the basis of the sensitivity could be attributed to a limited set of genes and those genes could be identified for future use. for example bg01v, while much more similar to bg01 than to any other cell type, could still be disting uished from a biological replicate of bg01 by the expression of a particular subset of differentiation markers ( figure 6 ). ec cells such as ntera2 could be disting uished from hescs by the expression of germ cell markers and the presence of a partially inactivated tgfβ ( bmp ) signaling pathway ( figure 6 ). disting uishing escs from ebs was relatively straightforward. we have confirmed the utility of previously identified markers for use in this platform as well as identified an additional set of markers that can serve as biomarkers to disting uish between the hesc and eb states. a subset of these markers have been used to develop a qpcr assay that shows such a high sensitivity that changes in cell behavior can be detected after as little as twenty - four hours and the development of ebs can be reliably staged [ 10, 20 ]. during the identification of es and eb specific markers, we have noticed that some known hesc markers, such as nanog, was not detected in all populations of hescs that were included in this analysis. several esc - specific gene, including lin41, sox2 and foxd3, were not detected in the array either ( table 3 ). we believe that the problem with lin 41, sox2 and foxd3 is a technical one as we were able to confirm expression using alternate methods. we are in progress of redesigning appropriate probes for these genes. in the case of the gene nanog, there are several pseudo genes in the genome for nanog and it has been a major technical challenge designing primers or probes that are specific and sensitive. we believe that a partial explanation for the variability in nanog expression is due to the lack of sensitivity to this gene. however, immunocytochemistry while not strictly quantitative shows similar variability when used to assess nanog expression in different cell lines [ 9, 27, 28 ]. this large comparison between samples allowed us to identify markers that disting uish human feeder cells from hesc. while we have listed 19 potential markers ( figure 3 ) and identified several hundred potential markers as shown in additional file 5, we suggest that as few as 3 – 4 genes may be sufficient. previously we found that as few as four were satisfactory to disting uish between hescs and hebs, which are two much more closely related samples [ 9 ]. in this study we have confirmed by qpcr the differential expression of four genes, thbs1, mmp3, tnfrsf11b and krth4, to separate human fibroblast feeders and hescs ( figure 3 ). several markers such as mmp3 and tnfrsf11b have commercially available antibod ies ( r & d systems ) that may be used to further confirm contamination of feeder cells by immunocytochemistry. efforts to identify other useful antibod ies based on these results continue [ 29 ]. while we have focused on the immediate utility of the illumina array platform, it is important to remember that this array provides a global snapshot of cell state and the data obtained can be readily compared in order to determine key signaling pathways. the ability to compare multiple samples in one run enhances data selectivity and reliability. to make such analysis more readily available, we utilized several software tools including the software package available through illumina. the beadstudio software provided with the beadlab and beadstudio genetic analysis systems for use with the bead array datasets provides a useful set of analytical and presentation tools that allow straightforward comparisons, which are sufficient for average users. for detailed analysis we recommend using more specific commercial tools or software packages developed by ncbi. in summary, the illumina bead array has several key strengths including high throughput, low cost and high sensitivity. by using this array, we can readily detect contaminating feeders and spontaneous differentiation, differentiate male and female lines and disting uish between one undifferentiated population and another. such a global analysis allows us to assess context dependent signaling and identify biomarkers of particular states of cells. our future efforts will focus on data mining and developing better cross platform comparison tools and generating focused high throughput arrays for quality control in clinical and research settings. hesc culturethe hesc lines h1, h7 and h9 ( wicell, madison, wi ) were cultured on feeder layers derived from mitotically inactivated hs27 human fibroblast cells ( hs27, atcc ), or mouse embryonic fibroblsts or under feeder - free conditions on matrigel ( bd, franklin lakes, nj ) coated plates for at least 10 passages. culture medium for all cultures was compose d of dmem / f12 - glutamax 1: 1, 20 % knockout serum replacement, 2 mm nonessential amino acids, 100 μm beta - mercaptoethanol, 50 μg / ml pen - strep ( all from invitrogen, carlsbad, ca ), and 4 ng / ml human recombinant basic fibroblast growth factor ( bfgf / fgf2; peprotech inc., rocky hill, nj. ) feeder - free cultures were prepared for gene expression analysis by manually harvesting individual colonies with uniform typical undifferentiated esc morphology. bg01 ( 46, xy ), bg02 ( 46, xy ), bg03 ( 46, xx ), i6 ( 46, xy ) and bg01v ( bg01 karyotypic variant: 49, xxy, + 12, + 17 ): cells were maintained for 3 ( bg01v ), 7 ( bg02 ), 8 ( bg01 ), or 21 ( bg03 ) passages under feeder - free condition on fibronectin - coated plates in medium that had been conditioned by mouse embryonic fibroblasts for 24 hours. culture medium was dmem / f12, 1: 1 supplemented with 20 % knockout serum replacement, 2 mm non - essential amino acids, 2 mm l-glutamine, 50 μg / ml pen - strep, 100 μm beta - mercaptoethanol, and 4 ng / ml of bfgf. different hesc lines were grown in slightly different culture conditions as described above. h lines were grown on matrigel coated dishes, while bg lines on fibronectin treated dishes. these coating substrata supported the growth of hescs similarly, as evaluated by colony morphology, immunocytochemistry and proliferation rate ( data not shown ). embryoid bodies ( ebs ) were prepared from bg lines as described in [ 5 ]. cells were aggregated and cultured on non - adherent substrata for fourteen days. other cellsntera2 cells were purchased from atcc and cultured in parallel with hescssamples using protocols described previously [ 21 ]. hs27 embryonic human newborn foreskin cells ( atcc crl - 1634 ) were grown in dmem with 10 % fbs. all samples included in this study can be found in additional file 6. bead array gene expression analysisrna was isolated from cultured cells using the qiagen rneasy kit ( qiagen, inc, valencia, ca ). sample amplification was performed using 100 ng of total rna as input material by the method of van gelder et al [ 30 ]. amplified rna synthesized from limited quantities of heterogenous cdna [ 30 ] was performed using the illumina rna amplification kit ( ambion, inc., austin, tx ) following the manufacturer instructions. labeling was achieved by use of the incorporation of biotin - 16 - utp ( perkin elmer life and analytical sciences, boston, ma ) present at a ratio of 1: 1 with unlabeled utp. labeled, amplified material ( 700 ng per array ) was hybri diz ed to a pilot version of the illumina humanref - 8 beadchip according to the manufacturer ' s instructions ( illumina, inc., san diego, ca ). amersham fluorolink streptavidin - cy3 ( ge healthcare bio - sciences, little chalfont, uk ) following the beadchip manual. arrays were scanned with an illumina bead array reader confocal scanner according to the manufacturer ' s instructions. array data processing and analysis was performed using illumina beadstudio software.