The methods were performed in accordance with relevant guidelines and regulations and approved by the Ethics Committee of the University Hospital and Faculty of Medicine Tuebingen. We confirm that participants provided a written informed consent to take part in the study. Inclusion and exclusion criteria for the selection of patients diagnosed with SCZ are described in Supplementary Table1. iPSCs were generated and fully characterized as described elsewhere (Table1; refs. 22,23). All experiments were carried out with all lines in parallel.

Patient-derived human fibroblasts were reprogrammed by nucleofection of non-integrative, episomal vectors encoding for OCT3/4, SOX2, LIN28, KLF4, c-MYC, p53 and EBNA1 (Addgene, catalog no. 41813, 41814, 41855, 41856, 41857). Electroporated fibroblasts were seeded onto Matrigel (Corning, catalog no. 354277) coated well plates and expanded in a feeder-free culture system for 2128 days until first iPS colonies appeared. iPS clones were manually picked and expanded on Matrigel in mTeSR Plus medium (STEMCELL Technologies, catalog no. 05825). Passaging was routinely performed non-enzymatically using Gentle Cell Dissociation Reagent (STEMCELL Technologies, catalog no. 100-0485) in early passage numbers or later enzymatically by using accutase (Sigma Aldrich, catalog no. A6964). All expanded iPS clones were routinely tested for expression of stem cell marker on protein and RNA level and pluripotency. All iPS clones used in this study were chromosomally intact.

For microglia differentiation, we modified a previously published protocol for the differentiation of iPSC into monocytes and macrophages55. iPSCs were dissociated using accutase and seeded at a density of 5104 cells per cm (day -2). Only wells containing equally distributed iPSC colonies of 1020 cells were considered for differentiation. For all differentiation steps, a 1:1 mix of IMDM without phenol red (Thermo Fisher Scientific, catalog no. 21056023) and Hams F12 Nutrient Mix (Thermo Fisher Scientific, catalog no. 21765029) was used. The basal medium was supplemented with 10g/mL poly vinyl alcohol (Sigma Aldrich, catalog no. P8136), 64g/mL ascorbic acid 2 phosphate (Sigma Aldrich, catalog no. A8960), 0.1x chemically defined lipid concentrate (Thermo Fisher Scientific, catalog no. 11905031), 2x ITS-X (Thermo Fisher Scientific, catalog no. 51500056), 0.0039% -Monothioglycerol (Sigma Aldrich, catalog no. M6145), 1x GlutaMAX (Thermo Fisher Scientific, catalog no. 35050061) and 1x non-essential amino acids. Additional growth factors and cytokines were always added freshly before usage. For mesoderm induction at day 0, the basal medium was supplemented with 50ng/mL BMP4 (Peprotech, catalog no. 120-05ET), 15ng/mL Activin A (Miltenyi Biotec, catalog no. 130-115-008) and 1.5M CHIR99021 (Axon Medchem, catalog no. 1386) for mesoderm induction. For the suppression of self-renewal in favor of stem cell differentiation at day 2, 10M of SB431542 and SCF (Peprotech, catalog no. 300-07), VEGF (Peprotech, catalog no. 100-20) and bFGF (Bio-Techne, catalog no. 233-FB) were added to the medium at a final concentration of 50ng/ml each. For hematopoietic patterning at day 5, 10ng/ml of IL-3 (Peprotech, catalog no. 200-03) and 50ng/ml of IL-6 (Peprotech, catalog no. 200-06), 50ng/ml of TPO (Miltenyi Biotec, catalog no. 130-095-747), 50ng/ml of bFGF, 50ng/ml of SCF and 50ng/ml of VEGF were supplemented to the basal medium. Medium was refreshed at day 7. At day 9, differentiated cells grew to full confluence with hematopoietic stem cells emerging into the supernatant. Adherent cells were dissociated by accutase treatment and added to non-adherent cells collected from the supernatant. After centrifugation at 300xg for 3min, cells were resuspended in microglia medium containing 100ng/ml of IL-34 (Peprotech, catalog no. 200-34), 50ng/ml of TGF-1 (Peprotech, catalog no. 100-21) and 25ng/ml of GM-CSF (Peprotech, catalog no. 300-03). Cells were subsequently plated on ultra-low attachment plates that were pretreated with Anti-Adherence Rinsing Solution for at least 5min and afterwards rinsed twice with DPBS. Microglia differentiation was allowed to proceed for further seven days with medium changes every other day.

Microglia were routinely characterized regarding expression of key markers like IBA1, SPI1 and TMEM119. Functionality was proven by active uptake of pHrodo-labelled bacteria and response to LPS as a pro-inflammatory stimulus. Microglia identity was confirmed by RNA sequencing. Transcriptome analysis and bioinformatical evaluation was performed by CeGaT GmbH (Germany) as previously described15. For characterization of microglial phenotypes, microglial genes were chosen according to previously published literature that identified panels of highly specific microglia signature genes21,56,57,58,59,60.

Secretion of the pro-inflammatory cytokine TNF was quantified by a standard sandwich-ELISA (human TNF-alpha DuoSet ELISA kit, R&D Systems, catalog no. DY210) according to the manufacturers instructions. Briefly, 96-well plates were coated with the capture antibody and incubated over night at room temperature. Wells were washed three times and blocked for at least 1h at room temperature. Wells were again washed and 100l of culture supernatant or standards were added and incubated for 2h at room temperature. After washing, detection antibody was added and incubated at room temperature for 2h. Wells were washed and the streptavidin / horse radish peroxidase (HRP) mix was added for 30min at room temperature. Afterwards, wells were washed again and substrate solution was added for 20min at room temperature in the dark, stop solution was added and the plate was tapped for mixing. Immediately afterwards, the optical density was determined using a microplate reader (Tecan Spark) set to 450nm with wavelength corrections set to 540nm.

Flow cytometry measurements were performed using BD FACS Chorus software on a BD FACS Melody and analyzed using FlowJo 10.6.1 (FlowJo Engine, Becton Dickinson & Company). Cells were detached, washed three times with DPBS and stained with conjugated antibodies for 30min at 4C. Subsequently, cells were washed three times with DPBS and resuspended in PBS+1% FCS for immediate analysis. The following conjugated antibodies were used: anti-human SSEA-4 PE-Vio770 (Miltenyi Biotech, catalog no. 130-105-081), anti-human CD11b FITC (Thermo Fisher Scientific, catalog no. 11-0118-42) and anti-human CD45 VioBlue (Miltenyi Biotech, catalog no. 130-110-775). Doublets were excluded in FSC and SSC. Unstained cells served as negative population.

Day 19 microglia were plated at a density of 1105 cells/cm on Matrigel-coated 96 well plates. Cells adhered within 24h and were subsequently fixed and stained against NFB p65 (Cell Signaling, catalog no. 6956T), Phalloidin CruzFluor 488 Conjugate (Santa Cruz Biotechnology, catalog no. sc-363791) and Hoechst (Sigma-Aldrich, catalog no. 911004450). Using confocal laser scan microscopy with a 63x plan-apochromatic oil immersion objective, at least ten 3D Z-stacks were acquired of microglia were taken within each experiment. During acquisition, all settings such as exposure time, laser intensity and gain were kept constant. Z-stacks were further processed using Imaris software (Bitplane, version 8.2.0). Therefore, a surface for the nucleus was generated covering the Hoechst signal. Within this mask, the mean fluorescence intensity of NFB p65 was determined and quantified.

Caspase-1 activity was determined using the Caspase-Glo 1 Inflammasome Assays (Promega, catalog no. G9951) according to the manufacturers instructions. Briefly, day 19 microglia were plated at a density of 1.2105 cells/cm on Matrigel-coated 96 well plates and incubated overnight. The next day, cells were treated with 100ng/ml of LPS (Sigma Aldrich, catalog no. L6529) for 3h and subsequently with 5mM ATP (Sigma Aldrich, catalog no. A2383) for 30min at 37C and 5% CO2. The culture supernatant was transferred into a white 96 well plate and Caspase-Glo 1 Reagent was added. The mixture was incubated at room temperature in the dark for 1h and luminescence was measured on a Tecan Spark microplate reader.

Day 19 microglia and nave iPSC as control were plated at a density of 2104 cells per well of a 96 well plate and cells adhered within 24h. pHrodo Red E. coli BioParticles (Thermo Fisher Scientific, catalog no. P35361) were resuspended in 2ml PBS to generate a stock suspension with a concentration of 1mg/ml. Bioparticles were vortexed rigorously to generate a homogenous suspension. 10l of pHrodo Red E. coli BioParticles were added to the wells. Cells were incubated at 37C and 5% CO2 for 4h in the Incucyte S3 live-cell imaging system (Sartorius). 9 images per well were acquired every 15min at x20 magnification. Finally, the relative red fluorescent units per image were analyzed over time.

For lentivirus production, HEK293FT were cultured at 37C and 8 % CO2 in culture medium consisting of DMEM (Thermo Fisher Scientific, catalog no. 10566016), 10% FCS (Thermo Fisher Scientific, catalog no. 10270106), 500g/ml G418 (Carl Roth, catalog no. 2039), 1% non-essential amino acids (Thermo Fisher Scientific, catalog no. 11140035) and passaged using 0.25% Trypsin/EDTA (Thermo Fisher Scientific, catalog no. 25200056) once or twice a week. For lentivirus production, cells were dissociated and seeded at a density of 3000 cells per cm. After four days of incubation, medium of HEK293FT cells was changed to a serum-reduced transfection medium of Opti-MEM (Thermo Fisher, catalog no. 11058021) supplemented with 5% FCS. 27g of pC-Pack2 Lentiviral Packaging Mix (Cellecta, catalog no. CPCP-K2A) were mixed with 108l of Lipofectamine 2000 Reagent (Thermo Fisher Scientific, catalog no. 11668019) in 4.5mL Opti-MEM, incubated at room temperature for 20min and added to the cells for further incubation at 37C and 5% CO2. After 24h, medium was changed, while after 48h and 72h post-transfection the supernatant was removed and stored at 80C. Lentiviral suspensions were filtered through a 22nm filter, transferred into ultracentrifugation buckets and centrifuged at 19,600rpm and 4C for 80min. Pellets were air dried for a few minutes and remaining liquid was removed with sterilized soft tissue papers. Finally, 100l of DPBS+1% BSA were added per tube without pipetting or resuspending. Tubes were sealed with Parafilm and left overnight at 4C. The next day, pellets were resuspended by pipetting several times and aliquoted for storage at 80C. Titer determination was performed using the Lenti-X p24 Rapid Titer Kit (Takara Bio, catalog no. 632200) according to the manufacturers instructions. Lentiviral suspensions were diluted 10-fold and 100-fold and quantified against a p24 standard curve. Yields ranged from 51010 to 51011 particles/ml.

Ectodermal patterning was induced using the STEMdiff Neural Induction Kit (STEMCELL Technologies, catalog no. 05835) according to the manufacturers instructions. iPSC were dissociated using accutase and 2106 iPSC were seeded into ultra-low attachment AggreWell 800 well plates (STEMCELL Technologies, catalog no. 34815) pretreated with Anti-Adherence Rinsing Solution (STEMCELL Technologies, catalog no. 07010). After cultivation at 37C and 5% CO2 for seven days with daily medium changes, embryoid bodies were harvested using 37m reversible strainers (STEMCELL Technologies, catalog no. 27215). Prior to seeding, 6-well plates were pretreated with 20% poly-L-ornithine (PLO, Sigma-Aldrich, catalog no. P4957) in Dulbeccos phosphate-buffered saline (Thermo Fisher Scientific, catalog no. 14190094), incubated for 2h at room temperature and washed three times with DMEM/F12 (Thermo Fisher Scientific, catalog. no. 21331020). Subsequently, wells were treated with 10g/ml laminin (Lam, Sigma-Aldrich, catalog no. L2020) diluted in DMEM/F12 and incubated overnight at 37C and 5% CO2. Harvested embryoid bodies were washed to remove remaining single cells and seeded onto PLO/Lam pre-coated well plates in STEMdiff Neural Induction Medium with daily medium changes. Neural rosettes were selected using the STEMdiff Neural Rosette Selection Reagent (STEMCELL Technologies, catalog no. 05832), resuspended in STEMdiff Neural Induction Medium supplemented with 1M Dorsomorphin dihydrochloride (Bio-Techne, catalog no. 3093), 10M SB 431542 (Bio-Techne, catalog no. 1614), 500ng/ml recombinant Human Noggin Fc Chimera Protein (Bio-Techne, catalog no. 719-NG) and cultivated in PLO/Lam coated 6-well plate. After the first passage, cultivation medium was changed to STEMdiff Neural Progenitor Medium (STEMCELL Technologies, catalog no. 05833). NPCs were passaged up to passage 10. All generated NPCs were routinely tested for progenitor marker expression, such as PAX6, NESTIN or SOX1.

Neuronal differentiation was achieved by lentiviral overexpression of human Neurogenin 2 following previously published protocols61,62. 3.15104 NPC were dissociated by accutase treatment and seeded in PLO/Lam-coated well plates at a density of 3104 cells per cm in STEMdiff Neural Progenitor Medium. For induction of neuronal differentiation62, NPC were co-infected with lentiviral vectors pLV-TetO-hNGN2-Puro (Addgene, catalog no. 79049), and FUdeltaGW-rtTA (Addgene, catalog no. 19780) at a final concentration of approximately 10ng/ml or 2108 particles/ml per lentivirus. After 24h, doxycycline (Sigma Aldrich, catalog no. D9891) was added to a final concentration of 10g/ml to induce tetracycline-dependent expression of the reverse tetracycline transactivator (rtTA) and hNGN2. 24h later, 2g/ml of puromycine (Thermo Fisher Scientific, catalog no. 11113803) was added to the medium to select for transduced NPC. After removal of selection medium at day 2 post transduction, cells were supplied with neuronal differentiation medium consisting of Neurobasal Plus Medium (Thermo Fisher Scientific, catalog no. A3582901) supplemented with 1x B27 Plus supplement (Thermo Fisher Scientific, catalog no. A3582801), 1x N2 supplement (Thermo Fisher Scientific, catalog no. 17502048), 1g/ml Laminin, 20ng/mL BDNF (Peprotech, catalog no. 450-02), 20ng/mL GDNF (Peprotech, catalog no. 450-10), 500g/mL dibutyryl cyclic adenosine monophosphate (Sigma Aldrich, catalog no. D0627), 35g/mL L-Ascorbic Acid (Sigma Aldrich, catalog no. A2078) and 10g/ml doxycycline. At this point, 3104 murine primary astrocytes per cm were added a 50% medium change was performed every other day until neurons were assayed or fixed after 1421 days in vitro.

Neuronal and microglial differentiation started separately from each other for 16 days. Subsequently, microglia were lifted from the ultra-low attachment plates, washed with DPBS, centrifuged and finally resuspended in microglia medium. In case of pretreatment, microglia were primed using 100ng/ml of LPS or 10M of Minocycline (STEMCELL Technologies, catalog no. 74112) at 37C and 5% CO2 for 60min. Subsequently, microglial cells were washed with DPBS and added to the neuronal cultures. For a final microglia:neuron ratio of approximately 1:5, microglia were seeded at a density of 5104 microglial cells per cm combined with 3104 initially seeded NPCs per cm. The co-culture plate was transferred to the incubator and left for 72h at 37C and 5% CO2. Co-cultures were maintained in microglia medium throughout the experiments.

iPSC-derived neurons and microglia, cultured in 96-well clear plates (Greiner Bio, catalog no. 655090), were fixed using paraformaldehyde (4% in PBS, Sigma Aldrich, catalog no. P6148) for 15min at room temperature. After fixation, cells were washed three times with PBS and then blocked and permeabilized at room temperature in 0.1% Triton X-100/PBS containing 1X Blocking Reagent for ELISA (Merck, catalog no. 11112589001) for 30min. After overnight incubation at 4C with primary antibodies diluted in blocking solution, cells were washed three times in PBS and exposed to fluorescently labeled secondary antibodies (1:500; Cy3 anti-rabbit (Jackson ImmunoResearch, catalog no. 111-165-144) or Cy5-coupled goat anti-mouse secondary antibodies (Jackson ImmunoResearch, catalog no. 115-175-146) and Alexa Fluor 488-coupled goat anti-chicken or 647-coupled goat anti-rat antibodies (Thermo Fisher Scientific, catalog no. A21247, A11039). Secondary antibodies were dissolved in blocking solution and incubated at room temperature for 2h. Nuclei were stained using Hoechst Dye 33258 (1:1,000 in PBS, Sigma-Aldrich, catalog no. 911004450). The following primary antibodies were used: mouse monoclonal anti-Beta-Tubulin III (STEMCELL Technologies, catalog no. 60100, 1:250), mouse monoclonal CX3CR1 (BioLegend, catalog no. 355701, 1:500), rabbit polyclonal anti-IBA1 (FUJIFILM Wako Chemicals, catalog no. 019-19741, 1:1000), CD11b monoclonal antibody (ICRF44), eBioscience (#14-0118-82), rat monoclonal anti-LAMP1 (Santa Cruz Biotechnology, catalog no. sc-19992, 1:100), chicken polyclonal anti-MAP2 (Invitrogen, catalog no. PA1-10005; 1:2500), mouse monoclonal anti-NFB p65 (Cell Signaling, catalog no. 6956), mouse monoclonal anti-PAX6 (BioLegend, catalog no. 862001, 1:200), Phalloidin CruzFluor# 488 (Santa Cruz Biotechnology, catalog no. sc-363791), rabbit monoclonal recombinant anti-PSD95 (Synaptic Systems, catalog no. 124008, 1:500), mouse monoclonal anti-SPI1 (PU.1, BioLegend, catalog no. 658002, 1:100), rabbit polyclonal anti-SOX1 (Abcam, catalog no. ab22572, 1:500), mouse monoclonal anti-Synapsin1 (Synaptic Systems, catalog no. 106011, 1:1000), rabbit polyclonal anti-Synaptophysin1 (Synaptic Systems, catalog no. 101002, 1:500), rabbit anti-TMEM119 (Synaptic Systems, catalog no. 400002, 1:400), rabbit monoclonal anti-TREM2 (Cell Signaling, catalog no. 91068, 1:400), mouse monoclonal anti-VGlut1 (Synaptic Systems, catalog no. 135511, 1:300), mouse monoclonal anti-Nestin (Synaptic System, catalog no. 312011, 1:1000). Antibody specificity was confirmed by analysis on differentiated cells and nave iPSC, and by secondary antibody only stainings.

To determine microglial pruning of synaptic structures, Z-stacks of neuronal networks were acquired with a confocal laser scan microscopy Cell Observer SD with a x63 plan-apochromatic oil immersion objective. Z-stacks were retrieved from regions of comparable fibre density, while the settings for acquisition (such as exposure time, laser intensity and gain) were unchanged for all conditions. Each image is a 3D reconstruction of a z-stack.

Images of neuronal cultures or neuron-microglia co-cultures were further processed by imaging using Imaris software. A surface was generated covering all MAP2 signals present in the whole stack. Next, the surface was masked using the Synapsin 1 (SYN1) signal creating a new channel for SYN1. After spot detection in the new SYN1 channel, SYN1-positive synaptic structures were counted after thresholding and referred to the volume of MAP2-positive structures to provide the density of SYN1-positive presynaptic terminals. The threshold for SYN1 spot detection was kept constant for each replicate. Data from multiple images were averaged to give yield to one datapoint for each biological replicate. The number of biological replicates is indicated in the figure legends. Within each image 2-3 microglial cells were analyzed on average. Within individual biological replicates, samples were normalized to the mean of CTR1.

Microglial uptake of synaptic structures was quantified by determination of the mean fluorescence intensity of SYN1 within IBA1 positive microglia. To this end, Z-stacks of microglia were acquired as described above and further processed using Imaris. A first surface was generated using the IBA1 signal to cover whole microglial cells and was subsequently masked with the signal for SYN1. Mean fluorescence intensities were measured for SYN1-positive spots identified within microglia. At least three independent experiments were performed for each donor combination.

Statistical analysis was performed using GraphPad Prism 9.2.0 (GraphPad Software Inc.). For non-Gaussian distribution in pairwise comparisons, the unpaired MannWhitney U test was performed and for group comparisons, KruskalWallis test with Dunns post-hoc multiple comparisons test was used. The type of statistical tests used and results are reported in the figure legends or main text.

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

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