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

我们对人类大脑发育和功能的探索需要对神经系统巨噬细胞小胶质细胞的显著可塑性有更深入的了解。小胶质细胞生物学经历了最近的范式转变，首先证实它们起源于卵黄囊原始巨噬细胞，在大脑中居住，并在原位翻转。与其他组织内巨噬细胞不同，小胶质细胞似乎只来源于这些早期祖细胞，并且不被单核细胞来源的巨噬细胞补充。它们呈现出不同的传感器和效应器表达谱，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外周和中枢免疫触发信号作出反应。由于他们终身居住，他们可以积累与年龄相关的脆弱性，并保留过去激活的记忆。遗传易感生物的小胶质细胞生命周期模型非常有用，但小胶质细胞生物学的某些方面可能是人类独有的。在小鼠和人类中，先天免疫及其与年龄相关的成分对不同的进化限制作出了反应。我们提出，小胶质细胞发育、居住建立和激活调节的人源化模型对于完善我们对人脑正常生物学的理解是必要的。我们之前描述了我们从人诱导多能干细胞中产生原始巨噬细胞的能力，并证明了它们填充三维共培养和类器官的能力。我们发现，这种驻留使它们进一步发展并采用成熟的小胶质分子特征和功能行为。我们针对多能干细胞生成报告细胞系，从而分离出不同成熟阶段或不同激活状态的小胶质细胞。利用这些工具，我们将进行无偏倚的CRISPR筛选，以发现参与人类小胶质细胞发育和反应性（AIM 1）的基因。在一些实际应用中，分离培养小胶质细胞是有利的，同时也为它们提供了主要的组织微环境线索。在合作者的帮助下，我们将对与小胶质细胞培养相容的生物材料进行货架筛选，重点是模拟组织输入的确定的、重组的和无xeno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，在适当的情况下以单细胞分辨率对体内样品进行额外的基准测试。在此过程中，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，为科学界创造技术平台，并为HQP提供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.