(W1091) A VERSATILE TOOLBOX OF HUMAN IPSC-DERIVED MICROGLIA FOR DISEASE MODELLING, CRISPR SCREENS AND MULTICELLULAR IN VITRO MODELS FOR NEURODEGENERATION DRUG DISCOVERY
Senior Product Manager Bit Bio Ltd, United Kingdom
Abstract: Microglia, the resident macrophages of the brain, are essential for neural homeostasis, regulating neurogenesis, synaptic remodelling, and serving as first responders to injury or infection. Dysregulated microglial function is implicated in neurodegenerative diseases such as Alzheimer’s disease (AD). Despite their critical role in disease progression, existing in vitro models fail to replicate the complexity of microglia, limiting advances in drug discovery. Therefore, new tools are needed to model disease, generate gene knockouts, and track cellular responses in co-culture systems. In this study, we used opti-ox™, a deterministic cell programming technology, to generate scalable human-induced pluripotent stem cell (hiPSC)-derived microglia from both male and female genetic backgrounds. These derived microglia express key markers, including CD45, P2RY12, CD11b, CD14, IBA1, and TREM2, and exhibit robust phagocytic activity, uptaking pHrodo labelled BioParticles and Amyloid-beta1-42 as well as pro-inflammatory cytokine secretion, with distinct responses based on genetic background. To advance AD modelling, we engineered hiPSC-derived microglia with AD-relevant mutations, such as TREM2 (R47H) and APOE (C112R), known to alter microglial function. In response to the time-intensive nature of CRISPR-compatible cell line development, we created CRISPRko-Ready ioMicroglia, which constitutively express Cas9, enabling high-throughput CRISPR screening and reducing workflow duration from months to days. Proof-of-concept experiments in these cells demonstrated efficient single-gene knockouts and successful pooled CRISPR screens. Additionally, we generated GFP-expressing ioMicroglia for live-cell imaging, antibody-free sorting, and tracking of the cells within complex multicellular neurobiology co-culture systems. Our cells were benchmarked against the HMC3 immortalised cell line, and exhibited superior phenotypic and functional characteristics, including more accurate gene expression profiles, enhanced phagocytosis and expression of key microglial markers. Our model offers a physiologically relevant tool for investigating AD mechanisms, providing critical advancements for drug discovery and neurodegeneration research.
