related microglia features and signature and 2) to characterize the ‘ground state’ and neuroinflammatory responses of control and AD iMG under a physiological or pathological microenvironment (i.e. wildtype vs. AD transgenic microtissue). Supported by our preliminary studies, our central hypothesis is that the iMG-microtissue growth system supports maturation and operation of human microglia and can be utilized to detect the nuances in microglial behaviors that may contribute to AD pathogenesis. Our specific aims are to test our working hypotheses that 1) iMG populated in cortical microtissues mimic homeostatic human microglia in vivo and can be phenotyped for microglia-intrinsic manifestations contributed by genotypes, and that 2) iMG maintained in AD mutant microtissues are affected by the AD-promoting extrinsic cues and show disease- associated characteristics. Our expected outcomes are to 1) establish a new-generation in vitro human microglia model system that uniquely allows dissecting the intertwined influences from AD-linked genetics and microenvironment on microglial functions and 2) to acquire a distinctive dataset that would offer a framework to pinpoint critical microglia characteristics for AD. We believe an expansion of this research beyond this award period to characterize in depth the microglial findings uncovered here will ultimately bring a valuable opportunity for the development of much-needed intervention and prevention strategies for AD.