Human microglia in brain assembloids display region-specific diversity and respond to hyperexcitable neurons carrying SCN2A mutation

Microglia critically shape neuronal circuit development and function, yet their region-specific properties and roles in distinct circuits of the human brain remain poorly understood. In this study, we generated region-specific brain organoids (cortical, striatal, and midbrain), each integrated with human microglia, to fill this critical gap. Overall design: Single-cell RNA sequencing was performed on human iPSC-derived cortical (hCO+Mg), striatal (hStrO+Mg), and midbrain (hMO+Mg) organoids containing microglia. For each condition, 4–5 organoids derived from three independent hiPSC lines (KOLF2.1J, B07 WT, B11 WT) were pooled into a single sample and processed using the PIPseq-T20 3' single-cell RNA-seq workflow. Libraries were sequenced on an Illumina NovaSeq S4 platform (2 × 150 bp), and the resulting data were used to compare microglial transcriptional states across distinct brain region–like environments.