Gene Expression Analysis of hESC Differentiation Figure 8. The gene regulatory network of 44 upregulated transcripts in neural rosette cells compared to hESCs. The STRING database was used to construct a regulatory network of differentially expressed transcripts. Then, a regulatory sub-graph was extracted from this network by selecting edges with inhibitory or activatory regulatory interactions. The visulation of networks was performed using Cytoscape. Green arrows indicate an activation mode, red arrows indicate an inhibitory mode, purple edges indicate an expression mode, and blue edges represent post- translational modification of source genes or target genes. doi:10.1371/journal.pone.0022856.g008 which works cooperatively with LEFTY2 to induce the transcrip- tion of ID2 and ID3. MSX1 is controlled by LMX1A and, in a positive feedback loop, can promote WNT1 expression. Recipro- cally, WNT1 can positively activate LMX1A expression, leading to dopaminergic progenitor proliferation [61]. Temporal gene expression analysis of 55 cell cycle signaling molecules with significant modulation during differentiation revealed that CDK2, CDK4, CDK6 and molecules involved in the G2-S phase transition, including CDC2, CDC25C and MAD2L1, have higher expression levels in NEs compared to hESCs. TFDP1, another transcription factor that binds to EF1 and controls the transcrip- tion of EF1 target genes [62], is also upregulated in NEs (Figure S4). In summary, transcriptional profiling during differentiation of hESCs to neural cells reveals systematic changes in the expression levels of transcription factors that control fate decisions, paracrine factors that coordinate the differentiation process, cell metabolism, cytoskeleton and genes in neurotransmitter secretion pathways. Our results clarify the gene expression changes that occur during differentiation of neural cells. Future studies will uncover functional changes in different neuronal subtypes and glia. Supporting Information Figure S1 The expression levels of approximately 6000 transcripts are depicted in pairwise plots and correlations among samples, calculated separately for each sample pair (A: hESC, B: NI, C: NE, D: DN). The distance of points from the regression line indicates the diversity of expression between two samples. The X and Y axes represent the fold changes of transcript levels. The number of genes up or downregulated by more than 2-fold in compared samples is shown on the other side of the matrix. (TIF) Figure S2 Western blot analysis of 25 ug of total protein extracts from Royan H6 cells. Equal amounts of protein from total cell lysates of human ESCs and neural differentiated cells were subjected to SDS-PAGE followed by western blotting and visualization with an ECL detection kit. Bands were visualized with hyperfilm ECL (GE Healthcare, USA) and scanned with a GS800 densitometer (Bio-Rad, USA). ESCs, neural rosette and differentiated neural samples (three replicates) were analyzed with antibodies against Nestin and tyrosine hydroxylase (TH) proteins. In panel B, western blot and qRT-PCR results for Nestin and TH were quantified to compare the mRNA and protein levels at the NE and DN stages. (TIF) Figure S3 A regulatory network of 96 nodes, modulated by a 1.5-fold or greater up or downregulation in rosette cells compared to hESCs. The downregulated nodes are shown in green, and the upregulated nodes are shown with red circles. Green arrows indicate an activation mode, red arrows indicate an inhibitory mode, purple edges indicate an expression mode, and blue edges represent post- translational modification of source genes or target genes. (TIF) Figure S4 Cyclin dependent kinase and other transcripts associated with cell cycle progression and control, according to BINGO results, were clustered with Expander. The resulting image shows that the genes involved in cell cycle control are differentially expressed in embryonic stem cells and the neural progenitor state. Some of the transcripts are overexpressed in differentiated neurons and inhibit transition from the G1 phase (e.g., CDKN1B, CDKN2D). (TIF) List of primers used for Real-Time PCR analysis. Table S1 (DOC) PLoS ONE | www.plosone.org 11 July 2011 | Volume 6 | Issue 7 | e22856