A human iPSC-derived platform to screen for modulators of microglial differentiation, residency and reactivity.

我们对人脑发育和功能的探索需要更多地了解小胶质细胞的显著可塑性，小胶质细胞是神经系统中驻留的巨噬细胞。小胶质细胞生物学最近经历了一次范式转变，首先证实它们来自卵黄囊原始巨噬细胞，在大脑中驻留，并原位翻转。与其他驻留在组织中的巨噬细胞不同，小胶质细胞似乎是由这些早期祖细胞独有的，而不是由单核细胞来源的巨噬细胞补充的。它们呈现不同的传感器和效应器的表达谱，并可能执行不同的功能。作为先天免疫细胞，它们准备对到达它们的信号的外周和中枢免疫触发做出反应。由于他们终生居住，他们可以积累与年龄有关的脆弱性，并保留对过去激活的记忆。小胶质细胞生命周期的模型在遗传上易驯化的生物中非常有用，但小胶质细胞生物学的某些方面可能是人类独有的。先天免疫及其与年龄相关的成分对小鼠和人类不同的进化限制做出了反应。我们认为，为了完成我们对人脑正常生物学的理解，小胶质细胞发育、住所建立和激活调节的人性化模型是必要的。我们之前描述了我们从人类诱导的多能干细胞中产生原始巨噬细胞的能力，并展示了它们在三维共培养和类器官中的能力。我们表明，这种驻留允许他们进一步发展和采用成熟的小胶质细胞分子特征和功能行为。我们以多能干细胞为靶点来产生报告细胞系，这样就可以分离出不同成熟阶段或不同激活状态的小胶质细胞。有了这些工具，我们将进行无偏见的CRISPR筛查，以揭示与人类小胶质细胞发育和反应有关的基因(目标1)。对于一些实际应用，分离培养小胶质细胞将是有利的，但为它们提供主要的组织微环境线索。在我们的合作者的帮助下，我们将对与小胶质细胞培养兼容的生物材料进行货架筛选，重点是模仿组织输入的已定义的、重组的和不含异物的材料(AIM 2)。我们的报告细胞系将使我们能够快速筛选最佳条件。除了蛋白质组学分析和功能分析外，还将使用下一代测序方法，在适当的时候以单细胞分辨率对体内样本进行额外的基准测试。在这个过程中，我们将对免疫系统和神经系统之间的相互作用产生宝贵的见解，为科学界创造造福于科学社区的技术平台，并为HQP配备面向未来的培训和知识。

Our exploration of the development and function of the human brain demands a greater understanding of the remarkable plasticity of Microglia, the resident macrophage of the nervous system. Microglial biology underwent a recent paradigm shift, starting with the confirmation that they derive from yolk sac primitive macrophages, take up residence in the brain, and turn over in situ. Unlike other tissue-resident macrophages, microglia appear to derive uniquely from these early progenitors, and are not replenished by monocyte-derived macrophages. They present distinct expression profiles of sensors and effectors, and likely perform different functions. As innate immune cells, they are poised to respond to peripheral and central immune triggers whose signals reach them. Owing to their lifelong residency, they can accumulate age-related vulnerabilities and retain memory of past activations. Models of the microglial life cycle in genetically tractable organisms have been very useful, yet some aspects of microglial biology may be uniquely human. Innate immunity, and its age-related components, have responded to different evolutionary constraints in the mouse and in the human. We propose that humanized models of microglial development, residence establishment, and activation modulation are necessary to complete our understanding of the normal biology of the human brain. We previously described our ability to generate primitive macrophages from human induced pluripotent stem cells, and demonstrated their capacity to populate three-dimensional co-cultures and organoids. We showed that this residency allows them to further develop and adopt mature microglial molecular signatures and functional behaviors. We targeted pluripotent stem cells to generate reporter cell lines, such that microglia of different maturation stages or different activation status can be isolated. With these tools, we will perform an unbiased CRISPR screen to uncover genes involved in human microglial development and reactivity (AIM 1). For several practical applications, it will be advantageous to grow microglia in isolation, yet provide them with their main tissue microenvironment cues. With input from our collaborators, we will perform a shelf screen for biomaterials compatible with microglial culture, focusing on defined, recombinant and xeno-free materials mimicking tissue inputs (AIM 2). Our reporter cell lines will allow us to quickly screen for the best conditions. Additional benchmarking against in vivo samples will be performed using next generation sequencing approaches, at single cell resolution when appropriate, in addition to proteomics profiling and functional analysis. In the process, we will develop invaluable insight into the interplay between the immune system and the nervous system, generate technological platforms for the benefit of the scientific community, and equip HQP with future-proof training and knowledge.