INTEGRATION OF HUMAN IPSC- DERIVED MICROGLIA IN ADHERENT CORTICAL ORGANOIDS: STRUCTURAL AND FUNCTIONAL INSIGHTS

The current study focuses on generating human microglia from human induced pluripotent stem cells (hiPSCs) and integrating them with an adherent cortical organoid model (ACOs) to elucidate the impact of such an integration on the different cells involved. The 3D adherent cortical organoids are obtained by differentiating hiPSC-derived neural progenitor cells (NPCs) in standard 384-wells to develop radially organised, functionally mature synaptic networks, with neurons, astrocytes, progenitors and sporadically oligodendrocytes. The reproducible small-scale format enables studying neural development over extended periods and makes them suitable for high-throughput studies. The hiPSC- derived microglia are generated via hematopoietic progenitor cells, and are sequentially seeded in the ACOs at different time points to observe their survival and integration. Using live cell imaging, we observed the highly motile microglia were detected upto 6 months post seeding and integrate efficiently in the ACOs, preferentially positioned in the densely populated regions. We report that microglia display dynamic and mature morphologies in co-culture with neural networks compared to monoculture, and show more pronounced response to known stimulants as studied by cytokine release. We are further investigating the effects of such an integration on the development and functionality of the model by studying cell composition, single cell RNA sequencing, laser ablation, and electrophysiological activity using calcium imaging. Supplementation of the neural cultures with microglia, enhances our understanding of the dynamic neuronal- microglial interactions and provides a valuable model for investigating neuroinflammation and early cortical development in health and disease.