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

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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.