Neurogenesis

hESCs or iPSCs were isolated from MEFs following dissociation to single cells with Accutase (Innovative Cell Technologies) by a 1 hr pre-plate on gelatin-coated dishes in hESC medium supplemented with 10 ng/ml FGF2 and 10 μM ROCK inhibitor (Calbiochem). The non-adherent pluripotent stem cells were harvested and plated on Matrigel (BD) coated 12-well plates in MEF-conditioned hESC medium with 10 ng/ml FGF2. Once the cell culture reached 95% confluence, neural induction was initiated by changing the culture medium to a culture medium that supports neural induction, neurogenesis and neuronal differentiation (referred to as 3N medium), a 1:1 mixture of N2- and B27-containing media. N2 medium: DMEM/F12, N2 (GIBCO), 5 μg/ml Insulin, 1mM L-Glutamine, 100 μm non-essential amino acids, 100 μM 2-mercaptoethanol, 50 U/ml Penicillin and 50 mg/ml Streptomycin; B27 medium: Neurobasal (Invitrogen), B27 with or without vitamin A (GIBCO), 200 mM Glutamine, 50 U/ml Penicillin and 50 mg/ml Streptomycin. 3N medium was supplemented with either 1 μm Dorsomorphin (Tocris) or 500 ng/ml mouse Noggin-CF chimera (R&D Systems), and 10 μm SB431542 (Tocris) to inhibit TGFβ signaling during neural induction ^{19 (link)}. Cells were maintained in this medium for 8-11 days, during which time the efficiency of neural induction was monitored by the appearance of cells with characteristic neuroepithelial cell morphology. Neuroepithelial cells were harvested by dissociation with Dispase and replated in 3N medium including 20 ng/ml FGF2 on poly-ornithine and laminin-coated plastic plates. After a further 2 days, FGF2 was withdrawn to promote differentiation. Cultures were passaged once more with Accutase, replated at 50,000 cells/cm² on poly-ornithine and laminin-coated plastic plates in 3N medium and maintained for up to 100 days with a medium change every other day.
For quantitative RT-PCR, total RNA was isolated from three cultures at each timepoint (days 5, 10, 15, 20 and 25) (Trizol, Sigma). Total RNA was reverse-transcribed and used for quantitative RT-PCR with primers specific to Foxg1 and Tbr2 using the Applied Biosystems 7000 system. Semi-quantitative RT-PCR with primers for Emx1, Dlx1, Nkx2.1, HoxB4 and Isl1 was carried out according to standard techniques on first strand, random-primed cDNA generated from total RNA extracted from cultures grown in the presence or absence of purmorphamine.

Transgenic and conditional mouse lines were used to recombine Bax in stem cells in the adult brain (Dranovsky et al., manuscript in preparation)21 (link). The impact of Bax ablation in stem cell on adult hippocampal neurogenesis and morphological maturation of adult-born neurons was characterized using various genetic reporter lines in combination with BrdU pulse-chase labeling and standard immunohistochemistry techniques and details can be found in Methods. Assessment of LTP at medial perforant path-granule cell synapses was performed as described previously 23 (link). Focal hippocampal x-irradiation was performed 23 (link) using sodium pentobarbital as the anaesthetic agent. Behavioural testing included hippocampal dependent learning paradigms (contextual fear conditioning, contextual fear discrimination learning 25 (link), object recognition, spatial and reversal learning) and tests for anxiety-like and depression-like behaviour. Experimental protocols were approved by the Institutional Animal Care and Use Committee at Columbia University and the New York State Psychiatric Institute. Details for all experimental techniques used in this study are available in Methods.