To characterize the 2D and 3D neurons derived from iPSCs, cells were immunostained using selected markers. 2D cells were transferred into an 8-chamber well slide (polystyrene vessels culture slides, Falcon) and postfixed with 4% paraformaldehyde (PFA). 3D cultures were also fixed with 4% PFA overnight followed by 30% sucrose solution for 3 days at 4°C. After fixation, 3D neuro-spheroids were transferred into embedding-medium (Tissue-Plus, O.C.T Compound 4585, Fisher HealthCare) and quickly frozen with dry ice. The cells were cut into 10μm thick sections using a cryostat (Leica). Sections were mounted in a superfrost slide and kept on dry ice until immunocytochemical (ICC) staining. Both 2D and 3D cells were blocked in 10% normal goat serum (NGS), 0.1% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS for 1h at room temperature, followed by overnight incubation at 4°C with primary antibodies. Then, cells were incubated with the appropriate secondary antibodies conjugated with fluorophores, examined and imaged using the confocal microscope (Leica TCS SP5 Confocal Laser Scanning Microscope). All antibodies were commercially available: Tau antibody BT-2, pTau181 antibody AT270, and PAX6
