Rapid and deterministic generation of microglia from human pluripotent stem cells - an in vitro platform for disease progression in neurodegeneration and neuroinflammation

• Applicant: Privatdozent Dr. Matthias Pawlowski
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 425898773

Microglia are the resident immune cells in the central nervous system (CNS) and implicated in the onset and progression of many neurological diseases. They originate from primitive yolk sac macrophages that colonise the developing CNS during early embryonic development. Once the embryonic microglial population is established, it is maintained throughout life by local proliferation, not replacement by bone-marrow-derived myeloid cells. Enormous research efforts are currently undertaken to establish faithful human microglia in vitro models as platform for disease modelling and drug discovery. Only most recently, the first protocols for the derivation of microglia-like cells from human pluripotent stem cells were reported. However, their long duration (35-80 days, depending on the protocol) and need for mechanical isolation steps to enrich certain developmental intermediate cell populations are likely to prohibit their widespread application. Here, I propose to establish a novel protocol for the generation of pure populations of microglia from human pluripotent stem cells at unprecedented speed and efficiency. I present preliminary data that this key step is feasible. Human microglia will be kept either in monoculture or placed in 2D coculture with human cortical neurons or 3D human brain organoids to establish a versatile human microglia platform. The different levels of complexity of the human microglia in vitro model will complement each other for diverse research questions ranging from reductionist studies in monoculture to studies of complex cellular interactions in an organoid setting. Microglia phenotypes are highly environment dependent. In the present application, we will apply the human microglia in vitro model to determine key cell-cell interactions that determine the human homeostatic microglia phenotype in brain organoids (as proxy of their in vivo counterparts) in contrast to the artificial in vitro phenotype. To this aim we will perform single cell RNA sequencing combined with the novel CITE-seq technology for simultaneous protein quantification. This will allow us to generate a comprehensive cellular interactome based on ligand receptor pair analysis. Finally, we will coculture microglia with tau-mutant cortical neurons and organoids to elicit and characterise a disease-associated microglia phenotype and unveil key cell-cell interactions that are associated with the phenotypic switch from homeostatic to disease-associated microglia.

• DFG Programme: Research Grants