genes plotted outside the two thin red lines were detected at > 2. 5 - fold difference. our results showed that, while ntera2 shared a high similarity with hescs [ 21 ], it did have important differences with hesc lines. examining these differences ( summarized in figure 6c and 6e ), we noted that some reflected the origin of the tumor cells from which this line was derived [ 14 ]. several germ cell markers such as gage2, gage7 and gage8 were highly expressed in ntera2 but were absent ( or present at low levels ) in any of the hesc lines examined ( see figure 6c and additional file 1. note that the gage genes are highly similar in sequence, making it difficult to disting uish one family member from another through hybri diz ation; thus, while all of these gage gene probes gave positive signal, it is difficult to say if the signal came from the specific gene itself or from cross - hybri diz ation from one of the other family members ). none of these were present in bg01v, indicating that the karyotypically abnormal variant is not the equivalent of a teratocarcinoma line such as ntera2. in addition to the expression of germ cell markers, we noticed a significant difference in the expression of genes in the tgfβ pathway, such as gdf3 ( figure 6c ), tgfbi, cdkn1a, igfbp7, igfbp3, nodal, cer1 and bmp2 ( figure 6e ). this is consistent with the postulated role of this pathway in germ cell differentiation [ 22, 23 ] and suggests that tgfβ pathway cannot be reliably tested using ntera2 as a model for hesc. the bg01v showed clear differences from its normal counterpart and some major changes are summarized in figure 6d and 6f. early markers of differentiation appeared to be present at higher levels in bg01v as compared to any of the hesc lines examined, although hesc specific genes continued to be expressed at high levels ( see additional file 4 ). in particular, the wnt pathway and the tgfβ signaling pathway ( figure 6d ), both of which involved in the early process of differentiation [ 24, 25 ], appeared to be activated ( additional file 4 ), suggesting that the role of growth factors and signaling in these early events cannot be readily studied in this cell line. in summary, the analysis highlighted the utility of the potential reference standards ntera2 and bg01v, demonstrated their general similarity and provided detail on potential caveats to their application. global arrays provide a snapshot of the state of the cells and identify core self - renewal pathwayswe have utilized a small fraction of the data to demonstrate the overall utility of this approach and its sensitivity in identifying small differences in cell populations. an additional potential application of such an analysis is the ability to examine the general state of a particular signaling pathway and determine whether it is active. by comparing across many samples, a procedure previously expensive and difficult in terms of the rna and replicate requirement, one can rapidly identify key regulatory pathways. to test whether we could use such multiple pairwise comparisons to elucidate the major regulatory pathways that may be required for hesc self - renewal, we examined several metabolic pathways. the results of the analysis of the insulin / insulin - like growth factor ( igf ) signaling pathway are shown in figure 7. using the same 4 groups of samples as in figure 1, we conducted pam ( prediction analysis of microarray ) [ 26 ], in search for biomarkers used in diagnost ic identification of these four groups, es, eb, ns, and fb. in pam, a list of significant igf pathway genes whose expression characterizes each diagnost ic class was obtained. the average gene expression level in each class was divided by the within - class standard deviation. the nearest centroid classification computed took the gene expression profile from a new sample and compared it to each of these class centroids. for cross - validation of prediction results, multiple classification processes were performed on two data sets randomly constructed each time from the entire gene expression dataset. the first dataset, consisting of 70 % of the total data, was used as the training dataset, and the other dataset, containing the remaining 30 % of data, was used for the data prediction and verification process. the final biomarkers were determined in such a way that the misclassification error rate was minimal. the resulting graph ( figure 7 ) showed the shrunken class centroids for genes that had at least one nonzero difference in each diagnost ic class. the genes with nonzero components in each class were almost mutually exclusive and represented candidate biomarkers for the diag nosis of each class. all data analyses were performed using the bioconductor package [ 17 ]. figure 7identification of diagnost ic markers by pam. the shrunken class centroids for genes which have at least one nonzero difference are shown. the genes with nonzero components in each class were almost mutually exclusive and were the candidate molecular markers for the diag nosis of the four groups of cell populations, including, ( from left to right ) hesc derived mesenchyme and human fibroblast feeder cells ( " fb ", n = 5 ), undifferentiated hescs ( " es ", n = 11 ), hesc derived neural cells ( " ns ", n = 3 ), and differentiated es cells and eb, ( " eb ", n = 6 ). the identified biomarkers can be used to disting uish the four groups of cell populations. undifferentiated hescs have been analyzed by est scan, mpss, sage and microarray [ 5, 10, 16 ]. the goal of these experiments including our own is to develop a low cost reliable method to assess multiple samples to generate a global database of markers and to provide a method of identifying core measures of similarities and differences across multiple laboratories. we and others have proposed three alternative methods of assessment: quantitative rt - pcr [ 9, 20 ], focused arrays [ 27 ] or a large scale array with bioinformatics tools being utilized to focus on appropriate subsets of genes [ 5, 7, 15, 16, 28 ]. each of these methods has its advantages and disadvantages. the present results suggest that the global illumina bead array retains the advantages of low cost per sample associated with focused arrays yet still has the strength of the global attributes of mpss or est scan while requiring much less rna and turnaround time. to test this array format we examined samples from a variety of laboratories in a blinded fashion to determine whether the array was sufficiently sensitive and rapid for routine assessment. duplicates using 100 ng of rna were run and results obtained forty - eight hours later. the resolution was sufficient that esc samples could be disting uished from one another and a variant karyotypically abnormal subclone could be disting uished from the parent population ( correlation coefficient = 0. 9043 ). aliquots of the pooled es and pooled eb samples, which we had prepared for mpss, were included in this run to compare these two methods directly. the current analysis confirms that comparison across platforms is difficult and that only positive results can be treated with any reliability. the absence of expression cannot be readily interpreted. in particular, genes expressed at low levels ( greater than 70 % of all genes detected ) should not be assessed in cross platform comparisons. the limited concordance at low levels raises a question as to how many genes are actually expressed by any one cell line and whether the cutoff of 3 tpm used for mpss or 100 intensity units for bead arrays is a reasonable cutoff. we used 100 units for our analysis and we would suggest that readers exercise similar caution. nevertheless even at this higher cutoff the arrays were remarkably sensitive and allowed us to readily disting uish between samples including cells grown in the same laboratory.