(bFGF, Royan Institute). The cells were grown in 5% CO2 and 95% humidity, and they were further passaged every 7 days. For passaging, hESCs were treated with collagenase IV (0.5 mg/ml, Invitrogen): Dispase (1 mg/ml, Invitrogen) at 37uC for 5–7 min then the enzyme was removed and washed with PBS. Cells were collected by gently pipetting and replated on matrigel coated dishes and the medium changed every other day [8]. Neural differentiation Neural differentiation was induced as described previously [8]. Briefly, seven-day hESCs were induced to neural phenotype by 20 ng/ml bFGF, retinoic acid (RA, 2 mM, Sigma-Aldrich), Noggin (500ng/ml, R&D), Shh (50 ng/ml, R&D), and leukemia inhibitory factor (LIF, 10 ng/ml, chemicon) for two days (NI stage). Cells were incubated for an additional seven days without Noggin and SHH until neuroectodermal islands with columnar cells, known as rosette structures, appeared (NE stage). For a further week RA removed from the medium and during this time, the rosette structures changed into neural tube-like structures (NT stage). These structures were separated manually from the surrounding flat cells with a sterile pulled-glass pipette under the phase contrast microscope (10X, Olympus, CKX41). The neural tube-like structures were dissociated into single cell by 0.01% trypsin (Invitrogen)/2 mM disodium EDTA and then replated on laminin (5 mg/ml, Sigma-Aldrich) and poly L-Ornithine (15 mg/ml, Sigma-Aldrich)-coated tissue culture dishes to generate differenti- ated neural cells (DN stage) in neurobasal medium, supplemented with 2% N2 and 2% B27, 2.5% fetal bovine serum (All from Invitrogen), ascorbic acid (AA, Sigma-Aldrich), fibronectin (5 mg/ ml, Sigma-Aldrich), laminin (1 mg/ml, Sigma-Aldrich), BDNF (20 ng/ml, R&D), GDNF(20 ng/ml, Sigma-Aldrich) and db- cAMP(1 ng/ml, Sigma-Aldrich) for up to 14 days. Immunofluorescence staining and flow cytometry analysis To perform immunofluorescence staining, cells were fixed in 4% paraformaldehyde for 20 minutes, permeabilized with 0.2% Triton X-100 for 10 minutes and blocked in PBS with 10% serum of host which secondary antibody was derived in PBS for 1 hour. Cells were incubated with primary antibody for 1 hour at 37uC, washed, and incubated with fluorescein isothiocyanate (FITC)- conjugated secondary antibodies for 1 hour at 37uC. The primary antibodies used were Oct4 (1:100, Santa Cruz Biotechnology, SC- 5279) and Nanog (1:100, Santa Cruz Biotechnology, SC-30331) for undifferentiated hESC determination and anti-Nestin, Sox1, Pax6, microtubule-associated protein monoclonal IgG (MAP2), glial fibrillary acidic protein (GFAP), serotonin, and tyrosine hydroxylase (TH) for differentiated cell determination. The complete information for and concentrations of the antibodies have been described previously [8]. The nuclei were counter- stained with DAPI or propidium iodide (PI). Cells were analyzed with a fluorescence microscope (Olympus, Japan). Flow cytometry analysis for Nestin, Sox1, and Pax6 was performed as described previously [8]. Electrophysiology analysis Whole cell patch clamp recording method was used for functional testing of differentiated cells in 30 days post induction which cultured on cover slip. The record was carried out in the room temperature (25uC) and cell currents were recorded in voltage clamp mode. Patch electrode (Filament borosilicate glass, 1.5 mm outer diameter, HARVARD apparatus, GC150F-10) resistance brought up to 3 to 5 M. and pulled used horizontal Gene Expression Analysis of hESC Differentiation puller (Sutter Instruments P-97 USA). Recorded signal amplified and filtered (2 KHz low-pass Bessel Filter) using Multiclamp 700B amplifier (Axon instrument, USA). Amplified signals were acquired at 10 KHz using a Digidata 1440 analog-to-digital (A/ D) board and PClamp 10 software (Axon instrument, USA). All voltage protocol will describe in result section. The signals analyzed by Clampfit10 software (Axon instrument, USA) in off- line mode. For recording cell currents the bath solution was used (mM): Nacl 160, CaCl2 2, HEPES 10, D-Glucose 10. The solution pH adjusted in 7.4 by using NaOH and osmolarity kept around 300 mOsm The pipette solution contained: CsCl2 (130 mM), MgCl2 (2 mM), TEA-Cl (20 mM), EGTA (10 mM), HEPES (10 mM), and D-Glucose (10 mM) adjusted to PH 7.2 by using KOH and osmolarity kept around 300 mOsm. Nifedipin (5 mM) had been used to block L type Ca++ channels that indicated and administrated via super fusion using a fast-exchange perfusion system (ALA instruments). Illumina bead chip hybridizations and analysis of expression data Differentiated cells that were representative of NI (day 2), NE (day 9) and DN (day 30) and control hESCs were generated in triplicate. Total RNA was isolated using Trizol reagent (Invitro- gen). Approximately 400 ng of total RNA from three biological replicates per time point served as the input to generate biotin- labeled cRNA using a linear amplification kit (Ambion, Austin, TX, United States). RNA and biotinylated cRNA concentrations were confirmed with Nanodrop ND-1000 and controlled for quality using a BioRad Experion electrophoresis station. Next, cRNA samples (750 ng) were hybridized onto Illumina SentrixH HumanHT-12 v3 Expression Bead Chips at 58 uC overnight (19 h). Chips were scanned with the Illumina Bead Array Reader (Factor = 1, PMT = 520, Filter = 100%), and the numerical results were extracted with GenomeStudio using the Gene Expression Module v.1.0.6. Raw data were background-subtracted and normalized using the quantile normalization method (lumi software package) [11]. Normalized data were filtered for genes with significant expression levels compared to negative control beads. Selection for differentially expressed genes was performed on the basis of arbitrary thresholds for fold changes plus statistical significance according to the Illumina t-test error model (limma software) [12]. The mRNA array data in MIAME compliant and has been submitted to the NCBI Gene Expression Omnibus (GEO) database (Accession: GSE28633). Data analysis All significantly expressed transcripts (P,0.05, FC$1.5) were clustered using a hierarchical clustering method. The determina- tion of the correct number of clusters was based on measuring the similarity of each gene to its own cluster compared to the similarity of the gene to genes in other clusters, which was measured using the average of the intracluster and intercluster distances. MATLAB software (v. 7.3) was used for clustering and correlation. Expander software (v. 5.07) [13] was used for the hierarchical clustering of transcripts overexpressed in each stage separately and cell cycle associated transcripts. Briefly, the fold changes of the expression values compared to the ESC stage were imported into the software and standardized with a mean of 0 and a variance of 1. Then, using the average linkage method, transcripts were clustered, and the expression matrix was visualized with a dendrogram. The STRING database (version 8.1) [14] was used to construct a regulatory network of differentially expressed transcripts. Then, a regulatory sub-graph was extracted from this network by PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22856