Reprogramming Fibroblasts to Induced Neurons

We generated induced neurons (iNs) from both control and mutant fibroblasts through direct reprogramming [24 (link),25 (link),26 (link)]. Cells were initially seeded in μ-Slide 4 Ibidi plates in DMEM + Glutamax medium (10566016, ThermoFisher Scientific, Waltham, MA, USA), supplemented with 1% penicillin/streptomycin and 10% FBS for 24 h. Next, the cells were infected with one-single lentiviral vector containing two shRNAs against the REST complex and two neural lineage-specific transcription factors (Achaete-Scute Family BHLH Transcription Factor 1 (ASCL1), POU class 3 homeobox 2 (BRN2)), obtained as previously described [27 (link)], at a multiplicity of infection of 30. Plasmids were provided as a gift from Dr. Malin Parmar (Developmental and Regenerative Neurobiology, Lund University, Sweden). The following day, DMEM + Glutamax medium was replaced with fresh DMEM, and 48 h later, neural differentiation medium (supplemented NDiff27 (Y40002, Takara-Clontech, San Jose, CA, USA) as described before [24 (link)]. Half of the neural differentiation medium was changed every 2–3 days. At 18 days post-cellular infection, the medium was replaced with NDiff27 only supplemented with growth factors. On day 21, cells were treated with polydatin and nicotinamide at 10 µM for seven days. By day 28 post-infection, neuronal purity and conversion efficiency were calculated, considering Tau+ cells as iNs.