![Characterization of hCOs and chimeric iMicroglia-hCOs (related to Fig. 6). (A) Evaluating endogenous IL-34 and CSF-1 secretion levels in hCOs and SV40 microglia and iMicroglia. (B) Schematics of inducing iMicroglia (iMacrophage) from ESC/hiPSCs and the timeline/strategies for generating and validating iMicroglia-hCOs (B). (C) Morphology of iMicroglia during induction and testing the purity of iMicroglia using flow cytometry with CD14, CD11b, CD86, and CD163 antibodies revealed the high purity of iMicroglia. (D) Immunostaining of daf 15 and 30 iMicroglia-hCOs with GFP and CSF-1R antibodies revealed the amoeboid morphology of iMicroglia in daf 15 (left two panels) and the ramified morphology of iMicroglia in daf 30 iMicroglia-hCOs (right two panels). Boxed, the middle magnified images (white arrowheads in the upper left, nuclear debris). Scale bars, 200 µm. (E and F) Size and density of iMicroglia in daf 15 and daf 30 iMicroglia-hCOs (hCOs, n = 4; data, mean ± SD; Mann–Whitney U test). (G) iMicroglia and iMicroglia area/total region in daf 15 and daf 30 iMicroglia-hCOs (hCOs, n > 7; repeat, n = 3; data, mean ± SD; Mann–Whitney U test). (H) Whole slide scanning of daf 30 iMicroglia-hCOs immunostained with Ki67 and IBA-1 antibodies revealed that a restricted number of intra-hCO iMicroglia express Ki67, and the iMicroglia included multiple DAPI-stained nuclear fragments. Right, the boxed region (yellow arrow, Ki67+ iMicroglia; white arrowheads in the lower panel, nuclear debris). Scale bars, 200 µm. (I) Percentage of Ki67+ microglia in daf 30 iMicroglia-hCOs (intra-organoid) and Ki67+ surface iMicroglia in daf 15 iMicroglia-hCOs (repeat, n = 3; total iMicroglia-hCOs, n = 8; Mann–Whitney U test; data, mean ± SD). (J) SV40 microglia in the hCO stained by GFP, PSD95, and MAP2 antibodies revealed PSD95+ and MAP2+ puncta in penetrated SV40 microglia (white dashed line, phagocytosed nuclear debris; white arrowheads, PSD95+ dots). Recording, SIM. Rendering, IMARIS software. Scale bars, 10 µm. (K) Immunostaining for SV40 microglia-hCO or hCO using PAX6 and TBR2 antibodies revealed that more TBR2+ cells are present in the SVZ region of hCO with SV40 microglia. Boxed (left), the magnified region (right). Percentage of TBR2+ or PAX2+ cells in SV40 microglia-hCO and hCO (organoid, n ≥ 3; all data, means ± SD; Mann–Whitney U test, *P < 0.05). Scale bars, 100 µm (left) and 20 µm (right). (L) Immunostaining with Ki67, pH3, and GFP antibodies in hCO with SV40 microglia or hCO. Percentage of Ki67 intensity of Ki67+ cells in hCO with SV40 microglia with hCO (organoids: n = 3; data, mean ± SD; Mann–Whitney U test. *P < 0.05; **P < 0.001). Scale bars, 100 µm (left) and 20 µm (right). (M) Immunostaining with CTIP2, GFP, and PAX6 antibodies in SV40 microglia-hCO and hCO. Percentage of CTIP2+ cells in SV40 microglia-hCO and hCO. Organoid, n ≥3; all data, means ± SD; Mann–Whitney U test, ns, P > 0.05. Scale bars, 100 µm (left) and 20 µm (right). (N) Schematic illustrates the generation of SV40-hCO chimeras in HD-MEA (left). Living imaging of SV40-hCO chimeras revealed the interaction of GFP + SV40 microglia with hCO. SV40-hCO chimeras were stripped from HD-MEA at the end of recordings (right). (O) Quantification of the mean firing rate [Hz], the mean spike amplitude [µV], and the mean ISI [ms] in hCO. n = 3 control group or microglia group; The mean ± SD; Kruskal–Wallis test; ns, no significant difference (left). Quantification of network features indicated SV40 microglia-guided remodeling of the neuronal network. The network features include mean spikes per burst, mean spikes per burst per electrode, mean burst duration [s], mean burst peak firing rate [Hz], mean IBI [s], mean ISI within burst [ms], and mean ISI outside burst [ms]. n = 3 control group or microglia group (right). (P) Raster plots show the dynamic neuronal network in chimeras after SV40 microglial plating (left). The quantification of network features at the burst level in the representative SV40-hCO chimera, including the number of spikes per burst, the duration of the burst [s], the peak firing rate [Hz] of the burst, the IBIs [s], ISI within burst [ms], and ISI outside burst [ms]. The mean ± SD; Kruskal–Wallis test; and statistical significance levels (ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) are presented (right). (Q) IL-34 and TUJ-1 antibody immunostaining of the hCOs infected by shRNA and hCO. The inner insert on the left (top) is a multichannel image. The rectangle in the inner insert is the magnified region on the right (left). The relative IL-34 intensity in the hCOs infected by shRNA lentivirus particles and control hCOs (Mann–Whitney U test; **P < 0.001) (right). Scale bars, 200 µm. (R) Immunostaining with the Ki67 antibody and EdU labeling of the surface aggregate (SA) in SV40-hCOs (left). Comparing Ki67+ surface aggregates with Ki67+ cells in the SMFC; Mann–Whitney U test; **P < 0.001 (right). Scale bars, 20 µm. IBI, interburst intervals. Refer to the image caption for details.](https://cdn.rupress.org/rup/content_public/journal/jem/223/6/10.1084_jem.20251801/2/jem_20251801_figs3.png?Expires=1782204300&Signature=JfvGPjeh-3V32-Kh7E6MjiB9afVM3Akc~6E9Z9rGBp0b-1d-xoOiCE8xjZ8L7AOw4AdGH4ELGJkEmWHd2yhB8pvjRacYxTtQdoJ6KriiKPneXF3Yug2AqXSoVfCET6ZTkunowoYENjUUwALBi2xLtqeOvhfPMYhe9MUxt-11MDFoAjBtuYOLEqzUT8bVpTlrfTR4vOLddQ6EPHSu1ox4i6YyfuPWRgaVyQfqAehZswMgcw2blod3feFD2dQ-3vq0Hd5IYdIfjpr1FunrHdBvnO5OjVhDlD67tsT1lIczji0pERn6f1S951gk6akXft2D1N~ysnNeca3r5RCzFtVGmA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A shows bar graphs measuring IL34 and CSF-1 secretion levels across hCOs, SV40 microglia, and iMicroglia conditions. Panel B shows a schematic diagram illustrating stepwise differentiation of iMicroglia from stem cells and generation timeline of chimeric organoids. Panel C shows brightfield images and flow cytometry plots demonstrating iMicroglia morphology and high purity based on marker expression. Panel D shows immunostaining images of GFP and CSF-1R revealing morphological transition from amoeboid to ramified microglia over time. Panel E shows scatter bar plot quantifying microglia size differences between early and later stages. Panel F shows scatter bar plot quantifying microglia density across developmental stages. Panel G shows scatter bar plot graphs measuring microglia area relative to total organoid area. Panel H shows immunostaining images of IBA-1 and Ki67 highlighting limited proliferation and presence of nuclear debris in microglia. Panel I shows a scatter bar plot quantifying percentage of Ki67 positive microglia across conditions. Panel J shows 3D rendering and sectional immunostaining images demonstrating interaction of microglia with neuronal structures and synaptic markers. Panel K shows immunostaining images with scatter bar plots quantification indicating increased progenitor marker expression in microglia-containing organoids. Panel L shows immunostaining images with scatter bar plot showing quantification of proliferative markers including Ki67 and pH3 showing enhanced proliferation. Panel M shows immunostaining images with scatter bar plot showing quantification indicating no significant difference in CTIP2 positive neuronal populations. Panel N shows schematic diagram and live imaging illustrating integration of microglia into organoids and interaction dynamics. Panel O shows scatter bar plots and heatmap analysis of neuronal network activity parameters indicating microglia-driven remodeling. Panel P shows raster plots and bar graph quantification of burst-level neuronal activity demonstrating dynamic network changes after microglia integration. Panel Q shows immunostaining images and scatter bar plot quantification demonstrating reduced IL34 expression following shRNA knockdown. Panel R shows immunostaining images with EdU and Ki67 labeling, and scatter bar plot shows quantification comparing proliferative activity in surface aggregates versus SMFC regions.
Characterization of hCOs and chimeric iMicroglia-hCOs (related to Fig. 6). (A) Evaluating endogenous IL-34 and CSF-1 secretion levels in hCOs and SV40 microglia and iMicroglia. (B) Schematics of inducing iMicroglia (iMacrophage) from ESC/hiPSCs and the timeline/strategies for generating and validating iMicroglia-hCOs (B). (C) Morphology of iMicroglia during induction and testing the purity of iMicroglia using flow cytometry with CD14, CD11b, CD86, and CD163 antibodies revealed the high purity of iMicroglia. (D) Immunostaining of daf 15 and 30 iMicroglia-hCOs with GFP and CSF-1R antibodies revealed the amoeboid morphology of iMicroglia in daf 15 (left two panels) and the ramified morphology of iMicroglia in daf 30 iMicroglia-hCOs (right two panels). Boxed, the middle magnified images (white arrowheads in the upper left, nuclear debris). Scale bars, 200 µm. (E and F) Size and density of iMicroglia in daf 15 and daf 30 iMicroglia-hCOs (hCOs, n = 4; data, mean ± SD; Mann–Whitney U test). (G) iMicroglia and iMicroglia area/total region in daf 15 and daf 30 iMicroglia-hCOs (hCOs, n > 7; repeat, n = 3; data, mean ± SD; Mann–Whitney U test). (H) Whole slide scanning of daf 30 iMicroglia-hCOs immunostained with Ki67 and IBA-1 antibodies revealed that a restricted number of intra-hCO iMicroglia express Ki67, and the iMicroglia included multiple DAPI-stained nuclear fragments. Right, the boxed region (yellow arrow, Ki67+ iMicroglia; white arrowheads in the lower panel, nuclear debris). Scale bars, 200 µm. (I) Percentage of Ki67+ microglia in daf 30 iMicroglia-hCOs (intra-organoid) and Ki67+ surface iMicroglia in daf 15 iMicroglia-hCOs (repeat, n = 3; total iMicroglia-hCOs, n = 8; Mann–Whitney U test; data, mean ± SD). (J) SV40 microglia in the hCO stained by GFP, PSD95, and MAP2 antibodies revealed PSD95+ and MAP2+ puncta in penetrated SV40 microglia (white dashed line, phagocytosed nuclear debris; white arrowheads, PSD95+ dots). Recording, SIM. Rendering, IMARIS software. Scale bars, 10 µm. (K) Immunostaining for SV40 microglia-hCO or hCO using PAX6 and TBR2 antibodies revealed that more TBR2+ cells are present in the SVZ region of hCO with SV40 microglia. Boxed (left), the magnified region (right). Percentage of TBR2+ or PAX2+ cells in SV40 microglia-hCO and hCO (organoid, n ≥ 3; all data, means ± SD; Mann–Whitney U test, *P < 0.05). Scale bars, 100 µm (left) and 20 µm (right). (L) Immunostaining with Ki67, pH3, and GFP antibodies in hCO with SV40 microglia or hCO. Percentage of Ki67 intensity of Ki67+ cells in hCO with SV40 microglia with hCO (organoids: n = 3; data, mean ± SD; Mann–Whitney U test. *P < 0.05; **P < 0.001). Scale bars, 100 µm (left) and 20 µm (right). (M) Immunostaining with CTIP2, GFP, and PAX6 antibodies in SV40 microglia-hCO and hCO. Percentage of CTIP2+ cells in SV40 microglia-hCO and hCO. Organoid, n ≥3; all data, means ± SD; Mann–Whitney U test, ns, P > 0.05. Scale bars, 100 µm (left) and 20 µm (right). (N) Schematic illustrates the generation of SV40-hCO chimeras in HD-MEA (left). Living imaging of SV40-hCO chimeras revealed the interaction of GFP + SV40 microglia with hCO. SV40-hCO chimeras were stripped from HD-MEA at the end of recordings (right). (O) Quantification of the mean firing rate [Hz], the mean spike amplitude [µV], and the mean ISI [ms] in hCO. n = 3 control group or microglia group; The mean ± SD; Kruskal–Wallis test; ns, no significant difference (left). Quantification of network features indicated SV40 microglia-guided remodeling of the neuronal network. The network features include mean spikes per burst, mean spikes per burst per electrode, mean burst duration [s], mean burst peak firing rate [Hz], mean IBI [s], mean ISI within burst [ms], and mean ISI outside burst [ms]. n = 3 control group or microglia group (right). (P) Raster plots show the dynamic neuronal network in chimeras after SV40 microglial plating (left). The quantification of network features at the burst level in the representative SV40-hCO chimera, including the number of spikes per burst, the duration of the burst [s], the peak firing rate [Hz] of the burst, the IBIs [s], ISI within burst [ms], and ISI outside burst [ms]. The mean ± SD; Kruskal–Wallis test; and statistical significance levels (ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) are presented (right). (Q) IL-34 and TUJ-1 antibody immunostaining of the hCOs infected by shRNA and hCO. The inner insert on the left (top) is a multichannel image. The rectangle in the inner insert is the magnified region on the right (left). The relative IL-34 intensity in the hCOs infected by shRNA lentivirus particles and control hCOs (Mann–Whitney U test; **P < 0.001) (right). Scale bars, 200 µm. (R) Immunostaining with the Ki67 antibody and EdU labeling of the surface aggregate (SA) in SV40-hCOs (left). Comparing Ki67+ surface aggregates with Ki67+ cells in the SMFC; Mann–Whitney U test; **P < 0.001 (right). Scale bars, 20 µm. IBI, interburst intervals.