For overexpression of miR-34a, hNPCs (unaffected male, derived from GM08330 iPSCs) were seeded at a density of 1.5×105 cells per well of a 12-well plate and transduced with the human Lenti-miR-34a or Lenti-miR-Ctrl expression system (Biosettia, San Diego, CA, USA) 2h after plating. The titer of the virus was ~1×107 infection units per mL and cells were transduced with a MOI = 33. Each miRNA precursor in the lentiviral vector was cloned from its native context, including its flanking and stem-loop precursor sequence within the intron of human EF1α promoter region. This ensured that each miRNA was properly expressed and processed, and would function similarly to its endogenous form. miR-Ctrl has no DNA sequence inserted into the EF1α intron. A complete medium change was performed after 7h of transduction. Then, selection of NPCs expressing miR-34a or miR-Ctrl started after three days of transduction for 10 days with medium containing 1 µg/mL puromycin (Sigma). hNPCs stably expressing miR-34a and miR-Ctrl were expanded and seeded for subsequent differentiation.
