Culture of hPSCs
The hPSC lines [human embryonic stem cell line WA-09 and human parthenogenetic stem cell lines LLC2P and LLC12PH (International Stem Cell Corporation)15,16
] were first maintained on a mitomycin-C inactivated mouse embryonic fibroblast (Millipore) feeder layer in embryonic stem cell medium: Knock Out DMEM/F12 (Life Technologies), 2 mM L-glutamine (GlutaMax-I, Invitrogen), 0.1 mM MEM nonessential amino acids (Life Technology), 0.1 mM β-mercaptoethanol (Life Technologies), penicillin/streptomycin/amphotericin B (100 U/100 μg/250 ng) (MP Biomedicals) and 5 ng/ml bFGF (Peprotech). Cells were passaged with dispase (Life Technologies) every 5–7 days with split ratio of 1:4 or 1:6. The hPSCs were then transferred to Matrigel (BD Biosciences) coated plates and grown with Stem Pro hESC SFM medium (Invitrogen): DMEM/F12 with GlutaMAX, 1× STEMPRO hESC SFM Growth Supplement, 1.8% Bovine Serum Albumin, 8 ng/mL bFGF and 0.1 mM 2-Mercaptoethanol.
Derivation and growth of hPSC-NSCs
hPSC-NSCs were derived by treating undifferentiated hPSCs growing under feeder-free culture conditions with a chemical combination consisting of SB218078 (5 μM) plus DMH-1 (1 μM) in N2B27 Medium [Knockout DMEM/F12, 1× GlutaMax, 1× N2/B27 Supplement (Invitrogen)] for 7 days. The neuralized hPSCs were then dissociated with Accutase (Sigma) and grown on Matrigel coated plates in NSC medium [KnockOut DMEM/F12, 2% StemPro Neural Supplement, 1× GlutaMAX, 20 ng/ml bFGF and 20 ng/ml EGF] for ≥ 4 passages to generate and highly pure and homogeneous population of hpSC-NSCs.
High-throughput screening assay for DA neuron differentiation
hPSC-NSCs were treated with purmorphamine (2 μM) and FGF8 (100 ng/mL) in NB medium [NeuroBasal medium, 1× GlutaMAX, 1× N2/B27 Supplement (Invitrogen)] for 7 days. The primed NSCs were then dissociated with Accutase (Sigma), and seeded at a concentration of 20,000 cells/mL into Matrigel- coated 96 well plates and then treated with small molecule library (Torcris 1120 Biologically Active Compounds) at 2.5 μM (final concentration) or control (0.1% DMSO) for 2 weeks. Two weeks after treatment with small molecules, all wells were visually observed and scored based on neurite density and dopamine release. For measuring neurite density, cells were fixed with 4% paraformaldehyde and phase contrast images were acquired using Cellavista Cell Imaging System (Roche Applied Science) from randomly selected fields. Neurite density was measured using the Cellavista density software image processing program. Each experimental condition was done in duplicate wells, and at least three independent experiments were conducted to acquire the final results.
Conditioned medium was collected from samples containing 50,000 cells per well and centrifuged at 3000 RPM to remove cell debris. Supernatant was then collected and analyzed for quantitative determination of dopamine using a commercial dopamine ELISA assay kit (Cusabio). Data analysis was performed using the microtiter plate reader (BIO-TEK Synergy 2). The results presented are from three independent experiments (n = 3) and represented as mean ± standard error of mean (s.e.m.) and statistical analysis was performed using the two-tailed Student's t-test with a confidence level of 95% (α = 0.05) with statistical significance of P < 0.05.
Whole-cell patch clamp electrophysiology
For electrophysiological recordings, the cells were transferred to a recording chamber mounted on an inverted Olympus microscope. During the recordings the temperature was maintained at 31–32°C, and the cells perfused at constant flow of 1 ml/min. with 150 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM Glucose at pH 7.30 and 315 mOsm. Whole-cell recordings were obtained using borosilicate electrodes with tip resistance of 2–3 MΩ, filled with 130 mM K-Gluconate, 5 mM NaCl, 5 mM KCl, 10 mM HEPES, 20 mM Sucrose, 0.3 mM EGTA, 3 mM MgATP at pH 7.30 and 290 mOsm. Data were acquired using an HEKA EPC-10 digitizer/amplifier. Once whole-cell access was achieved the resting membrane potential was immediately recorded; the cells were then allowed to equilibrate for 3–5 min. From a resting membrane potential of −70 mV, the cells were then depolarized in current-clamp mode with 200 ms current injections, delivered at 0.2 Hz., and with increasing amplitude from 50 pA to up to 1000 pA to induce action potentials. The presence of inward or outward currents was determined by applying a voltage ramp. Cells with unstable recording configuration or for which the series resistance became larger than 15 MΩ were excluded from the study. We recorded 20 different cells and obtained action potentials from 7 patch-clamped cells.
Total RNA was extracted from collected from duplicate sample pellets (RNeasy; Qiagen) according to the manufacturer's protocol. RNA quantity (Qubit RNA BR Assay Kits; Invitrogen) and quality (RNA6000 Nano Kit; Agilent) was determined to be optimal for each sample before further processing. 200 ng RNA per sample was amplified using the Illumina Total Prep RNA Amplification Kit according to manufacturer's protocol and quantified as above. 750 ng biotinylated RNA per sample was hybridized to Illumina HT-12v4 Expression BeadChips, scanned with an Illumina iScan Bead Array Scanner, and quality controlled in GenomeStudio and the lumi bioconductor package. All RNA processing and microarray hybridizations were performed according to manufacturer's protocols. In GenomeStudio, probes were filtered for those detected with a P value of 0.01 in at least one sample and exported for normalization in R. Raw probe expression values were transformed and normalized using the robust spline normalization (RSN) as implemented in the lumi R/Bioconductor package. To obtain the Venn Diagram, Qlucore Omni Explorer was used. Transcripts that were differentially expressed between pairs of cell types (hPSC vs Substantia Nigra, hPSC vs NSC, and hPSC vs Dopaminergic neurons) were identified using two-tailed Student's t-test with P-value cut-off of <0.05 and a variance cut-off of >0.005. The sets of differentially expressed probes were then compared to each other. The gene expression array data is available at the NCBI GEO database under the accession designation GSE42265.
The results are presented as mean ± standard error of mean (s.e.m.) and statistical analysis was performed using a confidence level of 95% (α = 0.05) with two-tailed Student's t-test for comparing two groups or one factor ANOVA with Dunnett test for comparing multiple groups against control. The criterion for statistical significance for all tests was P < 0.05.
Immunocytochemistry, flow cytometry, and RT-PCR have been previously described17
and details for these experimental procedures as well as for Microelectrode Array (MEA) System are given in the Supplementary Information