Amyloid precursor protein (APP) regulates gliogenesis and 2 neurogenesis of human neural stem cells by several signaling 3 pathways
we have evaluated the effects of APP overexpression in hNSCs at a global 16 level by a transcriptomic analysis using the massive RNA sequencing (RNA-seq) technology. Spe-17 cifically, we have focused on differentially expressed genes that are related to neuronal and glial 18 differentiation processes, as well as on groups of differentially expressed genes associated with dif-19 ferent signaling pathways, in order to find a possible interaction between them and APP Overall design: In this study, hNS1 cells were cultured adherently on plates pre-treated with poly-L-329 lysine (10µg/ml, Sigma) and proliferated on a chemically defined human stem cell (HSC) 330 medium supplemented with fibroblast growth factor (FGF2) (20ng/ml, Peprotech) and ep-331 idermal growth factor (EGF) (20ng/ml, Peprotech). Proliferating hNS1 cells at 70-80% con-332 fluence were dissociated with trypsin (0.25%, Gibco), centrifuged (4 minutes at 900 rpm) 333 and nucleofected (Nucleofector Amaxa 2B, Lonza) according to manufacturer's instruc-334 tion. Briefly, for the APP overexpression group, hNS1 cells were pre-mixed with 4µg of 335 pcDNA3-APP695 plasmid (contains the human gene of APP695 isoform and was kindly 336 provided by Dr. Daniel Lu, UCLA) in 100µl of nucleofection solution (Lonza).