Protocol for characterization of spatiotemporal network dynamics in cortical and hippocampal assembloids.

This protocol generates neural assembloids from human induced pluripotent stem cells (hiPSCs) to model human brain circuitry. It details the differentiation of excitatory-predominant hippocampal (Hc) and cortical (Cx) organoids and their fusion with inhibitory interneuron-predominant ganglionic eminence (GE) organoids. We then detail procedures for maintaining assembloids to promote interneuron migration and network integration, followed by functional assessment using two-photon calcium imaging to measure neuronal activity. For complete details on the use and execution of this protocol, please refer to McCrimmon et al.1.