as seen in table 4 and table 5, the neurostem chip identified numerous genes associated with stem cells. in particular, homeo box expressed in es cells 1 ( hesx1 ) gene was identified as the most up - regulated in the es cell preparation, compared to universal reference rna. highly expressed in pluripotent escs, hesx1 expression is down-regulate d upon embryonic stem cell differentiation [ 35, 36 ], as also clearly seen in differentiation experiment of our own ( table 4 ). similarly, gremlin 1 homolog, cysteine knot superfamily gene ( grem1, also known as cktsf1b1 and dand2 ) is a recognized factor of cell - fate determination of escs [ 37 ]. many more genes highly up - regulated in the hesc sample in comparison with universal reference rna are associated with stem cells: further examples include gap junction protein α1 ( gja1 ) and zic family member 3 heterotaxy 1 ( zic3 ) ( table 4 ) [ 20 ]. the expression of fibroblast growth factor receptor 2 ( fgfr2 ) is of particular interest. basic fibroblast growth factor ( fgf2, bfgf ) supports hesc proliferation and their ability to maintain undifferentiated phenotype when cultured in vitro [ 38, 39 ]. moreover, in some hesc lines a very high concentration of fgf2 could substitute for the need of feeder cells [ 40 ]. at the same time, genes listed in table 4 represent the most highly up - regulated entries in a relatively limited group of genes ( figure 5c ). many other genes involved in maintenance of esc phenotype ( i. e. established or candidate markers of stem cells ) have lower levels of expression ( table 5 ). examples include undifferentiated embryonic cell transcription factor 1 ( utf1 ), dna methyltransferase 3b ( dnmt3b ), developmental pluripotency associated 4 ( dppa4, a newly established pluripotency marker [ 41 ] ) and numerous candidate markers of " stemness ": e. g. genes for kiaa1573 protein, forkhead box o1a ( foxo1a ), high - mobility group box 1 ( hmgb1 ), c - terminal binding protein 2 ( ctbp2 ) and left - right determination factor 1 ( lefty1 ), as well as others. for numerous established or candidate markers of stem cells the expression levels were not considerably higher ( log2 ratio < 1 ) in the hesc sample compared to the universal reference rna. for example, the expression of nanog, dna ( cytosine - 5 - ) - methyltransferase 3α ( dnmt3a ), muts homolog 2, colon cancer, nonpolyposis type 1 ( e. coli ) ( msh2 ), thy - 1 cell surface antigen ( thy1 ), high - mobility group box 2 ( hmgb2 ), transcription factor 3 ( tcf3 ), nanos homolog 1 ( nanos1 ), myod family inhibitor ( mdfi ), calumenin ( calu ) and soluble thymidine kinase 1 ( tk1 ) was detected in hes sa02 cells with log2 ratio value < 1. expression levels of those genes range from being inconsiderably higher to nearly equal to that in universal reference rna sample. we believe that those findings could be explained by cellular composition of human universal reference rna sample [ 42 ], which includes pooled rna samples from proliferating cells ( e. g., skin and testis cell lines ). thus, the relative difference between gene expression of certain markers of stem cells in undifferentiated hescs and universal reference rna is naturally decreased. taken together, the gene expression signature of hes sa02 cell line profiled by neurostem chip is indeed characteristic for pluripotent stem cells, providing proof - of - concept. notably, comparison of expression profiles of undifferentiated hescs and hesc - derived cells committed toward dopaminergic differentiation pathway by co - culturing with sdia for 16 days have revealed that many of the stem cell marker genes mentioned above were down-regulate d in differentiation ( table 5 ). expectedly, hesx1, grem1, dnmt3b, utf1 and nanog could be listed among these. at the same time, numerous other genes, including pitx2, dlk1 and msx1 were up - regulated in the latter sample ( [ see additional file 2 ], figure 3 ). table 1 lists 24 dopaminergic system - related entries ( e. g., ptx3, th, lhx1 ) with gene expression up - regulated by day 16 of hesc differentiation protocol; few more genes have demonstrated less prominent up - regulation ( log2 ratio values in the range of 0. 7 / 0. 97 – 1. 0 ). the gene expression profiles generated are therefore consistent with the results of earlier studies utilizing hsc - derived samples with similar characteristics [ 43, 44 ]. diversity of neurostem chip entries responsive to hesc commitment toward neuronal / dopaminergic differentiation pathway clearly illustrates the complexity of that pathway. the cell population obtained after 16 day exposure to sdia is highly heterogeneous. only around 0. 2 % of the cells are th - positive cells ( figure 2 ). this heterogeneity, with an apparent presence of residual pluripotent cells explains the presence of stem cell marker genes, including homeobox transcription factor nanog, as revealed by rt - pcr data ( figure 3 ). it would be therefore impossible to apply the platform to identify novel genes associated with the process of differentiation; for that application, the genome - scale microarray platforms ( e. g., affymetrix ) are clearly superior. nevertheless, being based upon a moderate assay of pre - selected specific gene targets, the comparative analysis of microarray data derived from undifferentiated and dopaminergic differentiate pathway - committed hescs provides a valuable cross - cut of complex relationship between factors driving or indicative to neuronal / dopaminergic differentiation [ see additional file 2 ]. rt - pcr analyses have validated the overall reliability of neurostem microarray platform: all of the entries detected in the hybri diz ation experiments have demonstrated similar trends when analyzed by rt - pcr means ( figure 3 ). those entries include sox2, en1 and nanog ( ratio of differentiated / undifferentiated hesc sample normalized spot intensity < 0. 75, down-regulate d ), gadph, aldh1a1, sdha, tubb and nestin ( ratio. 1. 0, unchanged ), actb, th, msx1 and pitx2 ( ratio > 1. 25, up - regulated ). some of the housekeeping genes ( gapdh, sdha, tubb, actb ) have somewhat different expression in undifferentiated vs. differentiated cells, consistent with previous reports on certain established housekeeping genes ( including gapdh ) being variable in human samples [ 45 ]. importantly, all the observed gene expression trends were similar in both microarray and rt - pcr. our experiment therefore confirms that the neurostem chip microarray platform can still identify gene expression changes related to early stages of differentiation of hesc into dopaminergic neurons. recent technological advances have led to dna microarrays which contain over hundred thousand of spots of dna material, reaching a truly genomic scale. highly specialized dna microarrays of smaller scale ( e. g. the neurostem chip ) still have an important role in the directed studies in particular fields. since they are significantly less expensive, compared to many recognized large - scale platforms ( e. g. affymetrix human genome platforms ), they have a clear advantage in routine work involving samples from, e. g., multiple cell culture conditions. while there is a risk that one will miss out on changes in genes previously not believed to be relevant to neural differentiation, the restricted number of genes in the neurostem chip also simplifies analysis and adds power. neurostem chip is comparable to other stem cell - related focused microarray platforms in regards to manufacturing costs and technical simplicity of the recommended hybri diz ation protocols. at the same time, it currently implies an advantage in both the scale and the spectrum of pre - selected, specific gene targets assayed.