BACKGROUND
embryonic stem cells  have been isolated from multiple species  <cit>  including non-human primates  <cit>  and humans  <cit> .
currently, over hundred different human embryonic stem cell  lines have been established  <cit> .
hesc populations grow as tightly compacted colonies of undifferentiated cells on mouse  <cit>  or human  <cit>  feeders or as colonies in feeder-free conditions using matrix and conditioned medium  <cit> .
hesc has been shown to differentiate in vitro and in vivo to form derivates of all three germ layers.
in vitro differentiation can be induced by the process of embryoid body  formation, which involves aggregating the cells and preventing separation by plating on a non-permissive substrate.
cell to cell interaction and addition of differentiation agents such as retinoic acid  induces differentiation into derivatives of all three germ layers   <cit> .
heb can subsequently be induced to undergo further differentiation to generate a variety of cell types, including hematopoietic  <cit> , neuronal  <cit> , myogenic  <cit>  and cardiac muscle cells  <cit> .
thus, heb represent an early stage in the process of lineage specification and should differ from hesc or their more differentiated progeny in their profile of gene expression.
several different methods have been developed that can be used to assess the process of differentiation.
subtractive hybridization  <cit> , differential display polymerase chain reaction   <cit> , representational difference analysis   <cit> , analysis of expressed sequence tag   <cit>  and serial analyses of gene expression   <cit>  are but a few commonly used techniques.
perhaps the most commonly used however, is gene array   <cit> .
microarrays have been used by several investigators to assess the undifferentiated hesc state  <cit>  and provide a data set of useful information.
we for example utilized a large-scale oligonucleotide based array to identify a set of  <dig> genes that are highly upregulated in six hesc lines when compared against human universal reference rna derived from mature tissues  <cit> .
this set of "stemness genes" along with additional novel genes identified has served to assess the state of undifferentiated cells.
however, currently no similar data set is available for genes that may be used to define the embryoid body stage of hesc differentiation and no comparisons between the undifferentiated and differentiated populations have been performed utilizing microarray technology.
a recent study has characterized gene expression in embryoid bodies by massively parallel signature sequencing  and suggested that several candidate genes specific to heb may exist  <cit> .
mpss analysis however, is expensive and unavailable for most laboratories.
therefore, an alternate readily available and economical assay is needed to characterize embryoid bodies and compare them with available datasets on hesc.
microarray studies of heb's offer the possibility of such an assay that can be used for routine assessment of the state of es cell differentiation.
to determine if microarrays  can be used to distinguish between hesc and heb and to identify candidate markers of the process of differentiation we have compared the gene expression pattern of undifferentiated hesc and differentiated heb derived from them using a large-scale oligonucleotide based arrays.
the expression of selected genes was confirmed by another large-scale array, reverse transcriptase-polymerase chain reaction , comparison with an expressed sequence tag  enumeration database of esc  <cit> , mpss data from embryoid bodies  <cit>  and immunocytochemistry.
our results show that microarray studies can readily distinguish between hesc and heb and can be used to identify markers of the embryoid body stage.
RESULTS
microarray detects differences between hesc and heb derived from them
to assess alteration in gene expression in hesc and hebs, cells were cultured and induced to form ebs .
total rna was harvested from undifferentiated hescs and hebs derived from them and a time course of gene expression was performed to assess downregulation of known es cell specific genes.
day  <dig> of differentiation was chosen as a time point for subsequent analysis where there is a clear downregulation of known hesc markers and an upregulation of some early markers of differentiation .
the expression of known undifferentiated hesc markers including oct <dig>  nanog and esg <dig> showed reduction in expression in heb at day  <dig> while known markers of differentiation  showed a marked increase in expression.
several genes  did not show detectable change between hesc and heb cells.
for microarray studies each sample was compared to human universal reference rna  to maintain uniformity and allow comparisons across samples.
cdna from bg <dig> and pooled samples of hesc were labeled with cy <dig> and huurna with cy <dig>  and ~ <dig>  oligonucleotide arrays were hybridized.
similarly, cdna from heb derived from differentiated bg02-esc  and pooled wicell eb  was labeled with cy <dig> and huurna with cy <dig> and arrays were hybridized and data analyzed.
since the process of differentiation is relatively stochastic and cell lines may behave differently as they differentiate, data from different experiments  was not pooled and reported as the expression from single hybridization.
each sample was analyzed in duplicate obtained from two different independent cultures.
overall microarray results between technical replicates were similar and representative images from each experiment are available .
microarray analysis showed that  <dig>  of the ~ <dig>  features present on the array were detectable above background at a ≥  <dig> minimum intensity and target pixels of one standard deviation  above background  cutoffs.
heb and hesc expressed approximately  <dig> to  <dig> genes.
pooled wicell esc and bg <dig> esc showed over expression of  <dig> and  <dig> genes at ≥  <dig> fold respectively, compared to huurna .
as these cells differentiated to heb, the number of total genes that were detected remained similar to hesc .
for some experiments esc samples were pooled as we were interested in identifying differences between esc and ebs that would be common to multiple lines.
further, as eb formation is variable we felt pooling may allow us to focus on large differences which would not be lost in the averaging process.
once we obtained results we then tested if this was true by using cell line provided by a different provider and propagated under different culture condition.
in addition, since the purpose of day  <dig> differentiation was to study which genes persisted and which were modulated as a result of further differentiation we did not use day  <dig> pooled ebs but focused on results from a single line
we have previously shown that six hesc lines  express  <dig> genes in common at ≥  <dig> fold levels when compared with huurna .
we therefore examined the expression of these genes and in addition all genes overexpressed in bg <dig> and pooled hescs  .
eighty-seven of  <dig> genes were also detected in the present sample confirming the quality of the array hybridization and the fidelity of the samples used.
in addition, previous study identified several early markers of differentiation, that were present at low levels in hesc and when hesc cells were differentiated, these genes were upregulated and up regulation was confirmed by est enumeration technique  <cit> .
the present study confirmed these earlier observations by microarray experiments .
these differentiation genes included keratin  <dig>  keratin  <dig>  actc, and tubb <dig>  when we examined our current results, we found that these genes were also up regulated in heb cells derived from bg <dig> and wicell lines by microarray studies .
these results confirm the est enumeration data and further provide support for the quality of hebs used.
overall the technical replicates, the testing of the expression of known genes and the ability to detect expected changes in the current samples confirmed the suitability of the current data set for additional analysis.
modulation of "stemness genes by es cell differentiation
previously, we identified a set of  <dig> genes are expressed at high levels in most hesc and can be detected by microarray and est enumeration  <cit> .
to test if changes in their expression could be used to monitor differentiation, we examined the relative levels of these genes as es cell differentiated.
eighty-seven of the  <dig> genes expressed in all six hesc lines were also over expressed in day  <dig> bgo2-eb and day  <dig> pooled wicell eb compared to huurna .
however, as a result of differentiation of the bg02-esc line  <dig> out of  <dig> genes were down modulated in bg <dig> eb cells .
among these, known es cell markers e.g., pou5f <dig>  gtcm- <dig>  leftb, galanin, gja <dig>  tdgf <dig>  sfrp <dig>  fabp <dig> and lin- <dig> showed a marked decline in their expression compared to undifferentiated es cells.
similarly, other es cell specific markers such as cer <dig> and dnmt3b also showed marked decline in the expression in day  <dig> bg <dig> eb compared to esc .
however, three zinc finger proteins did not show any significant change and down modulation of numatrin, c20orf <dig> and laminin receptor was modest .
twelve novel genes that were overexpressed in all  <dig> hesc lines tested  <cit> , were all down modulated by differentiation.
among them mgc <dig> , gsh <dig>  ppat, kiaa <dig>  td- <dig>  c20orf <dig>  arl <dig> showed a marked decline in fold expression in bg <dig> eb compared to undifferentiated bg <dig>  while changes in other genes was modest.
pooled eb samples of es cells  showed a similar gene expression profile as observed in day  <dig> bg02-eb.
<dig> out of  <dig> "stemness" genes were down modulated compared to undifferentiated wicell esc .
these  <dig> genes were a subset of the  <dig> that were markedly downmodulated in bg <dig> derived eb compared to undifferentiated bg02-esc .
pou5f <dig>  leftb, fabp <dig>  galanin, gja <dig>  sfrp <dig>  nanog and tdgf <dig> es specific genes showed ≥  <dig> times lower expression in pooled wicell eb compared to pooled wicell esc .
in addition, other es cell specific markers like dnmt3b, cer <dig>  and sox <dig> also showed down modulation in pooled wicell eb.
novel genes such as ppat, mgc <dig>  gsh <dig>  arl <dig>  and kiaa <dig> showed marked down modulation in pooled eb compared to pooled es cells.
however, zinc finger protein znf <dig>  laminin receptor, c20orf <dig> and c20orf <dig> showed modest decline .
both pooled wicell eb and day  <dig> bg02-eb showed an upregulation of genes that we had previously identified as being markers of differentiation that were present in detectable levels in undifferentiated hesc lines .
these included early differentiation markers e.g., krt <dig>  krt <dig>  tubb <dig> and actc.
overall, the pattern of differentiation appeared similar with bg <dig> when compared to embryoid bodies derived from wicell esc.
among  <dig> genes, thirteen additional novel genes have been identified previously in hesc  <cit> .
however, only  <dig> of the thirteen genes were present on this array and their expression was analyzed.
our results show that dppa <dig> was expressed in bg <dig> es and pooled wicell es, however, its expression level was eight fold higher in day  <dig> bg <dig> eb compared to huurna and down modulated to  <dig> fold at day  <dig> bg02-eb.
similarly, pooled wicell hesc cells showed  <dig> fold over expression of dppa <dig> compared to huurna, however upon differentiation to eb the expression was decreased.
in sharp contrast, claudin- <dig> showed down modulation of expression from pooled wicell esc to eb by about  <dig> fold but was not present in detectable levels in either bg <dig> es or bg <dig> ebs.
in peb some genes that showed down modulation in bg <dig> ebs were in fact up regulated compared to esc.
the reason for this reverse pattern is not known.
although we did not check whether these were also up regulated in day  <dig> pooled ebs, our future studies are focused on addressing these issues.
it is possible that a mixture of three hesc lines behaved differently when they were mixed and differentiated.
overall, these data confirm that seventy-seven of the ninety-two genes we had identified as hesc specific could be used to assess the differentiation state of es cells as they differentiate.
a large number of these genes show a dramatic down modulation as early as day  <dig> and this is seen in both wicell and bresagen  cell lines and can be readily assessed by microarray analysis.
genes that are upregulated as es cells differentiated to form embryoid bodies
to analyze genes that were upregulated as hesc differentiated, embryoid bodies were prepared as previously described and their quality analyzed as described above.
samples, which showed a clear downmodulation of es cell specific genes, were subjected to microarray studies and their overall gene expression pattern compared with that of the undifferentiated population.
each individual experiment was analyzed separately and differences in gene expression observed were then compared.
when bg <dig> hesc were compared with embryoid bodies derived from them a total of  <dig> new genes were expressed at ≥  <dig> fold higher levels in day  <dig> bg02-eb compared to undifferentiated bg <dig> esc .
these genes included many genes known to be upregulated in eb cells and confirmed the quality of the array and the hybridization .
in addition, we identified numerous additional genes whose expression in hebs had not been documented previously.
since we were interested in identifying a core set of genes that are upregulated as es cells differentiated we compared the pattern of gene upregulation in a second distinct population of es cells  grown under different conditions using protocols described previously  <cit> .
out of  <dig> genes that were upregulated in day  <dig> bg <dig> eb  <dig> genes were also upregulated at ≥  <dig> fold in wicell derived eb  .
the remaining  <dig> genes were also upregulated but they did not meet the rigorous three fold cutoff criteria.
the expression of these  <dig> genes by other techniques will be subject of confirmation in future studies.
the common subset of genes, which showed upregulation at ≥  <dig> fold in both bg <dig> eb and pooled wicell ebs were classified by hierarchical clustering.
as shown in fig  <dig>  all listed genes  were upregulated in ebs derived either from bg <dig> or wicell lines and the expression levels can be readily distinguished from those in undifferentiated esc.
these results clearly show a marked difference in gene expression profile in differentiated compared to undifferentiated es.
more detailed examination of the genes that were differentially expressed in esc and ebs indicated multiple signaling pathways are altered.
bg <dig> eb showed over expression of keratin  <dig>  profilin- <dig>  fibronectin  <dig>  hand <dig>  col1a <dig>  zak, col4a <dig>  birc <dig>  nid <dig>  tubb <dig>  tmsb4x, plp <dig>  eno <dig> and col5a <dig> .
pooled wicell eb also showed upregulation of similar genes including hand <dig>  krt <dig>  fibronectin  <dig>  profilin <dig>  tmsb4x, vimentin, enolase, plp <dig>  col4a <dig>  capn <dig>  nid <dig>  ivl, zak, spta <dig> and col1a <dig>  which are related to cell differentiation or cytoskeleton .
several genes related to cell signaling, cell growth, cell cycle and metabolic activities were uniquely identified  and .
for example, glypican- <dig>  member of the glypican-related integral membrane proteoglycan family  contains a core protein anchored to the cytoplasmic membrane via a glycosyl phosphatidylinositol linkage and plays a role in the control of cell division and growth regulation was over expressed.
calreticulin , which can inhibit androgen receptor and retinoic acid receptor transcriptional activities in vivo, as well as retinoic acid-induced neuronal differentiation was over expressed by  <dig> fold.
cyclin-dependent kinase inhibitor 1c , an inhibitor of several g <dig> cyclin/cdk complexes  and to lesser extent of the mitotic cyclin-b-cdc <dig>  was over expressed in ebs.
finally,  <dig> hypothetical,  <dig> zinc finger proteins and  <dig> unknown genes whose functions are still to be determined were also over expressed in ebs  and .
overall, these results indicate that  <dig> genes identified as upregulated in two different eb cell lines may serve as early and sensitive markers to monitor es cell differentiation.
confirmation of gene expression profile by microarray, mpss, est-enumeration, rt-pcr and immunohistochemical analyses
to provide independent verification of the results we utilized three different strategies.
we compared gene expression patterns obtained using microarray studies with the mpss data set generated by our laboratory using the pooled es and ebs derived from them  <cit> .
in addition, we prepared duplicate samples of bg <dig> es and bg <dig> derived embryoid bodies and subjected them to a microarray analysis using a large scale oligonucleotide array based on a different set of oligonucleotides that were commercially available .
finally we examined a subset of genes that were not validated by either of these methods by rt-pcr.
of the  <dig> genes, which were uniquely over expressed at ≥  <dig> fold in both bg <dig> and wicell cell derived ebs  <dig> genes showed higher expression in an mpss analysis of wicell samples .
similarly, early es differentiation markers e.g., krt <dig>  krt <dig>  krt <dig>  actc and eb specific genes such as vimentin, afp, hand <dig>  and col1a <dig> showed higher expression in ebs compared to esc by both microarray and est enumeration .
when fold expression of  <dig> out of  <dig> unknown genes identified by microarray was compared with tpm level detected by mpss, a similar pattern of gene expression was observed .
all of these genes showed higher expression in bg02-eb and wicell derived eb compared to bg02-es and wicell esc samples.
six of these genes were also confirmed by rt-pcr analysis .
the reliability of microarray results was further confirmed by rt-pcr analysis of  <dig> genes for hypothetical proteins,  <dig> for zinc finger proteins,  <dig> for unknown proteins and  <dig> genes that were highly expressed in embryoid bodies .
this similarity in the results confirmed the reliability of the microarray studies.
as forty-six of the  <dig> genes  identified by microarray showed no significant difference by mpss analysis, it was concluded that microarray and mpss assays may be different in sensitivity or there was variability in the production of embryoid bodies.
to address this issue and to determine if the differences observed by microarray were reliable, we used a second microarray platform to examine gene expression in the same samples.
interestingly  <dig> of the  <dig> genes showed overexpression in wicell derived ebs using agilent human  <dig> k oligo-arrays .
this suggested that different methods have different sensitivities and it is important to use multiple methods to confirm expression.
the expression of the remaining  <dig> genes detected as overexpressed by microarray studies using a custom built microarray but not by mpss or by agilent microarrays was tested by rt-pcr analysis .
of the thirteen genes,  <dig> were confirmed by rt-pcr analysis further confirming the differential sensitivity of various arrays and other large-scale analytical methods.
gene expression profile of day  <dig> bg02-eb
our results identified a large set of genes that are differentially modulated as cells differentiate to form embryoid bodies over a period of two weeks.
to examine whether the same set of genes could be used to assess differentiation of hesc at twenty-one day of differentiation, we examined gene expression using rna prepared from day  <dig> embryoid bodies.
for these studies, undifferentiated hesc  cells were used to generate ebs by a brief exposure to collagenase iv and small clusters of cells were obtained by scraping with a pipette.
es cells were differentiated for  <dig> and  <dig> days, harvested to prepare total rna and analyzed by hybridization to microarrays.
we found that a majority of esc specific genes that were down modulated as cells differentiated for thirteen days were further down modulated as a result of differentiation to day  <dig> bg02-eb .
among  <dig> genes overexpressed in day  <dig> bg02-eb and peb, thirty-three genes showed a decrease in fold expression at day  <dig> bg02-eb.
however, other than col1a <dig>  which was down modulated from  <dig> fold to  <dig> fold, none showed any marked decrease.
the remaining genes were either not expressed or did not show any change in the expression .
we previously reported that eight early differentiation marker genes were expressed in hesc, which were further upregulated in hebs as determined by est enumeration  <cit> .
therefore, in this study we examined their expression at day  <dig> of differentiation.
for this experiment gene expression between bg <dig> es was compared with day  <dig> bg02-eb and day  <dig> bg02-eb.
we found that six of these genes were markedly down modulated in day  <dig> bg02-eb  compared to day 13eb and peb.
the other two genes showed minor or no significant change.
in addition, expression of  <dig> known eb specific genes that were shown to be overexpressed in both day  <dig> bg02-eb and pooled eb  also examined in day  <dig> bg02-eb.
interestingly, all of them were down modulated in day  <dig> bg02-eb
additionally, we examined the status of genes that were upregulated in day 21eb but not in es or day  <dig> bg02-eb.
genes related to cytokeratin and hair keratin , which are responsible for structural integrity showed higher expression in day 21eb compared to bg02es or day 13eb.
in addition, genes related to mature tissues were exclusively upregulated in day 21eb but not in es and day  <dig> bg02-eb  indicating that additional differentiation markers can be used to distinguish early vs. late ebs.
while most genes followed an expected pattern of change in expression in day <dig> eb, nanog showed a reverse pattern of expression.
on day  <dig> its expression level was significantly reduced compared to bg02-es, however, on day 21eb an increased expression was observed compared to day  <dig> bg02-eb.
levels were almost similar to those in bg02-esc samples .
downmodulation of nanog in day 13-eb was confirmed by rt-pcr analysis  though its upregulation in day  <dig> samples did not match the rt-pcr results .
the downregulation in nanog followed the downregulation of oct 3/ <dig> and tert and other es specific genes .
in addition, although sox  <dig> gene showed a decrease in expression in pooled eb compared to pooled esc but it didn't show any change in expression pattern from bg02es to day  <dig> bg02-eb and day  <dig> bg02-eb .
expression of sox- <dig> gene was also confirmed by rt-pcr and immunocytochemistry analyses .
both analyses demonstrated that sox <dig> is expressed in day <dig> and day 21ebs.
oct3/ <dig> protein expression was used as a control that did not show expression in sox- <dig> expressing cells confirming that the sox- <dig> expression represented induction in a newly differentiated population rather than in persistent esc.
these results indicate that krt <dig>  krt <dig>  tubb <dig>  actc, serpinh <dig>  tubb <dig>  krt <dig>  hand <dig>  fn <dig>  eno <dig>  col1a <dig>  col5a <dig>  col4a <dig> and other identified markers may serve as a indicators of early and late stages of differentiation as they were further markedly down regulated on day  <dig> compared to day  <dig> of differentiation.
in contrast, genes such as krt <dig>  krt <dig>  ivl, nphp <dig>  capn <dig>  and cntn <dig> are markers of later stage of differentiation as they are overexpressed in day  <dig> compared to day  <dig> of differentiation.
thus microarray studies can distinguish between es cells and embryoid bodies as well as between early and late stage embryoid bodies.
discussion
our results show that large-scale oligonucleotide based microarrays can be used to distinguish between undifferentiated hesc and differentiating hebs.
two sets of markers can be distinguished in a set of 'stemness" genes that are present at higher levels in undifferentiated cells whose levels are reduced as cells differentiate and a complementary set of genes that are absent or present at low levels in hesc and are upregulated as cells differentiate.
the set of genes that are upregulated as cells differentiate include known as well as unknown genes.
this pattern of gene expression was confirmed by a variety of independent means including microarray using a second independent array set, comparison with mpss using similar samples and by rt-pcr.
the combined set of upregulated and down regulated genes will serve as a sensitive indicator of the state of hesc and the novel sets of genes identified are likely candidates that may participate in regulating the process of differentiation.
in our previous study we demonstrated over expression of a set of  <dig> genes in six undifferentiated esc cell  <cit> .
expression of almost all of the genes was confirmed in the bg02-es and pooled hesc used in the present study.
the present study further refines the set of stemness genes to identify those that are rapidly down-regulated as es cells differentiate.
we show that  <dig> of these are down modulated when differentiated to embryoid bodies using undifferentiated bg02-es.
further differentiation of bg02-es to day  <dig> bg02-eb showed consistency in down modulation of many of these.
this refined subset of previously identified stemness genes can be used to monitor the transition of undifferentiated pluripotent human embryonic stem cells to differentiated embryoid bodies.
analyzing the pattern of gene expression, we would suggest that es specific genes such as lin- <dig>  psip <dig>  pitx <dig>  dnmt3b, and galanin whose downregulation is seen in both lines and confirmed by est scan and microarray represent good initial candidates to assess the es cell state.
furthermore, leftb and cer <dig>  inhibitors of nodal signaling, which were downmodulated as es cells differentiated  in addition to other members of the tgfβ signaling family may be sensitive indicators of esc differentiation.
oct3/ <dig>  sox- <dig> and nanog in contrast while expressed by es cells may not be as good in assessing es cell to embryoid body differentiation.
while oct-3/ <dig> expression declined markedly in both bg <dig> ebs and pooled es derived ebs compared to es the decline was slow and not as rapid as that of other genes.
this may be because oct3/ <dig> expression persists in germ cells that are derived from esc  <cit> .
likewise our analysis of nanog showed variable results and there was a discrepancy between the microarray and pcr results and we noted an increase in nanog levels in day  <dig> bg02-eb samples by microarray.
the reason for this difference is not clear.
it is possible that microarray is detecting one of the eleven processed pseudogenes for nanog and that its expression on day  <dig> represents crosshybridization.
alternatively, nanog expression may be increased as a result of differentiation.
this conclusion is supported by a recent report that nanog is expressed in mature tissues  <cit> .
we have confirmed that nanog expression is indeed present in some germ cells and early neuronal populations .
these tests were performed by rt-pcr using multiple primers and double label immunocytochemistry .
future studies will examine expression of nanog in ebs using specific antibody and its function in differentiated cells.
at present we recommend that nanog expression should be confirmed by pcr or immunocytochemistry and it should not be included as an initial indicator of the es cell state and differentiation.
in addition to confirming the downregulation of hesc specific genes our study identified a set  <dig> genes that were uniquely overexpressed at ≥  <dig> fold in day  <dig> bg <dig> eb compared to undifferentiated bg <dig> es cell lines.
out of these  <dig> genes  <dig> genes also showed ≥  <dig> fold over expression in eb samples prepared from wicell lines.
this significant similarity of unique genes in bg <dig> and wicell line derived ebs suggest that this subset of genes can be classified as eb specific  and that embryoid bodies derived from different es cells maintain significant similarity with each other.
these eb specific genes like the es cell specific genes may be useful in distinguishing ebs from es cells.
a detailed analysis of upregulated genes in eb indicated that a) differentiation genes, b) cell signaling, cellular process and cell cycle genes, c) cytoskeleton or cell motility genes and d) metabolism and dna and rna related genes were modulated.
in addition,  <dig> hypothetical genes,  <dig> unknown genes and  <dig> zinc finger genes were upregulated in bg <dig> derived eb and wicell lines derived eb.
the status of upregulated genes identified by microarray studies was confirmed by mpss analysis.
among  <dig> genes,  <dig> genes showed higher expression in eb by mpss analysis.
overexpression of  <dig> genes in ebs compared to huurna was confirmed using alternate large-scale array.
the expression of a subset of the remaining  <dig> genes was confirmed by rt-pcr analysis.
these results suggest that microarray analysis is a useful complement to mpss and may identify a overlapping but non-identical set of genes.
among the genes that were upregulated in both eb samples is hand <dig> .
hand <dig> was dramatically up regulated .
hand <dig> belongs to basic helix-loop-helix family of transcription factor, may be required for early trophoblast differentiation as well as the differentiation of heart tissue  <cit> .
additionally we noted that several markers of early differentiation such as keratin, actin and beta tubulin, which are present at low levels in esc, were dramatically upregulated as ebs differentiated.
we suggest that these markers can serve as sensitive indicators of the process of differentiation and when coupled with the down regulation of esc markers may reliably distinguish the state of es cell cultures.
future experiments will assess the sensitivity of a combination of markers identified in this study to temporally profile the process of differentiation.
a recent study  has compared gene expression of 24-marker genes between ebs differentiated with eight different growth factors and hesc.
similar to our observation by microarray analysis, this study showed over expression of genes related to ectoderm differentiation such as keratin- <dig>  keratin- <dig> and keratin- <dig> when differentiated by retinoic acid.
a highly significant upregulation of mesodermal related genes e.g., hand <dig> was also observed which confirms our observation.
our results further show that many genes that are induced or upregulated as ebs differentiate remain elevated at day  <dig> of differentiation.
these genes represent good markers for distinguishing differentiated cells from esc.
in addition we noted that several genes showed differential expression when levels of expression in day  <dig> and day  <dig> bg02-eb's was compared.
thirty-three of the  <dig> genes upregulated at day  <dig> were down modulated at day <dig> eb.
other genes such as nphp <dig>  capn <dig>  and cntn <dig> were up regulated in day  <dig> bg02-eb's when compared to day  <dig> eb.
these later appearing genes may reflect additional derivatives that appear as ebs undergo further differentiation.
these later appearing markers along with markers that are down regulated can be used to distinguish early from late ebs.
a recent study has characterized differences in gene expression between mouse and human esc .
this study identified differences between mouse and human esc and reported that differences were species specific rather than arising from differing culture conditions.
in our current study, while the expression of many genes was similar in rodent and human cells significant differences were found.
for example the expression of vimentin, eomesodermin, ssea <dig>  afp, il6st and heb was found in hesc but not in mouse es cells.
the large oligonucleotide arrays have allowed us to build a comprehensive data set that includes hundreds of genes  that are either upregulated or downregulated as hesc differentiate.
the list of genes has been validated by testing on different cells lines, testing using different oligonucleotide sequences to different regions of the genes and testing arrays used in different formats.
our results show clearly that while small differences exist as different techniques are used the core set of markers is quite robust, the differences in expression quite large and are readily detectable even using a relatively insensitive method such as a microarray.
our results show further that restricting the analysis to only this set of genes as would be necessary in focused array does not reduce the sensitivity of the assessment.
indeed, based on our results we would suggest that an order of magnitude fewer genes may be sufficient if they are appropriately selected from the lists.
we have recently developed a focused array that includes genes that are down regulated as hesc differentiate as well as those that are induced as cells differentiate  <cit> .
CONCLUSIONS
in conclusion, a similar pattern of gene expression profile was observed in two differentiated embryoid body samples derived from different embryonic stem cell lines.
a consistent and marked downmodulation of most of the "stemness" genes was observed in embryoid bodies indicating that relative levels of these genes can be used to assess the transition of es cells to embryoid bodies.
in addition, we show that  <dig> unique genes are over expressed in eb and that this subset serves as a complement to the previously characterized "stemness genes" in assessing the degree of differentiation of es lines and differentiating embryoid bodies.
further validation and confirmation of these data with mpss and rt-pcr documents the usefulness of high throughput microarray technology and identifies an additional set of previously unknown genes that are likely important in regulating the process of differentiation.
these eb enriched genes in combination with downmodulated stemness genes may serve as biomarkers to monitor the transition of undifferentiated human embryonic stem cells to differentiated embryoid bodies.

 embryonic stem cells have been isolated from multiple species including non-human primates and humans. currently, over hundred different human embryonic stem cell lines have been established. hesc populations grow as tightly compacted colonies of undifferentiated cells on mouse or human feeders or as colonies in feeder-free conditions using matrix and conditioned medium. hesc has been shown to differentiate in vitro and in vivo to form derivates of all three germ layers. in vitro differentiation can be induced by the process of embryoid body formation, which involves aggregating the cells and preventing separation by plating on a non-permissive substrate. cell to cell interaction and addition of differentiation agents such as retinoic acid induces differentiation into derivatives of all three germ layers. heb can subsequently be induced to undergo further differentiation to generate a variety of cell types, including hematopoietic, neuronal, myogenic and cardiac muscle cells. thus, heb represent an early stage in the process of lineage specification and should differ from hesc or their more differentiated progeny in their profile of gene expression. several different methods have been developed that can be used to assess the process of differentiation. subtractive hybridization, differential display polymerase chain reaction, representational difference analysis, analysis of expressed sequence tag and serial analyses of gene expression are but a few commonly used techniques. perhaps the most commonly used however, is gene array. microarrays have been used by several investigators to assess the undifferentiated hesc state and provide a data set of useful information. we for example utilized a large-scale oligonucleotide based array to identify a set of genes that are highly upregulated in six hesc lines when compared against human universal reference rna derived from mature tissues. this set of "stemness genes" along with additional novel genes identified has served to assess the state of undifferentiated cells. however, currently no similar data set is available for genes that may be used to define the embryoid body stage of hesc differentiation and no comparisons between the undifferentiated and differentiated populations have been performed utilizing microarray technology. a recent study has characterized gene expression in embryoid bodies by massively parallel signature sequencing and suggested that several candidate genes specific to heb may exist. mpss analysis however, is expensive and unavailable for most laboratories. therefore, an alternate readily available and economical assay is needed to characterize embryoid bodies and compare them with available datasets on hesc. microarray studies of heb's offer the possibility of such an assay that can be used for routine assessment of the state of es cell differentiation. to determine if microarrays can be used to distinguish between hesc and heb and to identify candidate markers of the process of differentiation we have compared the gene expression pattern of undifferentiated hesc and differentiated heb derived from them using a large-scale oligonucleotide based arrays. the expression of selected genes was confirmed by another large-scale array, reverse transcriptase-polymerase chain reaction, comparison with an expressed sequence tag enumeration database of esc, mpss data from embryoid bodies and immunocytochemistry. our results show that microarray studies can readily distinguish between hesc and heb and can be used to identify markers of the embryoid body stage.  microarray detects differences between hesc and heb derived from them to assess alteration in gene expression in hesc and hebs, cells were cultured and induced to form ebs. total rna was harvested from undifferentiated hescs and hebs derived from them and a time course of gene expression was performed to assess downregulation of known es cell specific genes. day of differentiation was chosen as a time point for subsequent analysis where there is a clear downregulation of known hesc markers and an upregulation of some early markers of differentiation. the expression of known undifferentiated hesc markers including oct nanog and esg showed reduction in expression in heb at day while known markers of differentiation showed a marked increase in expression. several genes did not show detectable change between hesc and heb cells. for microarray studies each sample was compared to human universal reference rna to maintain uniformity and allow comparisons across samples. cdna from bg and pooled samples of hesc were labeled with cy and huurna with cy and ~ oligonucleotide arrays were hybridized. similarly, cdna from heb derived from differentiated bg02-esc and pooled wicell eb was labeled with cy and huurna with cy and arrays were hybridized and data analyzed. since the process of differentiation is relatively stochastic and cell lines may behave differently as they differentiate, data from different experiments was not pooled and reported as the expression from single hybridization. each sample was analyzed in duplicate obtained from two different independent cultures. overall microarray results between technical replicates were similar and representative images from each experiment are available. microarray analysis showed that of the ~ features present on the array were detectable above background at a ≥ minimum intensity and target pixels of one standard deviation above background cutoffs. heb and hesc expressed approximately to genes. pooled wicell esc and bg esc showed over expression of and genes at ≥ fold respectively, compared to huurna. as these cells differentiated to heb, the number of total genes that were detected remained similar to hesc. for some experiments esc samples were pooled as we were interested in identifying differences between esc and ebs that would be common to multiple lines. further, as eb formation is variable we felt pooling may allow us to focus on large differences which would not be lost in the averaging process. once we obtained results we then tested if this was true by using cell line provided by a different provider and propagated under different culture condition. in addition, since the purpose of day differentiation was to study which genes persisted and which were modulated as a result of further differentiation we did not use day pooled ebs but focused on results from a single line we have previously shown that six hesc lines express genes in common at ≥ fold levels when compared with huurna. we therefore examined the expression of these genes and in addition all genes overexpressed in bg and pooled hescs. eighty-seven of genes were also detected in the present sample confirming the quality of the array hybridization and the fidelity of the samples used. in addition, previous study identified several early markers of differentiation, that were present at low levels in hesc and when hesc cells were differentiated, these genes were upregulated and up regulation was confirmed by est enumeration technique. the present study confirmed these earlier observations by microarray experiments. these differentiation genes included keratin keratin actc, and tubb when we examined our current results, we found that these genes were also up regulated in heb cells derived from bg and wicell lines by microarray studies. these results confirm the est enumeration data and further provide support for the quality of hebs used. overall the technical replicates, the testing of the expression of known genes and the ability to detect expected changes in the current samples confirmed the suitability of the current data set for additional analysis. modulation of "stemness genes by es cell differentiation previously, we identified a set of genes are expressed at high levels in most hesc and can be detected by microarray and est enumeration. to test if changes in their expression could be used to monitor differentiation, we examined the relative levels of these genes as es cell differentiated. eighty-seven of the genes expressed in all six hesc lines were also over expressed in day bgo2-eb and day pooled wicell eb compared to huurna. however, as a result of differentiation of the bg02-esc line out of genes were down modulated in bg eb cells. among these, known es cell markers e.g., pou5f gtcm- leftb, galanin, gja tdgf sfrp fabp and lin- showed a marked decline in their expression compared to undifferentiated es cells. similarly, other es cell specific markers such as cer and dnmt3b also showed marked decline in the expression in day bg eb compared to esc. however, three zinc finger proteins did not show any significant change and down modulation of numatrin, c20orf and laminin receptor was modest. twelve novel genes that were overexpressed in all hesc lines tested, were all down modulated by differentiation. among them mgc, gsh ppat, kiaa td- c20orf arl showed a marked decline in fold expression in bg eb compared to undifferentiated bg while changes in other genes was modest. pooled eb samples of es cells showed a similar gene expression profile as observed in day bg02-eb. out of "stemness" genes were down modulated compared to undifferentiated wicell esc. these genes were a subset of the that were markedly downmodulated in bg derived eb compared to undifferentiated bg02-esc. pou5f leftb, fabp galanin, gja sfrp nanog and tdgf es specific genes showed ≥ times lower expression in pooled wicell eb compared to pooled wicell esc. in addition, other es cell specific markers like dnmt3b, cer and sox also showed down modulation in pooled wicell eb. novel genes such as ppat, mgc gsh arl and kiaa showed marked down modulation in pooled eb compared to pooled es cells. however, zinc finger protein znf laminin receptor, c20orf and c20orf showed modest decline. both pooled wicell eb and day bg02-eb showed an upregulation of genes that we had previously identified as being markers of differentiation that were present in detectable levels in undifferentiated hesc lines. these included early differentiation markers e.g., krt krt tubb and actc. overall, the pattern of differentiation appeared similar with bg when compared to embryoid bodies derived from wicell esc. among genes, thirteen additional novel genes have been identified previously in hesc. however, only of the thirteen genes were present on this array and their expression was analyzed. our results show that dppa was expressed in bg es and pooled wicell es, however, its expression level was eight fold higher in day bg eb compared to huurna and down modulated to fold at day bg02-eb. similarly, pooled wicell hesc cells showed fold over expression of dppa compared to huurna, however upon differentiation to eb the expression was decreased. in sharp contrast, claudin- showed down modulation of expression from pooled wicell esc to eb by about fold but was not present in detectable levels in either bg es or bg ebs. in peb some genes that showed down modulation in bg ebs were in fact up regulated compared to esc. the reason for this reverse pattern is not known. although we did not check whether these were also up regulated in day pooled ebs, our future studies are focused on addressing these issues. it is possible that a mixture of three hesc lines behaved differently when they were mixed and differentiated. overall, these data confirm that seventy-seven of the ninety-two genes we had identified as hesc specific could be used to assess the differentiation state of es cells as they differentiate. a large number of these genes show a dramatic down modulation as early as day and this is seen in both wicell and bresagen cell lines and can be readily assessed by microarray analysis. genes that are upregulated as es cells differentiated to form embryoid bodies to analyze genes that were upregulated as hesc differentiated, embryoid bodies were prepared as previously described and their quality analyzed as described above. samples, which showed a clear downmodulation of es cell specific genes, were subjected to microarray studies and their overall gene expression pattern compared with that of the undifferentiated population. each individual experiment was analyzed separately and differences in gene expression observed were then compared. when bg hesc were compared with embryoid bodies derived from them a total of new genes were expressed at ≥ fold higher levels in day bg02-eb compared to undifferentiated bg esc. these genes included many genes known to be upregulated in eb cells and confirmed the quality of the array and the hybridization. in addition, we identified numerous additional genes whose expression in hebs had not been documented previously. since we were interested in identifying a core set of genes that are upregulated as es cells differentiated we compared the pattern of gene upregulation in a second distinct population of es cells grown under different conditions using protocols described previously. out of genes that were upregulated in day bg eb genes were also upregulated at ≥ fold in wicell derived eb. the remaining genes were also upregulated but they did not meet the rigorous three fold cutoff criteria. the expression of these genes by other techniques will be subject of confirmation in future studies. the common subset of genes, which showed upregulation at ≥ fold in both bg eb and pooled wicell ebs were classified by hierarchical clustering. as shown in fig all listed genes were upregulated in ebs derived either from bg or wicell lines and the expression levels can be readily distinguished from those in undifferentiated esc. these results clearly show a marked difference in gene expression profile in differentiated compared to undifferentiated es. more detailed examination of the genes that were differentially expressed in esc and ebs indicated multiple signaling pathways are altered. bg eb showed over expression of keratin profilin- fibronectin hand col1a zak, col4a birc nid tubb tmsb4x, plp eno and col5a. pooled wicell eb also showed upregulation of similar genes including hand krt fibronectin profilin tmsb4x, vimentin, enolase, plp col4a capn nid ivl, zak, spta and col1a which are related to cell differentiation or cytoskeleton. several genes related to cell signaling, cell growth, cell cycle and metabolic activities were uniquely identified and. for example, glypican- member of the glypican-related integral membrane proteoglycan family contains a core protein anchored to the cytoplasmic membrane via a glycosyl phosphatidylinositol linkage and plays a role in the control of cell division and growth regulation was over expressed. calreticulin, which can inhibit androgen receptor and retinoic acid receptor transcriptional activities in vivo, as well as retinoic acid-induced neuronal differentiation was over expressed by fold. cyclin-dependent kinase inhibitor 1c, an inhibitor of several g cyclin/cdk complexes and to lesser extent of the mitotic cyclin-b-cdc was over expressed in ebs. finally, hypothetical, zinc finger proteins and unknown genes whose functions are still to be determined were also over expressed in ebs and. overall, these results indicate that genes identified as upregulated in two different eb cell lines may serve as early and sensitive markers to monitor es cell differentiation. confirmation of gene expression profile by microarray, mpss, est-enumeration, rt-pcr and immunohistochemical analyses to provide independent verification of the results we utilized three different strategies. we compared gene expression patterns obtained using microarray studies with the mpss data set generated by our laboratory using the pooled es and ebs derived from them. in addition, we prepared duplicate samples of bg es and bg derived embryoid bodies and subjected them to a microarray analysis using a large scale oligonucleotide array based on a different set of oligonucleotides that were commercially available. finally we examined a subset of genes that were not validated by either of these methods by rt-pcr. of the genes, which were uniquely over expressed at ≥ fold in both bg and wicell cell derived ebs genes showed higher expression in an mpss analysis of wicell samples. similarly, early es differentiation markers e.g., krt krt krt actc and eb specific genes such as vimentin, afp, hand and col1a showed higher expression in ebs compared to esc by both microarray and est enumeration. when fold expression of out of unknown genes identified by microarray was compared with tpm level detected by mpss, a similar pattern of gene expression was observed. all of these genes showed higher expression in bg02-eb and wicell derived eb compared to bg02-es and wicell esc samples. six of these genes were also confirmed by rt-pcr analysis. the reliability of microarray results was further confirmed by rt-pcr analysis of genes for hypothetical proteins, for zinc finger proteins, for unknown proteins and genes that were highly expressed in embryoid bodies. this similarity in the results confirmed the reliability of the microarray studies. as forty-six of the genes identified by microarray showed no significant difference by mpss analysis, it was concluded that microarray and mpss assays may be different in sensitivity or there was variability in the production of embryoid bodies. to address this issue and to determine if the differences observed by microarray were reliable, we used a second microarray platform to examine gene expression in the same samples. interestingly of the genes showed overexpression in wicell derived ebs using agilent human k oligo-arrays. this suggested that different methods have different sensitivities and it is important to use multiple methods to confirm expression. the expression of the remaining genes detected as overexpressed by microarray studies using a custom built microarray but not by mpss or by agilent microarrays was tested by rt-pcr analysis. of the thirteen genes, were confirmed by rt-pcr analysis further confirming the differential sensitivity of various arrays and other large-scale analytical methods. gene expression profile of day bg02-eb our results identified a large set of genes that are differentially modulated as cells differentiate to form embryoid bodies over a period of two weeks. to examine whether the same set of genes could be used to assess differentiation of hesc at twenty-one day of differentiation, we examined gene expression using rna prepared from day embryoid bodies. for these studies, undifferentiated hesc cells were used to generate ebs by a brief exposure to collagenase iv and small clusters of cells were obtained by scraping with a pipette. es cells were differentiated for and days, harvested to prepare total rna and analyzed by hybridization to microarrays. we found that a majority of esc specific genes that were down modulated as cells differentiated for thirteen days were further down modulated as a result of differentiation to day bg02-eb. among genes overexpressed in day bg02-eb and peb, thirty-three genes showed a decrease in fold expression at day bg02-eb. however, other than col1a which was down modulated from fold to fold, none showed any marked decrease. the remaining genes were either not expressed or did not show any change in the expression. we previously reported that eight early differentiation marker genes were expressed in hesc, which were further upregulated in hebs as determined by est enumeration. therefore, in this study we examined their expression at day of differentiation. for this experiment gene expression between bg es was compared with day bg02-eb and day bg02-eb. we found that six of these genes were markedly down modulated in day bg02-eb compared to day 13eb and peb. the other two genes showed minor or no significant change. in addition, expression of known eb specific genes that were shown to be overexpressed in both day bg02-eb and pooled eb also examined in day bg02-eb. interestingly, all of them were down modulated in day bg02-eb additionally, we examined the status of genes that were upregulated in day 21eb but not in es or day bg02-eb. genes related to cytokeratin and hair keratin, which are responsible for structural integrity showed higher expression in day 21eb compared to bg02es or day 13eb. in addition, genes related to mature tissues were exclusively upregulated in day 21eb but not in es and day bg02-eb indicating that additional differentiation markers can be used to distinguish early vs. late ebs. while most genes followed an expected pattern of change in expression in day eb, nanog showed a reverse pattern of expression. on day its expression level was significantly reduced compared to bg02-es, however, on day 21eb an increased expression was observed compared to day bg02-eb. levels were almost similar to those in bg02-esc samples. downmodulation of nanog in day 13-eb was confirmed by rt-pcr analysis though its upregulation in day samples did not match the rt-pcr results. the downregulation in nanog followed the downregulation of oct 3/ and tert and other es specific genes. in addition, although sox gene showed a decrease in expression in pooled eb compared to pooled esc but it didn't show any change in expression pattern from bg02es to day bg02-eb and day bg02-eb. expression of sox- gene was also confirmed by rt-pcr and immunocytochemistry analyses. both analyses demonstrated that sox is expressed in day and day 21ebs. oct3/ protein expression was used as a control that did not show expression in sox- expressing cells confirming that the sox- expression represented induction in a newly differentiated population rather than in persistent esc. these results indicate that krt krt tubb actc, serpinh tubb krt hand fn eno col1a col5a col4a and other identified markers may serve as a indicators of early and late stages of differentiation as they were further markedly down regulated on day compared to day of differentiation. in contrast, genes such as krt krt ivl, nphp capn and cntn are markers of later stage of differentiation as they are overexpressed in day compared to day of differentiation. thus microarray studies can distinguish between es cells and embryoid bodies as well as between early and late stage embryoid bodies. discussion our results show that large-scale oligonucleotide based microarrays can be used to distinguish between undifferentiated hesc and differentiating hebs. two sets of markers can be distinguished in a set of'stemness" genes that are present at higher levels in undifferentiated cells whose levels are reduced as cells differentiate and a complementary set of genes that are absent or present at low levels in hesc and are upregulated as cells differentiate. the set of genes that are upregulated as cells differentiate include known as well as unknown genes. this pattern of gene expression was confirmed by a variety of independent means including microarray using a second independent array set, comparison with mpss using similar samples and by rt-pcr. the combined set of upregulated and down regulated genes will serve as a sensitive indicator of the state of hesc and the novel sets of genes identified are likely candidates that may participate in regulating the process of differentiation. in our previous study we demonstrated over expression of a set of genes in six undifferentiated esc cell. expression of almost all of the genes was confirmed in the bg02-es and pooled hesc used in the present study. the present study further refines the set of stemness genes to identify those that are rapidly down-regulated as es cells differentiate. we show that of these are down modulated when differentiated to embryoid bodies using undifferentiated bg02-es. further differentiation of bg02-es to day bg02-eb showed consistency in down modulation of many of these. this refined subset of previously identified stemness genes can be used to monitor the transition of undifferentiated pluripotent human embryonic stem cells to differentiated embryoid bodies. analyzing the pattern of gene expression, we would suggest that es specific genes such as lin- psip pitx dnmt3b, and galanin whose downregulation is seen in both lines and confirmed by est scan and microarray represent good initial candidates to assess the es cell state. furthermore, leftb and cer inhibitors of nodal signaling, which were downmodulated as es cells differentiated in addition to other members of the tgfβ signaling family may be sensitive indicators of esc differentiation. oct3/ sox- and nanog in contrast while expressed by es cells may not be as good in assessing es cell to embryoid body differentiation. while oct-3/ expression declined markedly in both bg ebs and pooled es derived ebs compared to es the decline was slow and not as rapid as that of other genes. this may be because oct3/ expression persists in germ cells that are derived from esc. likewise our analysis of nanog showed variable results and there was a discrepancy between the microarray and pcr results and we noted an increase in nanog levels in day bg02-eb samples by microarray. the reason for this difference is not clear. it is possible that microarray is detecting one of the eleven processed pseudogenes for nanog and that its expression on day represents crosshybridization. alternatively, nanog expression may be increased as a result of differentiation. this conclusion is supported by a recent report that nanog is expressed in mature tissues. we have confirmed that nanog expression is indeed present in some germ cells and early neuronal populations. these tests were performed by rt-pcr using multiple primers and double label immunocytochemistry. future studies will examine expression of nanog in ebs using specific antibody and its function in differentiated cells. at present we recommend that nanog expression should be confirmed by pcr or immunocytochemistry and it should not be included as an initial indicator of the es cell state and differentiation. in addition to confirming the downregulation of hesc specific genes our study identified a set genes that were uniquely overexpressed at ≥ fold in day bg eb compared to undifferentiated bg es cell lines. out of these genes genes also showed ≥ fold over expression in eb samples prepared from wicell lines. this significant similarity of unique genes in bg and wicell line derived ebs suggest that this subset of genes can be classified as eb specific and that embryoid bodies derived from different es cells maintain significant similarity with each other. these eb specific genes like the es cell specific genes may be useful in distinguishing ebs from es cells. a detailed analysis of upregulated genes in eb indicated that a) differentiation genes, b) cell signaling, cellular process and cell cycle genes, c) cytoskeleton or cell motility genes and d) metabolism and dna and rna related genes were modulated. in addition, hypothetical genes, unknown genes and zinc finger genes were upregulated in bg derived eb and wicell lines derived eb. the status of upregulated genes identified by microarray studies was confirmed by mpss analysis. among genes, genes showed higher expression in eb by mpss analysis. overexpression of genes in ebs compared to huurna was confirmed using alternate large-scale array. the expression of a subset of the remaining genes was confirmed by rt-pcr analysis. these results suggest that microarray analysis is a useful complement to mpss and may identify a overlapping but non-identical set of genes. among the genes that were upregulated in both eb samples is hand. hand was dramatically up regulated. hand belongs to basic helix-loop-helix family of transcription factor, may be required for early trophoblast differentiation as well as the differentiation of heart tissue. additionally we noted that several markers of early differentiation such as keratin, actin and beta tubulin, which are present at low levels in esc, were dramatically upregulated as ebs differentiated. we suggest that these markers can serve as sensitive indicators of the process of differentiation and when coupled with the down regulation of esc markers may reliably distinguish the state of es cell cultures. future experiments will assess the sensitivity of a combination of markers identified in this study to temporally profile the process of differentiation. a recent study has compared gene expression of 24-marker genes between ebs differentiated with eight different growth factors and hesc. similar to our observation by microarray analysis, this study showed over expression of genes related to ectoderm differentiation such as keratin- keratin- and keratin- when differentiated by retinoic acid. a highly significant upregulation of mesodermal related genes e.g., hand was also observed which confirms our observation. our results further show that many genes that are induced or upregulated as ebs differentiate remain elevated at day of differentiation. these genes represent good markers for distinguishing differentiated cells from esc. in addition we noted that several genes showed differential expression when levels of expression in day and day bg02-eb's was compared. thirty-three of the genes upregulated at day were down modulated at day eb. other genes such as nphp capn and cntn were up regulated in day bg02-eb's when compared to day eb. these later appearing genes may reflect additional derivatives that appear as ebs undergo further differentiation. these later appearing markers along with markers that are down regulated can be used to distinguish early from late ebs. a recent study has characterized differences in gene expression between mouse and human esc. this study identified differences between mouse and human esc and reported that differences were species specific rather than arising from differing culture conditions. in our current study, while the expression of many genes was similar in rodent and human cells significant differences were found. for example the expression of vimentin, eomesodermin, ssea afp, il6st and heb was found in hesc but not in mouse es cells. the large oligonucleotide arrays have allowed us to build a comprehensive data set that includes hundreds of genes that are either upregulated or downregulated as hesc differentiate. the list of genes has been validated by testing on different cells lines, testing using different oligonucleotide sequences to different regions of the genes and testing arrays used in different formats. our results show clearly that while small differences exist as different techniques are used the core set of markers is quite robust, the differences in expression quite large and are readily detectable even using a relatively insensitive method such as a microarray. our results show further that restricting the analysis to only this set of genes as would be necessary in focused array does not reduce the sensitivity of the assessment. indeed, based on our results we would suggest that an order of magnitude fewer genes may be sufficient if they are appropriately selected from the lists. we have recently developed a focused array that includes genes that are down regulated as hesc differentiate as well as those that are induced as cells differentiate.  in conclusion, a similar pattern of gene expression profile was observed in two differentiated embryoid body samples derived from different embryonic stem cell lines. a consistent and marked downmodulation of most of the "stemness" genes was observed in embryoid bodies indicating that relative levels of these genes can be used to assess the transition of es cells to embryoid bodies. in addition, we show that unique genes are over expressed in eb and that this subset serves as a complement to the previously characterized "stemness genes" in assessing the degree of differentiation of es lines and differentiating embryoid bodies. further validation and confirmation of these data with mpss and rt-pcr documents the usefulness of high throughput microarray technology and identifies an additional set of previously unknown genes that are likely important in regulating the process of differentiation. these eb enriched genes in combination with downmodulated stemness genes may serve as biomarkers to monitor the transition of undifferentiated human embryonic stem cells to differentiated embryoid bodies. 
in order to identify biomarkers and potential regulators of the process of differentiation, a high quality microarray containing seventy base pair oligonucleotides was used to compare gene expression profiles of undifferentiated hesc lines and differentiating embryoid bodies. previously identified "stemness" genes in undifferentiated hesc lines showed down modulation in differentiated cells while expression of several genes was induced as cells differentiated. these results present a detailed characterization of a unique set of genes, which can be used to assess the hesc differentiation. a hierarchical clustering analysis clearly depicted a distinct difference in gene expression profile among undifferentiated and differentiated hesc and confirmed that microarray analysis could readily distinguish them. the identification of molecular pathways of differentiation of embryonic stem cells is critical for the development of stem cell based medical therapies. gene expression was validated by a variety of techniques including another large scale array, reverse transcription polymerase chain reaction, focused cdna microarrays, massively parallel signature sequencing analysis and immunocytochemisty. in addition, a subset of genes showed overexpression of greater than ≥ folds in human embryoid bodies.
 the identification of molecular pathways of differentiation of embryonic stem cells is critical for the development of stem cell based medical therapies. in order to identify biomarkers and potential regulators of the process of differentiation, a high quality microarray containing seventy base pair oligonucleotides was used to compare gene expression profiles of undifferentiated hesc lines and differentiating embryoid bodies.  previously identified "stemness" genes in undifferentiated hesc lines showed down modulation in differentiated cells while expression of several genes was induced as cells differentiated. in addition, a subset of genes showed overexpression of greater than ≥ folds in human embryoid bodies. these included novel and known genes. gene expression was validated by a variety of techniques including another large scale array, reverse transcription polymerase chain reaction, focused cdna microarrays, massively parallel signature sequencing analysis and immunocytochemisty. several novel heb specific expressed sequence tags were mapped to the human genome database and their expression profile characterized. a hierarchical clustering analysis clearly depicted a distinct difference in gene expression profile among undifferentiated and differentiated hesc and confirmed that microarray analysis could readily distinguish them.  these results present a detailed characterization of a unique set of genes, which can be used to assess the hesc differentiation. 