Extended Data Fig. 1: Phenotypic characterization of iMicro.

a, Full Schematic overview of the generation of microglia-sufficient brain organoids. iMac and brain organoid are obtained 26 days after initial iPSC culture and constitute day 0 iMac and organoids. Co-cultured organoids are analysed from 15 days onwards (day 15) of co-culture of day 0 iMac and organoids. iMac co-cultured with brain organoids are characterised as co-iMac initially and then iMicro. Unless otherwise stated, experiments throughout the study are performed using the KYOU1 iPSC line. b, Flow cytometry analysis of activation markers by iMac in various conditions: treated for 24 h with fresh iMac medium, fresh cerebral organoid medium, cerebral organoids conditioned medium, or fresh cerebral organoids medium supplemented with LPS (100 ng/ml) for 24 hrs (representative of n = 3). c, RT-qPCR data showing the expression levels of microglia-specific markers in iMac grown for 15 days in iMac medium, fresh cerebral organoid medium and cerebral organoid conditioned medium (n = 3 each). Statistical analysis, one-way ANOVA. Error bars, mean +/− s.e.m. d, Sectioning and immunofluorescence analysis of brain organoids co-cultured with (n = 2) or without (n = 2) iMac for 19 days for NESTIN, SOX2 and IBA1. Individual staining profile are shown below each merged staining. Scale bar, 100 µm. e, Immunofluorescence staining for SOX2, NESTIN and IBA1 of day 18 co-culture organoids (3 represented from n = 4). Boxed areas are shown on the right in higher magnification. Scale bar, 100 µm and 20 µm. f, Sectioning and immunofluorescence staining of brain organoids co-cultured with iMac for 18 days showing the positivity of IBA1⁺ iMac for Ki67. Quantification performed using the Imaris Imaging software. Scale bar, 100 µm (n = 4). g, Immunofluorescence staining for expression of P2RY12 by IBA-1⁺ co-iMac (day 18). Scale bar, 20 µm. Representative of 3 independent staining. h, EGFP-expressing iMac elongates its dendrites towards the neuronal injury induced by two-photon laser ablation. Scale bar, 20 µm. (Representative of multiple experiments with same observation). i, Live imaging showing EGFP-expressing iMac that actively moved on the organoid and contained Ab1-42 peptide-TAMRA suggesting their ability to survey the organoid and phagocytose the peptides. Scale bar, 50 µm. Boxed area highlighted below. Scale bar: 10 µm (n = 3). Quantification performed using the Imaris Imaging software.