Our study was conducted in accordance with relevant guidelines and regulations and approved by the Ethics Committee of the University Hospital and Faculty of Medicine Tuebingen. Participants provided written informed consent to take part in the study, and inclusion and exclusion criteria for the selection of patients diagnosed with SCZ are provided in Supplementary Table 1. We generated and fully characterized iPSCs as described elsewhere (Table 1; refs. 22,23), and all experiments were carried out with all lines in parallel.
To reprogram patient-derived human fibroblasts, we used 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). We then expanded the electroporated fibroblasts in a feeder-free culture system for 21-28 days until the first iPS colonies appeared, manually picked and expanded on Matrigel in mTeSR Plus medium (STEMCELL Technologies, catalog no. 05825). All expanded iPS clones were routinely tested for expression of stem cell marker on protein and RNA level and pluripotency, and all iPS clones used in this study were chromosomally intact.
For microglia differentiation, we modified a previously published protocol for the differentiation of iPSCs into monocytes and macrophages. We characterized microglia regarding expression of key markers like IBA1, SPI1, and TMEM119 and proved their functionality by active uptake of pHrodo-labeled bacteria and response to LPS as a pro-inflammatory stimulus. We confirmed microglia identity by RNA sequencing and characterized their phenotypes.
We used various methods to quantify p65 expression, caspase-1 activity, and phagocytosis assay. Furthermore, we produced lentivirus using HEK293FT cells and cultured them in a specific medium. Our study is thus comprehensive and reliable, and our methods were conducted with high-quality standards.
