since differentiated cells are often scattered within or at the edge of colonies [ 11 ] and the differentiation is so subtle that morphological characteristics and even immunoh istochemistry are insufficient to detect it, larger scale methods of analysis need to be developed. our strategy was to compare a variety of different hesc lines that were derived and expanded by three different institutions ( wicell research institute, bresagen, inc., and technion - israel institute of technology ), and cultured in two separate laboratories ( burnham institute and nia ) to a baseline set of data against which cell samples can be compared. by using cells grown in different conditions we expected to be able to identify core commonalities and by comparing feeders and embryoid bodies ( eb ) with hesc identify measures of contamination and early markers of differentiation. further, by comparing embryonal carcinoma cell ( ec ) and karyotypically variant lines with hesc, we would be able to directly assess their utility as surrogates ( for quality control purposes ) for hesc. we employed a pre - commercial prototype of the illumina humanref - 8 beadchip [ 12 ], a genome - scale bead based array technology that combines the sensitivity and low cost of a focused array with the coverage of a large scale array, while requiring much smaller sample sizes than mpss, est scan or sage. we show that the illumina bead based array correctly identified blinded duplicates as the closest related samples and readily disting uished between hesc lines, as well as between escs and ebs derived from them. this array allowed us to estimate the degree of feeder contamination present in the cultures. similarities and differences between ec line ntera2 and hesc lines could be determined and verified, and the database comparisons allowed us to identify core self - renewal pathways that regulate hesc propagation. multiple hesc lines can be assessed by illumina bead arrayforty - eight samples were selected from multiple laboratories and gene expression profiles were examined using a bead array containing 24, 131 transcripts derived from the human refseq database that included full length and splice variants. each gene was represented by sequences containing an average of thirty beads to provide an internal measure of reliability. samples included 7 hesc lines bg01, bg02, bg03, i6, h1, h7 and h9, ebs that were differentiated from hescs of the three bg lines, human fibroblast feeder hs27 ( atcc ), hesc - derived fibroblasts, karyotypically abnormal hesc line bg01 variant ( bg01v ) [ 13 ] and ec line ntera2 [ 14 ]. samples were blinded and biological and technical repeats were examined at the same time. a single slide contained eight replicates and six such slides were used for the present set of samples. results were normalized to average following illumina beadstudio manual and the quality of each sample was assessed by immunocytochemitsry and rt - pcr prior to subjecting them for analysis ( data not shown ). results from the entire sample set are available for download as an excel spreadsheet ( additional file 1 ) and a cd of the results is available upon request. the total number of genes identified as expressed at > 0. 99 confidence is summarized in table 1. intensity results are reported in arbitrary units and ranged from 10 to 20, 000 ( a two thousand fold range ). although the sensitivity of the array has been reported to be high, in the present report we have restricted our analysis to expression of at least 100 units in any one sample. using this cutoff, on average cells expressed approximately 8, 000 transcripts ( table 1, 2 ), a number similar to the number detected by sage, mpss and est analysis [ 5 - 7, 10, 15, 16 ]. as with other analysis, genes with the highest abundance were housekeeping genes, ribosomal genes and structural genes ( table 2 and additional file 1 ). these genes were similar in most samples though relative levels varied. table 1correlation coefficients of paired samples in this bead array in order to test the reproducibility and reliability of the bead array, duplicate samples of hesc lines h9, i6, and ec line ntera2 and human fibroblast feeders ( hs27 ) were run at the same time and correlation coefficients ( r2 ) of duplicates were generated using the entire data of all genes with expression level > 0 ( § ), or genes with detection confidence > 0. 99 ( * ), or genes with detection confidence > 0. 99 and expression level > 100 arbitrary units ( # ). note that the correlation coefficients are in the range of 0. 9382 – 0. 9761 and the number of genes was in the range of 10, 000 – 14, 000. duplicate samplesno. of all genes ( expr. > 0 ) § r2of all genes ( expr. of genes ( > 0. 99 ) * r2 of genes ( > 0. 99 ) * no. ( > 0. 99, level > 100 ) # h918, 8990. 868113, 6720. 97087, 408i619, 1390. 866312, 5700. 97616, 826ntera219, 1620. 874114, 0360. 93827, 147feeder18, 1570. 872410, 6060. 97517, 021table 2distribution of genes with expression levels < 50 and > 50 – 10, 000 as detected by illumina bead array in 8 hesc populations all human esc samples were hybri diz ed in one experiment and the relative detection levels of genes were binned to obtain a global overview of transcription, approximately 8, 000 genes ( ~ 50 % ) were greater than 100 arbitrary units. the numbers are similar to results obtained by other large scale analysis such as mpss. abundance ( relative detection levels ) h9h9 on human feedersi6bg01bg02bg03bg01vpooled ( h1, h7, h9 ) no. % no. % no. % no. % no. % no. % no. % no. % < 502, 90921. 26, 06738. 43, 55925. 34, 52830. 74, 25630. 54, 70632. 24194. 35, 80334. 4 > 5010, 81778. 89, 74761. 610, 48474. 710, 23269. 39, 69469. 59, 91567. 89, 43895. 711, 08565. 6 > 1008, 12659. 27, 53947. 77, 40952. 87, 70352. 27, 49653. 77, 43050. 87, 23073. 38, 21748. 7 > 5003, 07722. 42, 94718. 62, 62618. 73, 06520. 82, 95021. 12, 85219. 52, 85128. 92, 94117. 4 > 10001, 62911. 91, 62510. 31, 49010. 61, 63811. 11, 58911. 41, 51710. 41, 56615. 91, 5549. 2 > 50002481. 82571. 62561. 82561. 72631. 92621. 82752. 82511. 5 > 10000900. 7940. 61120. 8920. 61000. 71070. 71011. 0940. 6total no. of genes detected at > 0. 99 confidence13, 72615, 81414, 04314, 76013, 95014, 6219, 85716, 888one of the advantages of the illumina arrays is the ability of running multiple samples simultaneously thus allowing multiple pairwise comparisons to be performed readily. to show the similarity of relative gene expression between samples, we have used illumina beadstudio and clustering software packages pcluster [ 17 ] and treeview [ 18 ] to generate a heat - map ( figure 1 ) and a dendrogram ( figure 2 ). based on their properties, we classified some of our samples into four groups, ( a ) undifferentiated hescs ( including a sample from karyotypically abnormal variant, designated as " es ", n = 11 ); ( b ) differentiated es cells and ebs ( designated as " eb ", n = 6 ); ( c ) hesc derived neural cells ( designated as " ns ", n = 3 ); and ( d ) hesc derived mesenchyme and human fibroblast feeder cells ( designated as " fb ", n = 5 ) and these groups were shown in the heat - map. comparing the overall pattern of expression, we made several important observations: 1 ) duplicates clustered close to each other and were more related to each other than to any other sample; 2 ) escs appeared more similar to each other than to ebs; 3 ) ntera2 cells appeared more similar to escs while differentiated ntera2 and ebs can be readily disting uished from their parent populations ( figure 2 ); 4 ) bg01v appeared similar to undifferentiated bg01 cells; 5 ) in general esc lines grown in one laboratory appeared more similar than samples grown in other laboratories, suggesting that culture conditions affected gene expression but that this effect was much smaller than the effect of differentiation. figure 1unsupervised two - way hierarchical cluster analysis of differentially expressed genes illustrated in a heat - map.