each row represents the relative levels of expression of a single gene. each column represents a sample. the samples include four groups of cells, es designates 11 samples of hescs, eb contains 6 samples of differentiated escs and ebs, ns consists of 3 hesc derived neural cells and fb is a collection of hesc derived mesenchyme and fibroblasts. high expressions relative to mean are colored red. low expressions are colored green. black represents no significant change in expression level between mean and sample. samples cluster closer within their own group than samples from other groups. figure 2dendrogram of unsupervised one - way hierarchical clustering analysis of relative expression of genes in selected samples. the clustering analysis was based on the average linkage and euclidean distances as the similarity metric using differentially expressed genes identified by anova ( p < 0. 05 ). hescs clustered together and bg lines cultured in the same laboratory shared the largest similarities. ebs were separated from hescs from which they were derived. ec line ntera2 and feeder cells can be disting uished from hescs respectively. the global analysis suggested that the bead arrays used were sufficiently sensitive such that individual subsets of genes could be analyzed, different populations of cells could be readily disting uished and that a subset of candidate genes could be sufficient to disting uish between groups of cells. the comparison across multiple samples will allow a set of core stem cell markers to be identified. in subsequent sections we have performed such analysis. readers are urged to analyze the expression of desired genes directly as it is impossible to test every gene given the large body of data generated. comparison between mpss and illumina bead array resultswe have previously used est scan and mpss to analyze pooled samples of escs and ebs from three different wicell lines ( h1, h7 and h9 ) [ 5 ]. comparison between the two methodologies indicated that while there is good concordance for genes expressed at high levels, this does not hold for genes expressed at lower levels. as a test of the quality of the data generated in these experiments and to evaluate whether comparisons can be made across different methodologies, we re - ran the identical samples on the bead array platform. the complete comparison of gene expression is shown in additional file 2 and is summarized in tables 3 and table 4. overall, concordance in illumina array was better than that evident between est scan and mpss datasets [ 9 ], but clearly showed much wider differences than that seen with running duplicates in the same assay format. nevertheless, this comparison provides an independent verification of the data and suggests that if a sample is detected in more than one large - scale analysis, the reliability of the gene expression detection is high, which also reduces the number of individual genes needed to be verified. caution should be observed in comparing different samples run on different platforms, especially when there has not been rigorous bioinformatic matching of the source sequences used to identify genes in the platforms. often genes called by the same symbol originate from different database records, which may originate from different splice variants or contain sequence differences due to polymorphisms or outright error [ 19 ]. table 3expression of hesc specific markers in pooled hesc sample as detected by illumina bead array the expression of previously identified hesc markers was examined in all hesc samples ( the values displayed represent the expression level of pooled h1, h7 and h9 ). most of the genes were also identified using illumina bead array in all 8 hesc populations in this study ( 1 * ), the gene cer1 was detected in all except one duplicate of h9 ( 2 * ), nanog was not detected in all populations ( 3 * ) and sox2, lin41, nr6a1 and foxd3 were not detected in the array although they were present in the chips for hybri diz ation ( 4 * ). accessionsymbolpooled es ( h1, 7, 9 ) commentsnm _ 003641. 1ifitm188441 * nm _ 020997. 2leftb6579. 61 * nm _ 024674. 3lin283944. 11 * nm _ 175849. 1dnmt3b33911 * nm _ 003212. 1tdgf131691 * nm _ 001769. 2cd92930. 81 * nm _ 000165. 2gja12404. 41 * nm _ 021195. 2cldn62247. 21 * nm _ 004360. 2cdh11972. 91 * nm _ 021127. 1pmaip11601. 51 * nm _ 032805. 1znf2061504. 51 * nm _ 003577. 1utf11444. 11 * xm _ 050625. 2sfrp21353. 91 * nm _ 006548. 3imp - 212061 * nm _ 152312. 2gyltl1b1066. 61 * nm _ 015973. 2gal1043. 31 * nm _ 003240. 2ebaf944. 11 * nm _ 054023. 2scgb3a2890. 51 * nm _ 020990. 2ckmt1742. 41 * nm _ 033668. 1itgb1694. 71 * nm _ 003744. 3numb618. 31 * nm _ 007015. 1lect1597. 41 * nm _ 021912. 2gabrb3482. 71 * nm _ 006729. 2diaph2467. 11 * nm _ 000222. 1kit188. 91 * nm _ 005454. 1cer1151. 52 * nm _ 024865. 1nanog56. 93 * nm _ 002701. 1pou5f1694. 41 * nm _ 003106sox2nd4 * nm _ 006458. 2lin41nd4 * nm _ 001489. 3nr6a1nd4 * nm _ 012183foxd3nd4 * table 4comparison of mpss and illumina bead array results the samples were analyzed by mpss and bead array. the number of genes detected by each method and the degree of overlap is summarized. note much higher degree of overlap when the top 2000 hits were compared. *: most of the genes detected by mpss were novel genes not included in the bead array. esno. % common in both ( top 2000 hits ) 1, 62281. 1common in both ( all hits ) 5, 07146. 0by bead array only3, 46231. 4by mpss only * 2, 50422. 7total11, 037100ebno. % common in both ( all hits ) 5, 16843. 1by bead array only4, 13134. 4by mpss only * 2, 69422. 5total11, 993100 human feeders and hescs can be readily disting uished and contamination can be readily assessedfor all samples, we conducted an unsupervised one - way hierarchical clustering analysis. the clustering analysis was based on the average linkage and euclidean distances as the similarity metric using differentially expressed genes identified by anova ( p < 0. 05 ). the analysis revealed the underlying features and variation patterns of gene expression in each cell types. figure 2 shows results of the cluster analysis of relative gene expression in selected samples. as one of our purposes of this study was to disting uish between human fibroblast feeders cells and hescs and hebs, wishing to readily detect feeder contamination in hescs, we included one of the human feeder cells hs27 ( atcc ) in this study. we have been using hs27 as feeder cells for h9 hescs for more than two years and all hescs grown on hs27 had normal karyotype, expressed all undifferentiated markers, and made teratomas with all germ layers ( data not show ). the global pairwise comparison clearly showed that human feeders were far more dissimilar to hescs than hescs grown in different laboratories, hescs compared to their differentiated ebs that contained mesodermal tissue, and hescs compared to the karyotypically variant hesc line bg01v. pairwise comparisons of human feeders with hescs resulted in a correlation coefficient of 0. 66, which was less than the correlation coefficient of 0. 71 – 0. 74 observed between hescs and their corresponding ebs. the large difference between human feeders and hescs suggested that it would be possible to identify markers that were robust and reliable in disting uishing the two populations, and these markers would be sufficiently sensitive in detecting contamination of feeders. we examined the data to develop a list of genes that had high levels of expression in human feeder cells maintained in hesc medium but whose expression was low or absent in either escs or ebs. the absence of expression in ebs was used as a control for spontaneous differentiation of esc colonies ( including mesodermal differentiation ) which may occur and the markers selected should be able to disting uish between these two events. a complete list of genes expressed at least ten - fold higher in human feeders is provided in figure 3.