Cell culture and high throughput chemical screen
The mouse ESC lines E14Tg2a and 46C were used. 46C was a generous gift from Dr Austin Smith, in which GFP was knocked into the sox1
locus (Ying and Smith, 2003
). mESCs were cultured in GMEM media (G5154, Sigma, St. Louis, MO) supplemented with glutamine, sodium pyruvate, 0.1 mM MEM non-essential amino acids, 10% (vol/vol) fetal bovine serum (characterized, Hyclone, Thermo Scientific, Waltham, MA), beta-mercaptoethanol, and 500–1000 units per ml of leukocyte inhibitory factor (ESG1107, Chemicon, Billerica, MA), on gelatinized cell culture surface without feeder cells.
To induce neuronal differentiation, the monolayer differentiation protocol developed by Ying et al. (Ying et al., 2003
) was used. Briefly, E14 cells was dissociated with Trypsin-EDTA (TE) into single cells and plated onto gelatinized cell culture dish at a density of 1.0 × 104
in N2B27 media. Cells were cultured in N2B27 media for 7 days, with media change every other day. On day 7, cells were dissociated with TE again and re-plated onto poly-L-ornithine-laminin coated 96-well plate in N2B27 media, at a density of 2~5 × 104
. Media was changed every 2 days after re-plating.
For high throughput screening, on Day 7, cells were dissociated in TE and re-suspended in fresh N2B27 media. The cell suspension was then dispensed into 96-well micro-clear imaging plates (Greiner cat.no 655956) with the WellMate liquid dispenser (Thermo Matrix), at a density of 1.5 × 104/ well. These assay plates were incubated in 37°C overnight for cell settling and adherence to the surface. On Day 8, screening compounds were dispensed into assay plates with Biomek FXP Laboratory Automation Workstation (Beckman Coulter, Brea, CA), at a final concentration of 1 μM. Chemical treatment lasted for 3 days from Day 8 to Day 11. On Day 11, chemical treatment was withdrawn via change of media. Cells were cultured in assay plates for additional 3 days until Day 14 before automated immunostaining using PlateMate Plus (Thermo Matrix) and image acquisitioning with INCell 1000 or 2000 (G.E. Healthcare, Little Chalfont, UK).
The chemical library was obtained from the UCSF SMDC, which is composed of FDA-approved drugs, bioactive compounds, and natural products (Microsource Spectrum Collection).
Primary antibodies for immunocytochemistry include: Rabbit-anti-TH (AB152, Millipore, Billerica, MA); Mouse-anti-NeuN, (MAB377, Millipore); mouse anti-sox2 (MAB2018, R&D); Rabbit-anti-Lmx1 (a generous gift from Dr. German, UCSF); mouse-anti-Nestin (MAB353, Chemicon); mouse-anti-islet (DSHB 39.4D5); Rabbit anti-GABA (A2052, Sigma); Rabbit-anti-Olig2 (AB9610, Millipore). After immunostaining, images were taken using the automatic system INCell 1000 or 2000 (GE). 20 field of views on three different channels (For TH, NeuN and DAPI) were taken for each well. Images were analyzed using the INCell Developer software (G.E. Healthcare). The percentage of TH in each well was expressed as a ratio. Fold change of chemical-treated well was calculated relative to the average of DMSO control wells. The percentage of other neuronal types was calculated similarly.
The following pharmacological compounds were used in this study: selamectin (01503720, Microsource), avermectin (31732-100 MG, Sigma), taurine (T8691-25 G, Sigma), muscimol (M1523-5MG, Sigma), Chlordiazepoxide (C2517, Sigma), picrotoxin (R284556-50 MG, Sigma), Pentylenetetrazole (P6500-25 G, Sigma), bicuculine (14340-25 MG, Sigma), STY (Strychnine, S0532-5G, sigma). Drugs were prepared as 10 mM stock and diluted to appropriate concentrations as indicated in the text.
EsiRNA knockdown of GABAA receptor subunits
Two rounds of PCR were done to obtain the template for in vitro synthesis of double-stranded RNAs. cDNAs from day 3 mESC-derived neural progenitors was used as template for the first round PCR. Primers for first round PCR begin with T7 ‘anchor’ sequence: 5′ GGGCGGGT 3′, to which the T7 Anchor primer will anneal in the second round PCR. T7 promoter was incorporated in the primers for the second round PCR. The product from the second round PCR was used as template for in vitro transcription with T7 RNA polymerase. Annealing is done in the same program immediately after in vitro transcription: 1) 37°C, 5.5 hr; 2) 90°C, 3 min; 3) Ramp (0.1°C/s) to 70°C; 4) 70°C, 3 min; 5) Ramp (0.1°C/s) to 50°C; 6) 50°C, 3 min; 7) Ramp (0.1°C/s) to 25°C; 8) 25°C, 3 min. Double-stranded RNA was digested with Shortcut RNAse III (NEB) and purified for transfection. The EsiRNAs targeting different GABAA receptor subunits were transfected into mESC-derived neural progenitors on day 6 and day 9 using Lipofectamine 2000 Reagent (Invitrogen) following the manufacturer’s protocol.
BrdU incorporation and TUNEL staining
Cells were labeled with 10 μM BrdU for 6 hr before immunostaining. Rat-anti-BrdU antibody (ab6326, Abcam, Cambridge, MA) was diluted 1:2000. An in situ cell death detection kit from Roche (Cat. # 12,156 792 910) was used. Staining was performed following manufacturer’s instruction.
Real time RT-PCR
Total RNA was isolated using TRIzol reagent (Invitrogen) and qPCR was carried out following manufacturer’s instructions (Applied Bio-systems). Primer sequences are: ascl1 (GenBank accession number NM_008553.4), forward, 5′-GAAGCAGGATGGCAGCAGAT-3′, reverse,5′-TCGGGCTTAGGTTCAGACAC-3′; neuroD1 (GenBank accession number NM_010894.2), forward, 5′-AGCCACGGATCAATCTTCTC-3′, reverse, 5′-ACTGTACGCACAGTGGATTC-3′; lmx1a (GenBank accession number NM_033652), forward, 5′- ACCCCTATGGTGCTGAACCT- 3′, reverse, 5′- CAGCAACCCTTCACACAGTA -3′; lmx1b (GenBank accession number NM_010725), forward, 5′-CTGGGCCAAGAGGTTCTGTC-3′, reverse, 5′-GAAGAGCCGAGGAAGCAGTC-3′; nurr1 (GenBank accession number NM_013613), forward, 5′-CTGGCTATGGTCACAGAGAGACAC-3′, reverse, 5′-GGTACCAAGTCTTCCAATTTCAGG-3′; β-actin (GenBank accession number NM_007393), forward, 5′-TCCTTCTTGGGTATGGAATCCTG-3′ reverse, 5′-GGAGGAGCAATGATCTTGATCTTC-3′. Ct values were the means of triplicate replicates. Each sample was normalized with loading reference β-actin (ΔCt), and then normalized with expression on Day 8. For relative expression level comparison, the difference in cycle threshold (ΔΔCt) between D11 and D8 was evaluated.
Clonal analysis of mESC-derived neural progenitor cells
Day 5 mESC culture was transfected with low concentration pCAG-GFP
plasmid to sparsely label mESC-derived neural progenitors. Transfection was done with Lipofectamine 2000 Reagent (Invitrogen) following the manufacturer’s protocol. Briefly, 0.8-μg pCAG-GFP plasmid DNA was used for a single confluent well of a 6-well plate. On day 7, cells were dissociated with Trypsin-EDTA and re-plated onto poly-L-ornithine-laminin coated 96-well plate in N2B27 media, at a density of 2~5 × 104
. Time-lapse live imaging was performed with a 2-hr interval from Day 8 to Day 14, using a third generation automated robotic microscopy system that incorporated several advances over earlier systems (Arrasate et al., 2004
; Arrasate and Finkbeiner, 2005
; Sharma et al., 2012
). Multiple images were taken for each condition and stitched together using a custom designed plugin for the open source image processing package Fiji (Schindelin et al., 2012
). The cells were fixed for immunostaining on day 14.
Electrophysiology in mESC-derived neural rosette progenitors
Neural rosettes are recognized because of the cells’ characteristic bipolar morphology and their radial floral-like arrangement. We used cell line that expresses GFP under SOX1 promoter. For majority of neural rosettes, we also verified their GFP signals. There are also non-rosette GFP positive cells, among which are new born neurons. New born neurons were identified based on their characteristic morphology, round-shaped cell body. The whole-cell recordings were performed at room temperature. Pipette electrodes (Sutter, Novato, CA) were fabricated using a Sutter P-97 horizontal puller and fire-polished and had final tip resistances of 2–4 MΩ. All recording have been performed using gap free protocol while the membrane potential was holding at −70 mV. The bath solution contained (in mM) NaCl 110, KCl 30, CaCl2 1.8, MgCl2 0.5, HEPES 5, and glucose 10, pH adjusted to 7.4 with NaOH. The internal solution for patch recordings contained (in mM) NaCl 10, KCl 130, MgCl2 0.5, HEPES 5, EGTA 1,and MgATP 5, pH adjusted to 7.3 with KOH. The applications of the activation and inhibition reagents were performed by a pressurized micro-perfusion system. The pressure was typically 7–10 kPa. The stock solutions were made by dissolving the reagents in the bath solution (GABA) or DMSO (bicuculline and selamectin). The stock solutions were kept at −80°C and were diluted to the working concentration using bath solution before each experiment. Unless otherwise indicated, we used 100 μM GABA, 100 μM bicuculline and 8 μM selamectin. The working solutions of bicuculline and selamectin contained up to 0.5% DMSO, therefore the bath solutions containing the corresponding concentration of DMSO were routinely used as control solution prior to the applications of bicuculline or selamectin.
Differentiation of multiple neuronal lineages from human pluripotent stem cells
The human ESC lines H9 (Thomson et al., 1998
) and a human iPSC line (a gift from Dr Bruce Conklin) (Kreitzer et al., 2013
) were used. Stem Cells were cultured on growth factor reduced Matrigel (BD Biosciences, Franklin Lakes, NJ) in mTeSR1 media (Stemcell Technologies, Vancouver, Canada) with the media changed daily. To initiate differentiation, H9 and WTC-10 cells were plated at a density of 2 × 104
in N2B27 media (DMEM/F12:Neurobasal [1:1], N2 supplement (1:100), B27 supplement without vitamin A (1:50), Glutamax, Insulin (20 μg/ml), beta-mercaptoethanol (110 μM), BSA Fraction V (20 μg/ml), bFGF (20 ng/ml) supplemented with Rock Inhibitor Y-27632 (10 μM, Millipore)). Media was changed with fresh N2B27 media every other day until Day 11. Cells were plated at 3 × 104
in N2B27 media supplemented with Rock inhibitor on Day 11. Media was changed on Day 12 to neuronal differentiation media (Neurobasal, B27 without vitamin A (1:50), BDNF (20 ng/ml, Peprotech, Rocky Hill, NJ), GDNF (10 ng/ml, Peprotech), cAMP (500 μM, Sigma Aldrich), Ascorbic Acid (200 μM, Sigma Aldrich)). Cells were treated with DMSO or Selamectin from Day 12 to Day 19, and were fixed on Day 26 for immunostaining. All reagents were purchased from LIfe Technologies, unless otherwise stated.
In vivo zebrafish treatment and immunostaining
A transgenic line (Hu-GFP) marking nascent neurons was used. At 10 hpf, embryos were de-chorionated with forceps and transferred to a glass vial with 3 ml Embryo Solution, and Selamectin was added into solution to a final concentration of 2 μM. Embryos were incubated at 28°C until 22 hpf and then fixed with 4% PFA and mounted onto slides for confocal imaging.
Embryos were also incubated at 28°C until 48 hpf to evaluate the effect of selamectin on TH differentiation (selamectin treatment lasted from 8 hpf to 48 hpf). At 48 hpf, embryos were stained with anti-TH antibody (custom made, 1:1000).