dings were obtained using Axon Instruments Multiclamp 700B amplifier and digitized at 1020 kHz. Extracellular solution was as follows: 129 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 30 Glucose, 25 HEPES, pH 7.4. Whole-cell patch-clamp electrodes were filled with 120 mM K D-gluconate, 25 mM KCl, 4 mM MgATP, 2 mM NaGTP, 4 mM Na2-phospho-creatin, 10 mM EGTA, 1 mM CaCl2 and 10 mM HEPES. Biocytin was 
added to the pipette solution to mark the recorded neuron for later cytochemical characterization. Spontaneous postsynaptic currents measured under this experimental condition at 270 mV are a superposition of mEPSCs. Further analysis and statistics were performed using IgorPRO with custom-written scripts. Ca2+ Imaging Neurons were loaded with Fluo 4-AM for 60 min in HBSS supplemented with 10 mM HEPES. Fluorescence microscopy was performed using a Nikon TE 2000 inverted AVE-8062 site microscope and an EMCCD camera imaging fluorescence emission at a frame rate of 100 s21 at 37uC. Fluorescence measurements are expressed as a ratio of the mean change in fluorescence relative to the resting fluorescence before stimulation. Mean values of Fo were obtained by averaging over several scans/ frames before stimulation. Western Blot Analysis Western blotting was carried out on whole cell lysates that had been extracted using RIPA buffer. Before loading, samples were denatured. Protein PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19648649 separation was achieved using SDS polyacrylamide gel electrophoresis and transferred onto PVDF membrane. Antibodies used as follows: TH, Dopa decarboxylase, DAT. Measurements of Mitochondrial Membrane Potentials Mitochondrial DY was measured by loading cells with 20 nM tetramethyl rhodamine methyl ester for 30 min at 37uC. Images were taken by the same inverted microscope described above equipped with objective lenses. TMRM excitation and emission was measured. Carbonyl cyanide ptrifluoromethoxyphenylhydrazone was added to collapse membrane potential. High Performance Liquid Chromatography Dopamine content was analysed using HPLC with electrochemical detection. Differentiated neurons were harvested in 0.1 M perchloric acid. Lysate was filtered and injected into the HPLC system via an autosampler and monoamines separated on a 250 mm Microsorb C18 reverse-phase column and detected with an LC-4B electrochemical detector. The mobile phase consisted of methanol, NaH2PO4, EDTA, and sodium octane sulfonate at pH 3.27. The flow rate was fixed at 1 ml/ min and dopamine content determined by comparing samples to standard solutions. Corresponding dopamine content normalised to protein. Results hiPSC Generation and Characterisation A Physiological Model of Human Dopamine Neurons 4 A Physiological Model of Human Dopamine Neurons and iPS-NHDF-2 cell lines are competent at differentiation to three germ layers in vitro. Left column shows embryoid body formation 4 days after forced aggregation of hiPSCs using Aggrewells. Second column, directed differentiation to neuroectoderm, showing neurons stained for bIII tubulin, using a TujI antibody. Third column, directed differentiation to mesoderm, showing cells stained with antibody against a-sarcomeric actin. Fourth column, directed differentiation to endoderm, showing nuclei stained with an antibody against the transcription factor, FoxA2. Nuclei are counterstained with DAPI. Scale bar:100 mm. D) PluriTest analysis of Illumina HT12v4 transcriptome array data shows iPS-NHDF-1 and iPSNHDF-2 to cluster with pluripotent stem cells and not with partly- or differentiated cells.