Generation of Microglia-like Cells from PBMCs

To generate iMGs from patients and controls, peripheral blood mononuclear cells (PBMCs) from whole blood were used to differentiate monocytes into microglia-like cells according to a previously published method [25 (link)]. Briefly, PBMCs were isolated by density gradient centrifugation using Ficoll (GE Healthcare, Uppsala, Sweden) and resuspended in RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotic/antimycotic (Invitrogen, Carlsbad, CA, USA) and incubated at 37 °C overnight with 5% CO₂. The next day, adherent cells (monocytes) were cultured in RPMI-1640 Glutamax (Gibco) supplemented with 1% antibiotic/antimycotic, recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF) (R&D Systems, Minneapolis, MN, USA), and recombinant interleukin (IL)-34 (IL-34) (R&D Systems) for 14 days to cultivate iMGs cells. To generate fibroblasts from identical FTD–GRN patient, adult human fibroblasts were extracted from forearm skin by punch biopsy and cultured at 37 °C with 5% CO₂ in Dulbecco’s modified Eagles’ medium (DMEM) supplemented with non-essential amino acids (Gibco), sodium bicarbonate (Sigma-Aldrich), 1% (vol/vol) penicillin/streptomycin/Fungizone (Cellgro), and 20% FBS.