Once hiPSC reached approximately 90% confluence, neural differentiation was performed according to a modified dual SMAD protocol (Shi et al., 2012 (link)). Neural induction was initiated by changing the media into neural induction media containing 50% DMEM/F12 (Thermo Fisher Scientific, 11330057), 50% advanced neurobasal medium (Thermo Fisher Scientific, 21103049), 1% N2 (Thermo Fisher Scientific, 17502048), 1% B27 without retinoic acid (Thermo Fisher Scientific, 1258010), 1% Glutamax™ (Thermo Fisher Scientific, 35050061), 1% non-essential amino acid (NEAA, Thermo Fisher Scientific, 11140-050), 0.1% Pen/Strep (Sigma-Aldrich, United States, P0781-100ML), supplemented with the inhibitors 10 μM SB431542 (SMAD inhibitor, SMS-gruppen, S1067) and 0.1 μM LDN193189 (Noggin analog, Sigma-Aldrich, SML0559). The cells were maintained in induction media for 12 days with daily media change. On day 12, a uniform neuroepithelial sheet appeared, and the neural progenitor cells (NPCs) were passaged with Accutase (Thermo Fisher Scientific, A1110501) into neural expansion media containing growth factors 10 ng/ml FGF2 (ProSpec, CYT-557) and 10 ng/ml EGF (ProSpec, CYT-217) instead of the inhibitors. NPCs were expanded and banked. Following expansion, NPCs were plated onto Poly-L-Ornithine (PLO, Sigma-Aldrich, P4957)/laminin (Sigma-Aldrich, L2020-1 mg) coated dishes with a seeding density of 50,000 cells/cm2, and terminal neural differentiation was performed in neural maturation media, supplemented with 50 μM db-cAMP (Sigma Aldrich, D0627-100 mg), 200 μM Ascorbic acid (Sigma Aldrich, A4403-100MG), 20 ng/ml BDNF (ProSpec, CYT-207) and 10 ng/ml GDNF (ProSpec, CYT-305). The maturation process was carried out for 5 weeks for MitoTracker™ and Golgi ICC analysis and 7 weeks for assessment of Aβ secretion and Tau phosphorylation as well as MitoTracker™, Golgi and synaptic evaluation, with partial media change every third day, before the neurons were fixed or harvested for further analyses.
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