Dibutyrl cyclicamp

All iPSCs were characterized as previously described (Brennand et al., 2011 (link)). EBs were formed by mechanical dissociation of iPSC colonies using collagenase and plating onto low-adherence dishes. For EB differentiation, floating EBs were treated in STEMdiff™ Neural Induction Medium + SMADi (StemCell Technologies) for 20 days. To obtain neural progenitor cells, EBs were then plated onto polyornithine/laminin (Sigma)-coated dishes in DMEM/F12 plus 1% N2 and 1% B27. Rosettes were manually collected and dissociated with accutase (Chemicon) after 1 week and plated onto PLO (50 µg/ml) and laminin-coated dishes in neural progenitor cell media (DMEM/F12, 1% N2, 1% B27 (Invitrogen), and 20 ng/ml  EGF, and 20 ng/ml  FGF2 (Invitrogen)). To obtain hippocampal mature neurons, neural progenitor cells were plated onto dPGA or PLO (50 µg/ml) and laminin (1 µg/ml) coated-dishes and differentiated in BrainPhys neuronal medium (StemCell Technologies) supplemented with 1 × N2, 1 × B27, 20 ng/ml BDNF (Peprotech), 1 mM dibutyrl-cyclicAMP (Sigma), 200 nM ascorbic acid (Sigma), 1 μg/ml laminin and 620 ng/ml Wnt3a (R&D) for 2 weeks. After 2 weeks post differentiation, media was replaced by STEMdiff™ Forebrain Neuron Maturation Kit (StemCell Technologies) in BrainPhys media until use. All cells used in the present study were verified as free from mycoplasma contamination.

RPMI, HBSS, DMEM, EMEM, Trypsin-EDTA and Fetal Bovine Serum (FBS) were purchased from Wisent (Saint-Jean-Baptiste, Canada). Cy3 and Cy5 labeled DNA oligomers were ordered from IDT (Coralville, Iowa). #1.5 thickness round cover slips and 16% paraformaldehyde (PFA) were purchased from Electron Microscopy Supplies (Hatfield, Pennsylvania). HEK 293 and HeLa cells were purchased from ATCC (Manassas, Virginia), SN56 cells were a kind gift from Jane Rylett (University of Western Ontario, Canada). Lipofectamine 2000, Alexa Fluor-labeled secondary Fab antibodies and anti-Golgin97, were purchased form Life Technologies (Burlington, Canada). Dylight-labeled secondary Fab antibodies were purchased from Cedarlane (Burlington, Canada). Cysteamine, mouse anti-HA antibody, rabbit anti-FLAG antibody, Isoproterenol, dibutyrl cyclic AMP, Bovine Serum Albumin (BSA), anti-AP-1γ, and anti-TGN46 were purchased from Sigma-Aldrich (Oakville, Canada). Rat anti-LAMP1 antibody was purchased from the Developmental Studies Hybridoma Bank (Iowa City, Iowa). PolyJet was purchased from Frogga Bio (North York, Canada). All other chemicals were purchased from ThermoFisher (Toronto, Canada). Matlab software was purchased from MathWorks (Natick, Massachusetts). Prism software was purchased from Graphad (La Jolla, California).