Understanding Neuroinflammation through advanced in vitro 3D modelling using human Pluripotent Stem Cells

小胶质细胞--驻留在大脑中的巨噬细胞--在大脑中执行动态平衡监测功能，清除死亡细胞、细胞外碎片，并在发育过程中修剪突触。然而，碎片的积聚，特别是聚集的蛋白质，会导致慢性小胶质细胞激活，导致神经退化和精神障碍。小胶质细胞炎症损伤可能是直接的，或由星形胶质细胞介导，星形胶质细胞通常支持神经元代谢，但在小胶质细胞的指导下发生损伤。显然，了解小胶质细胞的正常功能，它们与神经元和星形胶质细胞的相互作用，以及这种三者相互作用的机制，对于我们理解大脑健康、心理健康和衰老疾病至关重要--而真正的人类细胞模型是实现这一目标所必需的。我们的团队开创了从人类诱导的多能干细胞生成巨噬细胞和小胶质细胞的先河，并在iPS-神经元模型方面进行了广泛的工作。然而，我们目前使用的2D模型并没有形成成熟的、广泛的突触网络，也没有发展出蛋白质聚集的病理特征。在3D培养中，神经元进一步成熟，聚集性蛋白病理可以发展。这个D.Phil项目将开发和开发一个相对简单的3D三维培养系统，包括iPS-小胶质细胞、星形胶质细胞和皮质神经元。该研究项目将使学生接触到尖端的细胞和分子生物学、成像、生物化学和测序技术。将部署一套神经元、星形胶质细胞和小胶质细胞分析来评估细胞功能和成熟度，为了充分利用3D系统，可以使用几种先进的成像方法，包括共焦、多光子和光片显微镜、Incell和Opera Phoenix高含量共焦成像系统。这名学生将主要在牛津大学威廉·邓恩爵士病理学学院的学术合作机构工作，必要时还会借调到工业合作伙伴礼来公司。礼来公司的地理位置非常近(距离牛津1小时车程)，因此根据研究进展，学生可以非常灵活地借调到工业合作伙伴，每次为期1-3周，在4年的D.Phil项目中总共至少3个月。位于温德尔沙姆的礼来研究中心拥有现代生物化学(自动液体处理系统、AlphaLISA、MSD、SiMoA、自动Western印迹系统、DLS、流式细胞仪)、细胞生物学(OPERA和OPERETTA Phoenix高含量成像系统)和分子生物学(纳米串、RT qPCR)实验室，在这些实验室中，学生将接受适当的专业培训，以支持项目工作。

Microglia - brain-resident macrophages - carry out homeostatic surveillance functions in the brain, clearing dying cells, extracellular debris, and pruning synapses during development. However, build-up of debris, especially aggregated proteins, causes chronic microglial activation, contributing to neurodegeneration and psychiatric disorders. Microglial inflammatory damage may be direct, or mediated by astrocytes, which normally support neuronal metabolism but become damaging under instruction from microglia. Clearly, understanding the normal function of microglia, their interactions with neurons and astrocytes, and the mechanisms by which this triumvirate becomes perturbed, is crucial to our understanding of brain health, mental health and diseases of ageing - and authentic human cellular models are necessary to achieve this.Our team have pioneered the generation of macrophages and microglia from human induced Pluripotent Stem Cells, and worked extensively with iPS-neuronal models. However, the 2D models we work with currently do not form mature, extensive synaptic networks or develop the pathological features of protein aggregation. In 3D culture, neurons mature further and aggregated protein pathology can develop. This D.Phil project will develop and exploit a relatively simple 3D triculture system, containing iPS-microglia, astrocytes and cortical neurons. The research project will expose the student to cutting-edge cell and molecular biology, imaging, biochemistry and sequencing technologies. A suite of neuronal, astrocytic and microglial assays will be deployed to assess cellular function and maturity, and for full exploitation of the 3D system, several advanced imaging methodologies can be employed, including confocal, multiphoton and Lightsheet microscopy, InCell and Opera Phoenix high-content confocal imaging systems. The student will be primarily based at the academic partner institution, the Sir William Dunn School of Pathology, University of Oxford, with secondments to the industrial partner, Eli Lilly, as necessary. Eli Lilly is geographically very close (1 hour travel from Oxford), so the student can be seconded to the industrial partner very flexibly, according to the research progress, for periods of 1-3 weeks at a time, and for a minimum of 3 months in total across the 4 year D.Phil project. The Lilly Research Centre in Windlesham has modern biochemistry (automated liquid handling systems, AlphaLISA, MSD, SiMoA, automated Western blot systems, DLS, Flow cytometry), cell biology (Opera and Operetta Phoenix high-content imaging systems) and molecular biology (Nanostring, RT qPCR) labs, in which the student will receive appropriate professional training in support of the project work.