Gene Expression Analysis of hESC Differentiation DNs; (D) 98 genes that were upregulated in Nis compared to the other stages; and (E) 2234 genes that were mainly down-regulated in differentiated neurons (Table S2). doi:10.1371/journal.pone.0022856.g005 ment and pattern specification in the neural tube (Fig. 5, 6B). Differentially expressed genes in this group may be involved in basic molecular changes that underlie the conversion of progenitor cells to neural cells, including cell fate decisions and triggers for differentiation. The expression of several progenitor marker genes, including LMX1a, MSX1, ALDH1A1 (dopaminergic progenitors), SOX10 (oligodendroglial precursors) and PCP4 (Purkinje neuron protein), as well as genes involved in tight junctions among epithelial cells (AMOT), and proliferation of neural progenitors, such as WNT1 and WNT3a, were higher in NEs. Transcripts of genes involved in neural tube development and patterning, such as ALDH1A2, FOXA2, VANGL2, ZNF358, are also among the genes that were up-regulated in NEs (Fig. 6B). Several genes that were overrepresented in both hESCs and NIs were grouped together in cluster C (Fig. 5). These include genes involved in the negative regulation of cell migration and endothelial proliferation, the regulation of cell adhesion, histone methylation in the H3K9 position and the regulation of FGF and BMP signaling (Fig. 6C). Also represented in this group were genes for several adhesion molecules, including COL1A1 and COL6A3, neuroplastin, neuroligin and thrombospondins. These genes are involved in cell-ECM interactions, cell migration and axon guidance, which are crucial for hESC derived neural cells maintenance. The expression pattern of these genes revealed that the extracellular environment of hESCs might be more intimately related to the developmental lineage than to the biological properties of the neural plate. Neuroplastin (NPTN) is a glycoprotein that belongs to the immunoglobulin superfamily of cell adhesion molecules (CAMs). This gene is also involved in the long-term potentiation of hippocampal excitatory synapses through the activation of p38MAPK [22]. Recently, it was demonstrated that neuroplastin binds to and activates fibroblast growth factor receptor 1 (FGFR1) [23], and it may have a function in FGF signaling in hESCs. Neuroligin (NLGN4X) is a putative neuronal cell surface protein involved in cell-cell-interactions and may be involved in the formation and remodeling of central nervous system synapses [24]. It may also play a role in cell-cell interactions in hESC colonies. Thrombospondin family members (THBS4 and THBS2), which are down-regulated at the NE and DN stages, are adhesive glycoproteins that are involved in cell-cell and cell-ECM interactions [25]. THBS4 forms a pentamer and can bind to heparin and calcium, suggesting that this protein might be involved in local signaling in the developing and adult nervous system; its effect on the proliferation of endothelial cells is clear [25]. Another enriched protein in cluster C was JARD2, which modulates histone methyltransferase activity and promotes the recruitment of histone methyltransferase complexes [26] to their target genes. It also has a function in the neural tube fusion process [27]. The JARD2 protein binds DNA and mediates the recruitment of the PRC2 complex to target genes in ESCs. In ESCs, JARD2 associates with the PRC2 complex and inhibits trimethylation of Lys-27 of histone H3 (H3K27me3) by the PRC2 complex, playing a key role in the differentiation of ESCs and normal embryonic development [26]. Genes that are up-regulated in NIs (cluster D) were enriched in organogenesis, cell fate commitment, cell-cell signaling and developmental induction (Fig. 5, 6D). These include several well- Figure 6. Expression patterns of representative genes from different expression clusters were presented in Figure 3. (A) transcripts that are highly up-regulated in DNs compared with the other stages; (B) transcripts that are enriched in NEs; (C) transcripts that are mainly up- regulated in hESCs and NIs; (D) genes that are enriched in NIs; and (E) highly abundant genes in hESCs. doi:10.1371/journal.pone.0022856.g006 PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e22856