Neuronal differentiation of hESCs
hESCs were differentiated into neurons as described . Briefly, hESC colonies were detached from the feeder layer with 0.5 mg/ml dispase and cultured as aggregates in suspension for 4 days in hESC medium (Dulbecco's modified Eagle medium [DMEM]/12 with 20% knockout serum replacement) without FGF2. Cells were transferred to new culture dishes in the first 2 days to remove adherent MEFs. On day 5, EBs were transferred to flasks precoated with poly-L-ornithine/laminin (20 μg/ml) in neural induction medium (DMEM/F12/N2) consisting of 33% F12, 66% DMEM, 1X N2, 1% NEAA, 10 ng/ml FGF2, and 2 mg/ml heparin. Twelve days after neural induction, neuroepithelial cells in rosettes were isolated from the surrounding cells with 0.2 mg/ml dispase and transferred to new flasks for 2-3 h to allow non-neuronal cells to attach. Floating cells were transferred to a flask coated with poly-HEME (Sigma) to prevent cell attachment.
Terminal differentiation was induced as described  with some modifications. At 3-4 weeks of age, neurospheres were dissociated with Accutase (Sigma), plated on glass coverslips coated with poly-D-lysine and laminin (BD Biosciences), and cultured in neurobasal medium supplemented with 2% B27, 1% nonessential amino acids solution, 0.5 mM L-glutamine, 1 μg/ml laminin (Sigma), 10 ng/ml BDNF, and 10 ng/ml GDNF (R&D Systems) for 1-3 weeks. Half of the culture medium was changed every other day.
On day 7 or 14 after terminal differentiation, human neurons were fixed with 4% paraformadehyde for 10 min at room temperature (RT) and permeabilized with 0.1% Triton × 100 for 5 min at RT. After blocking with 3% BSA (Sigma), cells were incubated with primary antibodies [anti-nestin (1:100), anti-MAP2 (1:100), anti-BF1 (1:100), anti-GFAP (1:100), anti-PSD95 (1:100)] for 1 h at RT and then with secondary antibodies (anti-mouse or anti-rabbit conjugated with Cy3 or Alexa 488) for 45 min at RT. Anti-NR1 antibody recognizes the synthetic peptide RAEPDPKKKATFRA, corresponding to amino acids 849-862 of NMDAR1.
Gene expression analysis during neuronal differentiation of hESCs
Total RNA was isolated from human neurons at 3 or 14 DIV and incubated with DNase I for 30 min at 37°C. Reverse transcription was performed with Taq-Man reagent (Applied Biosystem). qRT-PCR was performed with an ABI Prism 7700 Thermocycler with fluorescence detection (Applied Biosystems) as described . The primers were Snf7-1 (sense 5'-cctatgggctttggagatga-3', antisense 5'-ttgtcgcccacatttaacaa-3'); Snf7-2 (sense 5'-cgaaacctgtagggtttgga-3', antisense 5'-ctgtttcgggtccactgatt-3'); Snf7-3 (sense 5'-gaactccacagcaatgagca-3', antisense 5'-ccagcatctcctcagtctcc-3'); NR2B (sense 5'-tctttggagatggggagatg-3', antisense 5'-tcgcagatgaaggtgatgag-3'); mGluR1 (sense 5'-ggaagggaattatggggaga-3', antisense 5'-tgtcatgccttcacaggagc-3'). The primers for GAPDH (Gene ID: NM_00204.3) were from ABI.
Electrophysiology of human neurons
Cells targeted for whole-cell patch-clamp recordings were visualized with infrared differential interference contrast microscopy (Olympus BX51WI). Cells were immersed in a HEPES-buffered saline solution containing 115 mM NaCl, 2 mM KCl, 10 mM HEPES, 3 mM CaCl2, 1.5 mM MgCl2, and 10 glucose. All experiments were performed at RT. Resistance of micropipettes was 2.5-4.0 MΩ when filled with the following: 130 mM KMeSO3, 10 mM NaCl, 2 mM MgCl2, 0.16 mM CaCl2, 10 mM HEPES, and 0.5 mM EGTA. Current- and voltage-clamp recordings were obtained with a Multiclamp 700B amplifier (Molecular Devices), filtered at 2 kHz, and digitized at 10 kHz. All data were acquired and analyzed with custom programs written in Igor Pro.
Induction of depolarization of human neurons
To induce depolarization, human neurons (14-21 DIV) were incubated in neurobasal medium (Invitrogen) with 1 μM tetrodotoxin (Sigma) and 10 μM 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (Sigma) for 30 min at 37°C. In some experiments, 2X depolarization solution (10 mM HEPES pH 7.4, 1.8 mM CaCl2, 110 mM KCl, 0.925 mM NaH2PO4, 0.39 mM MgSO4, 5.36 mM NaCl) was added. Depolarization-induced gene expression was determined by qRT-PCR.
Gene knockdown in human neurons
For generation of virus, each RNA was subcloned into the pSicoR vector (McManus lab, University of California, San Francisco). To knock down hSnf7-1 and hSnf7-2 in human neurons, specific sequences were selected as targets of siRNA (Additional file 1). For immunoblot analysis, proteins extracts were separated by SDS-PAGE and transferred to a PDVF membrane. After blocking with 5% non-fat milk in 1% Tween 20 in PBS, the membrane was incubated first with primary antibody at 4°C overnight and then with HRP-conjugated anti-mouse or anti-rabbit secondary antibody at RT for 1 h.
Survival assay for human neurons
To investigate the effect of loss of hSnf7-1 or hSnf7-2 in human neurons, we infected mature cortical neurons (14-16 DIV) with lentivirus expressing siRNA. PI- and GFP-positive neurons were counted as surviving neurons. Three independent cell survival assays were done.