selecting edges with inhibitory or activatory regulatory interac- tions. The visualization of networks was performed using Cytoscape (version 2.6.3) [15]. We used BiNGO (a Cytoscape plugin) [16] to find statistically over- or underrepresented Gene Onthology (GO) categories in the biological data as a tool to enrich the analysis of the transcriptome dataset. Enrichment was determined in reference to all human Entrez GeneIDs that were annotated in the Biological Process branch (14,394 genes total). P-values were derived from a hypergeometric test followed by the Benjamini and Hochberg false discovery rate [17]. A P-value cutoff of 0.01 was used to identify significantly enriched categories. Pathway analyses were assigned with the ClueGO (v. 1.2) plugin to all of the genes using the KEGG database. A two sided hypergeometric test was used as statistical test for the probability of each gene falling into a pathway. Real-Time PCR Reverse transcription of the isolated RNA was carried out using the MMLV reverse transcriptase (USB) and oligo-dT primer according to the manufacturer’s instructions. Real-time PCR was carried out on an Applied Biosystems 7900 instrument in 25 ml reactions containing 12.5 ml of SYBR Green PCR mix (Applied Biosystems, Foster City, CA, United States) and each primer at 0.375 mM. All primers used for the assays were tested for specificity and amplification efficiency. The sequences of the primers used are listed in Table S1. Relative mRNA levels were calculated using the comparative CT method, as described by the manufacturer (Applied Biosystems, Foster City, CA, United States), with GAPDH as an internal control for normalization. Western blot analysis For each sample, 25 mg of protein was separated by 12% SDS- PAGE (120 V for 1 h) using a Mini-PROTEAN 3 electrophoresis cell (Bio-Rad), and the proteins were transferred to a PVDF membrane (Amersham) using a semi-dry blotting method (Bio- Rad) and Dunn carbonate transfer buffer (10 mM NaCHO3, 3 mM Na2CO3, 20% methanol). Membranes were blocked for 1.5 h using Western Blocker solution (Sigma, W0138) and then incubated overnight at 4uC with the respective primary monoclo- nal antibodies, mouse anti-TH (Sigma, 1:10,000), rabbit anti- OTX2 (Abcam, 1 ug/ml) or mouse anti-Nestin (Chemicon, 1:1,000). Membranes were then incubated with peroxidase- conjugated secondary antibodies, anti-mouse IgG (1:5,000, Sigma, A9044) or anti-rabbit IgG (1:10,000, Sigma, A2074), for 2 hr at room temperature. Finally, the blots were visualized using ECL detection reagents (Sigma, CPS-1-120). Subsequently, the films were scanned with a densitometer (GS-800, Bio-Rad). To insure that a uniform amount of protein was loaded onto the gels, the membranes were stained for total protein with Fast Green (FCF, Sigma-Aldrich, F7252). Results and Discussion Differentiation of hESCs into neural cells The morphology of feeder-free adhesive hESCs was compact cells with a high nucleus:cytoplasm ratio, resulting in cells with clear borders (Fig. 1A); they had a typical morphology and the key pluripotency markers OCT4 and NANOG (Fig. 1B, 1C). The hESCs were differentiated into NEs by Noggin and RA-mediated induction [8], which led to the appearance of rosettes with columnar cells (Fig. 1D) and NT formation. The differentiated cells, 9 days after induction (NE stage), expressed Nestin (Fig. 1E) (83.764.9%), Sox1 (Fig. 1F) (46.463.3) and Pax6 (Fig. 1G) Gene Expression Analysis of hESC Differentiation (50.669.5%) (flowcytometry data Fig. 1H), which are neuroecto- dermal markers expressed during neural plate and NT formation [18]. We isolated hESC-generated NTs with a pipette (Fig. 1I). After dissociation, the cells were replated onto laminin/poly-L- ornithine-coated plates. Neural cell maturation was promoted by bFGF withdrawal and the addition of ascorbic acid, BDNF, GDNF, and cAMP (Fig. 1J). Within a few days, numerous processes grew, and neuronal migration began (Fig. 1K). The differentiated cells expressed neural markers (MAP2, GFAP, serotonin, and TH) that were detected by immunofluorescence staining (Fig. 1L-O). Quantification of the markers was performed as described previously [8]. To determine the functionality of the hESC-derived neurons we studied their electrophysiological properties (Fig. 2). In voltage clamp mode we evaluated voltage- activated ionic currents after plating. Voltage steps (25 ms duration) from a holding potential of 270 mV to a range of test potentials between 290 and +100 mV (10 mV increments) demonstrated inward currents (Fig. 2A). Inward currents that inactivated rapidly and were sensitive to Nifedipine, an L type Ca++ blocker (5 mM, student t test, P,0.001, n = 30) (Fig. 2B, 2C) Current-voltage relationships showed that inward currents activated near 240 mV and peaked at 210 mV in matured cell two weeks after platting. (Fig. 2D) (n = 30). The excitability of hESCs-derived neurons was assayed by whole cell patch clamping in the current clamp mode. Action potentials like responces were recorded after plating in 60% of matured cells (Fig. 2E). cDNA microarray analysis of hESC-derived neural cells Three independent biological replicates of undifferentiated hESCs and cells from the NI, NE and DN stages were analyzed using whole genome microarray technology. As a first level of quality control, the transcriptional profiles of all replicate samples were assessed for biological reproducibility (Figure S1). All triplicate mRNA samples clustered together. Real time PCR was carried out to confirm the expression levels of genes selected from four different groups: (i) pluripotency markers, such as OCT4, SOX2, ALP, and NANOG (ii) genes that were upregulated during neural induction (e.g., Nestin, SOX1, PAX6), (iii) neural rosette cells (e.g., FOXA2), and (iv) genes that were upregulated in differentiated neurons (e.g., HOXA5, HOXB5, HOXA2, and TH). In 13 of 14 reactions, microarray-derived differential expression was confirmed at a confidence level of 92%. For one gene, ACTB, the background mRNA expression at maturation, detected by microarray, could not be confirmed by real time PCR (Fig. 3). For confirmation of the Nestin as a neural progenitor marker and Tyrosine hydroxylase (TH) as a transcript that was highly expressed in the differentiated neurons, protein expression level of Nestin and TH were analyzed by western blotting and results revealed that differences in mRNA levels actually reflect differences in protein expression in both cases (Figure S2). Distinct classes of transcripts are differentially expressed during neural differentiation An analysis of transcriptome dynamics during differentiation revealed that 5955 transcripts were modulated during differenti- ation in at least one stage compared with hESCs (Table S2). As expected, the numbers of modulated genes increased during the differentiation of hESCs to MNs. While 505 and 1785 transcripts showed differential expression patterns in NIs and NEs, respec- tively, compared with hESCs, 5134 transcripts were modulated in MNs compared with hESCs (Fig. 4). While most (73%) of the modulated genes in NIs were up-regulated, only 48% (2,505) of regulated genes in the MNs were up-regulated. The minimum PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22856