correlation of the expression patterns between stages was between the hESC and NI stages and between the NI and MN stages (rNI/ MN = 0.68, rhESC/MN = 0.7) (Figure S1). The 5955 differentially expressed transcripts were categorized into five expression groups (Fig. 5), including (A) 2589 transcripts that were up-regulated in DNs compared to other stages, (B) 747 transcripts that were up-regulated in NEs, (C) 346 transcripts that were up-regulated in hESCs and NIs compared with NEs and DNs (D) 95 transcripts that were up-regulated in NIs compared to other stages, and (E) 2520 transcripts that were down-regulated in differentiated neurons (Table S2). We performed a GO analysis of differentially expressed genes in different clusters using BINGO (v. 2.3) software; the hypergeo- metric test and the Benjamini and Hochberg false discovery rate (FDR) were used for statistical tests and multiple testing corrections, respectively. According to the expression patterns in the preliminary results, transcripts were categorized in two main clusters, first cluster contains transcripts that were up regulated in neural differentiation and second cluster contains undifferentiated associated transcripts, transcripts involved in neurogenesis, axonogenesis and gliogenesis were over represented in first class while cell cycle, DNA replication and repair, Mitosis and cell proliferation processes were over represented in undifferentiated cluster (Table S3). Genes that were up-regulated in DNs (cluster A, Fig. 5, 6A) were mainly involved in neurogenesis, axogenesis, neuron Gene Expression Analysis of hESC Differentiation migration, axon guidance, neurotransmitter secretion and synaptic transmission. These include the up-regulation of various genes related to differentiated neurons and glia, such as MAPT (microtubule associated protein tau) and other tubulin associated genes, synapse formation (SNAP25, SNAPB), septins, stathmins, intermediate filaments and axon guidance (ROBO-SLIT) mole- cules (Table S2). We also observed up-regulation of axon guidance cues and neurotrophins, including ROBO2 and ROBO3 and their receptors SLIT2 and SLIT3, Eph receptors (EFNB3, EPHA3, EPHA5, EPHA8, EPHB1, EPHB3) and their ligands, and semaphorin receptors (SEMA3E, SEMA4F, SEMA5B, SEMA6C) (Table S2). The protachykinin gene (TAC1), tyrosine hydroxylase (TH) and the somatostatin gene (SST) were also among the highly expressed transcripts in differentiated neural cells. As the expression data show, peptide-releasing neurons (tachykinin, somatostatin, neuro- tensin peptides) coexist in culture with dopaminergic (DA) and gamma aminobutyric acid producing (GABAnergic)neurons, although their development may be different but this coexistence might be the result of neurotransmitter colocalization. There are many reports of colocalization of neurotransmitters in one neuron, especially neuropeptides, neurotensin and dopamine (DA) in dopaminergic neurons and also for somatostatin and GABA in GABAergic neurons [19,20,21]. The genes that were up-regulated in NEs (cluster B) were mainly involved in appendage development, epithelium develop- Figure 2. Inward currents recording in the differentiated neurons on two and four weeks after plating at DN stage. The electrophysiological protocol used for eliciting currents was as follows: voltage steps (25 ms duration) from a holding potential of -70 mV to a range of test potentials between -90 and +100 mV (10 mV increments) (A). Representative current tracing recorded from differentiated neurons before and after blocked by Nifidipine demonstrating the presence of Ca++ currents in these cells (B and C). I-V relationships for inward currents are shown (D). Action potential- like recordings in the differentiated neurons (E). b: P,0.01, c: P,0.001. doi:10.1371/journal.pone.0022856.g002 PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e22856