Recapitulation of the SMFC in chimeric microglia–human hCOs. (A) NeuN, IBA-1, and IL-34 immunostaining in the SMFC of a 15-gw fetal brain demonstrating that IL-34⁺ cells are NeuN⁺. Scale bar, 100 µm. (B) NeuN, SOX2, and IL-34 antibody immunostaining in hCOs at 35 (first row), 56 (second row), 80 (third row), and 120 (fourth row) days, indicating uniform IL-34 expression in hCO neurons. Scale bar, 200 µm. The rectangle, the magnified region in the middle and right. (C) Schematic of iMicroglia (iMacrophage) induction from ESC/hiPSCs and the timeline for generating chimeric iMicroglia-hCOs. (D) Longitudinal tracking of the surface microglial aggregate after introducing iMicroglia derived from ES-GFP from daf 12 to daf 15. Bottom, EdU labeling (red) and Ki67 immunostaining (green) in the iMicroglia-hCOs of the upper panel revealed that the surface aggregates harbor a high percentage of proliferative cells (the inner insert in the second panel; phase-contrast image of surface aggregates; inner insert in the lower, spliced Ki67⁺ and EdU⁺ signals). Scale bars, 200 µm. (E and F) Quantification of EdU⁺/Ki67⁺ cells in iMicroglia aggregates versus nonaggregate regions of hCOs (hCOs, n = 6; data, mean ± SD; Mann–Whitney test U test; **P <0.01). (G) Distribution patterns of iMicroglia in iMicroglia-hCOs stained by IBA-1 and CSF-1R antibodies. The white dashed line circled the necrotic regions. Below is the magnification of the necrotic region (inner insert, a highly ramified microglia in the rectangle). Scale bars, 200 µm (top) and 100 µm (bottom). (H) Schematic detailing the integration of SV40-immortalized microglia into hCOs. Microglia are introduced on day 30 by suspending the medium with SV40 microglia. (I) Heatmap displaying upregulated genes encoding microglia-related cytokines and proteins in SV40-hCOs. (J) Concentrations of IL-1β, IL-8, TGF-β, CCL2, and CX3CL1 in the supernatants of SV40-hCOs versus control hCOs (data, mean ± SD; Mann–Whitney U test; **P < 0.01). (K) Representative whole-view images of SV40 microglia in SV40-hCOs (left) and hCOs subjected to IL-34 knockdown via shRNA lentivirus particles (right) (below, the magnified surface aggregate; white arrowheads, surface aggregate). Recording, the long-distance object of Olympus SpinSR10 spinning disk confocal microscopy with Z-stack (intervals, 1 µm). Control, CTR. Scale bars, 200 µm (top) and 50 µm (bottom). (L) Quantification of microglial aggregates (>5 or >8 cells) in control versus IL-34-shRNA SV40-hCOs (n > 3 repeats; hCOs: CTR, n = 8; shRNA, n = 15; Mann–Whitney U test; **P < 0.01). (M) Immunostaining with Ki67/CSF-1R and GFP antibodies in SV40-hCOs shows that most microglia in the surface aggregate expressed Ki67 (top) and CSF-1R (bottom). Scale bars, 20 µm. (N) Reconstructed whole-view images of SV40 microglia in SV40-hCOs treated with PLX5622 (administered before introduction) and 20 ng/ml IL-34 (administered during coculture). Recording, 20× long objective of the Olympus SpinSR10 spinning disk confocal super-resolution microscope with 4-μm intervals in Z-stacks. Scale bar, 500 µm. (O and P) Quantification of aggregate size and count in treated SV40-hCOs (data are presented as the mean ± SD; one-way ANOVA with Tukey’s multiple comparisons; **P < 0.01).