markers associated with pluripotency and stem cell phenotype are often expressed in malignant tissues [ 1 , 2 ] . these pluripotent markers can serve as useful diagnostic tools [ 3 , 4 ] or as potential targets for cancer stem cell - directed therapies . stage - specific embryonic antigen ( ssea ) 5 is a novel cell surface marker of pluripotency . although ssea-5 immunoreactivity has been established for ipscs , it is unknown whether ssea-5 is also expressed in human malignancies . for comparison with ssea-5 , we chose l1cam , a cell surface adhesion molecule involved in self - renewal and pluripotency of embryonic stem cells . shrna targeting of l1cam expression in glioma cells inhibits tumor growth in vivo and increases the survival time of tumor - bearing animals . since both ssea-5 and l1cam are cell surface markers of pluripotency , they may also be useful together in monitoring the presence of pluripotent cells in ipsc populations that undergo differentiation . therefore , we examined human tumors , tumor cell lines , teratomas , and ipscs for immunoreactivity to ssea-5 and l1cam . the goals of this study were to ( 1 ) evaluate immunoreactivity of ssea-5 in human tumors , ( 2 ) assess potential colocalization of ssea-5 with another cell surface marker of pluripotency ( l1cam ) , and ( 3 ) to monitor cell populations for the presence of residual undifferentiated cells in ipscs that undergo retinal differentiation . the imr90 - 4 ips cell line was maintained in mtesr1 medium ( stem cell technologies , vancouver , ca ) on matrigel ( bd biosciences , san jose , ca ) and differentiated according to published protocols . retinoblastoma cell line rb143 was grown in rpmi 1640 ( life technologies , grand island , ny ) with 10% calf serum ( hyclone laboratories , inc . , logan , utah ) , and prostate carcinoma cell line lncap clone fgc cells were grown in rpmi 1640 with 10% fetal bovine serum ( american type culture collection atcc , manassas , va ) . u87-mg and t98 g , glioblastoma cell lines , hct 116 , a colorectal carcinoma cell line , and ht-29 , a colon adenocarcinoma cell line , were maintained in mccoy 's 5a medium ( atcc ) with 10% fetal bovine serum . capan-1 pancreatic adenocarcinoma cells were cultured in iscove 's modified dulbecco 's medium ( atcc ) with 20% fetal bovine serum . pc-3 prostate adenocarcinoma cells were grown in f-12k medium ( atcc ) with 10% fetal bovine serum . panc-1 , a pancreatic carcinoma cell line , was grown in dmem ( atcc ) with 10% fetal bovine serum . john ludlow ) and y79 ( atcc ) were cultured in dmem with 10% calf serum . mda - mb-231 , a human breast adenocarcinoma cell line , was grown in leibovitz 's l15 medium ( atcc ) with 10% fetal bovine serum at 37c in 100% room air . rb143 human retinoblastoma cells were magnetically separated using abcg2-fitc antibody ( stem cell technologies , vancouver , british columbia ) . we chose these two cell populations ( abcg2 + ( stem - like cells ) versus abcg2 cells in retinoblastoma ) based on our previous studies [ 1012 ] . the resulting abcg2 + and abcg2 cells were compared using a microarray ( agilent technologies ) , and a list of genes with differential expression was generated . l1cam was selected from the list for this study based on differential expression , relevance to stem cell biology and cell surface localization for comparison with ssea-5 . cells were rinsed with pbs , spun onto slides in cytospin solution : 72% isopropanol , 19% acetone , and 7.6% glycerol using a shandon cytospin ii and allowed to dry .   for diaminobenzidine immunostaining , the max poly one kit ( syd labs , malden , ma ) was used . slides were blocked for 10 minutes with 3% h2o2 , followed by blocking with protein block for 10 minutes . primary antibody diluted in pbs ( or isotype control ) was added to the slides and incubated for 1 hour at room temperature . louis , mo ) used at 0.25  ug / ml , ssea-5 ( genetex , irvine , ca ) used at 10  ug / ml , and aldh1a1 ( abcam , cambridge ma ) used at 5.0  ug / ml . slides were washed three times with pbs and incubated in polymer hrp secondary antibody for 15 minutes . after washing in pbs , slides were incubated in dab ( diaminobenzidine ) for 5 minutes . , grand island , ny ) was used to coverslip the slides , and three groups of 100 cells were counted for positive cells and graphed using prism software ( graphpad , la jolla , ca ) . retinoblastoma , adult , and embryonic tumor tissue arrays with corresponding normal tissues were obtained from us biomax , inc . prior to immunofluorescence staining of teratoma ( applied stem cell , inc , menlo park , ca ) and tumor arrays , slides were baked for 60 minutes at 60c , treated with xylene and alcohols , boiled in sodium citrate ( ph 6.0 ) , and blocked for one hour at room temperature in 1% bsa , 0.5% triton - x100 in pbs . for ipsc cytospin slides , the xylene , alcohol , and sodium citrate steps were omitted . primary antibodies in blocker ( or isotype control ) were added to the slides for one hour at room temperature . slides were washed three times in pbs and then incubated in tritc and/or fitc conjugated secondary antibody ( sigma , stock concentration listed as ranging from 36.5  mg / ml ) , diluted 1  :  400 in pbs . slides were cover - slipped using prolong gold mounting medium containing dapi ( invitrogen , inc . ) and photographed using spot advanced software .   for dab staining of tumor cell lines , three groups of 100 cells were counted for immunopositive cells and averaged and graphed using prism software . we evaluated immunoreactivity of ssea-5 and l1cam on a variety of human tumors to determine staining patterns and potential regions of colocalization . immunofluorescence staining with ssea-5 and l1cam was carried out on tissue arrays of retinoblastoma , embryonic tumors , and adult tumors . as shown in figure 1 , ssea-5 exhibited strong immunoreactivity in embryonal carcinoma and primitive neuroectodermal tumors , while l1cam was much less intense . in urothelial carcinoma , both l1cam and ssea-5 were seen , with many regions of colocalization . l1cam ( yellow bars ) are plotted against the left y - axis ; ssea-5 ( blue bars ) are plotted against the right y - axis . as seen in figure 2(a ) , capan-1 , a pancreatic adenocarcinoma cell line , and ht-29 , a colorectal adenocarcinoma cell line , showed the strongest ssea-5 staining . capan-1 displayed the greatest number of ssea-5 + cells , while u87 mg demonstrated the highest numbers of l1cam+ cells . for all bars not visible on the graph , the number of positive cells was less than 1% , as were isotype controls . in figure 2(b ) , examples of immunofluorescent staining are shown . t98 g cells have very few l1cam+ cells ( less than 1% ) and no detectable ssea-5 . since ssea-5 is a marker of teratoma - forming potential for ipscs , we examined ipsc - induced teratomas for ssea-5 and l1cam immunoreactivity . paraffin sections of teratomas grown in mice from undifferentiated ipscs were obtained from applied stemcell ( menlo park , ca ) and immunostained for ssea-5 ( red ) and l1cam ( green ) . since both ssea-5 and l1cam antibodies were specific for human markers , they bound specifically to human ipscs that became part of the teratoma . in figure 3(a ) , immunostaining of teratoma slides with ssea-5 and l1cam indicated that both of these markers were expressed , revealing the presence of ssea-5- and l1cam - expressing pluripotent cells . there were distinct areas of colocalization for ssea-5 and l1cam within the teratoma . to further validate the presence of pluripotent stem cell markers , we chose aldh1a1 , a stem cell marker , for a double labeling experiment with ssea-5 , seen in figure 3(b ) . colocalization of ssea-5 and aldh1a1 was evident in the teratoma tissue . since ssea-5 is expressed in pluripotent ipscs , as well as ipsc - induced teratomas ( figure 3 ) , we investigated the possibility that ipscs differentiated to a retinal progenitor cell fate would contain persistent ssea-5 + or l1cam+ cells . figure 4 illustrates the presence of both l1cam and ssea-5 in ipscs , with undifferentiated ipscs ( day 0 ) exhibiting strong immunofluorescence staining and colocalization . after 20 days of differentiation ( day 20 ) , ssea-5 and l1cam immunoreactive ipscs remain , also with extensive colocalization . this report represents the first demonstration of ssea-5 immunoreactivity in human tumors and tumor cell lines and partial colocalization with the cell surface marker l1cam . ssea-5 immunoreactivity in embryonic and neuroectodermal tumors is consistent with the pluripotent nature of the marker . although there was some colocalization with l1cam in human tumors , there were distinct areas of ssea-5 immunoreactivity alone . the regions of single versus double staining for ssea-5 and l1cam require further analysis to determine other important regional differences within these tumors . we find biological variation in the percentage of positive cells for most markers in a variety of tumors and cell lines . the percentage of positive cells may depend on the source of the tumor ( different patient samples ) , regional variation within a particular tumor , primitive versus differentiated state of the tumor , and so forth . so we are very careful not to overgeneralize the presence of a marker in all tumors of a particular type . since these samples contained all tumor cells , it was not possible to identify specific tissue cell types as being immunoreactive . additional studies are necessary with a large repository of tumors in order to determine whether ssea-5 and/or l1cam may be useful for diagnostic histopathology in a clinical setting . the heterogeneity of these cell lines is not surprising , considering that in our previous studies , abcg2 was expressed in 4% of retinoblastoma cell lines [ 1012 ] . capan-1 and ht-29 , both originating from adenocarcinomas , were the only two cell lines in our study to exhibit significant ssea-5 immunoreactivity . interestingly , capan-1 , a pancreatic adenocarcinoma cell line , had a high percentage of ssea-5 + cells , while panc-1 ( a pancreatic carcinoma cell line ) was immunonegative for ssea-5 . at the same time , panc-1 , lncap , weri - rb27 , y79 , and u87 mg cell lines contained l1cam immunoreactive cells , yet none of these cell lines displayed high levels of ssea-5 immunoreactivity . this list of cell lines is not exhaustive and further investigation into the presence these markers in a variety of tumor cell types is warranted to determine the significance of ssea-5 and l1cam expression . once a sequence is established for ssea-5 , pcr primers can be designed for further analysis . stem cells have considerable therapeutic potential , especially with the advent of induced pluripotent stem cells ( ipscs ) . the potential of ipscs as therapeutic agents for disease treatment is great but comes with the risk of uncontrolled growth of residual pluripotent cells . ipscs share common pathways with oncogenic foci , and there are recent reports of mutations and epigenetic changes in ipscs that warrant further investigation [ 17 , 18 ] . in this study , we demonstrated ssea-5 and l1cam immunoreactivity with areas of colocalization in ipsc - induced teratomas , as well as ipscs before and after 20 days of differentiation to a retinal progenitor cell fate . our results indicate that residual l1cam+ and ssea-5 + cells remain in ipscs after 20 days of differentiation . these results confirm the importance and point to a potential means of evaluating the persistence of undifferentiated ipscs cells following differentiation protocols . future studies will reveal the utility of ssea-5 and l1cam in diagnostic and therapeutic strategies in both cancer biology and regenerative medicine .
pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors . 
 the novel pluripotent marker stage - specific embryonic antigen-5 ( ssea-5 ) is expressed in undifferentiated human induced pluripotent cells ( ipscs ) , but little is known about ssea-5 expression in other primitive tissues ( e.g. , human tumors ) . 
 we evaluated ssea-5 immunoreactivity patterns in human tumors , cell lines , teratomas , and ips cells together with another pluripotent cell surface marker l1 cell adhesion molecule ( l1cam ) . 
 we tested two hypotheses : ( 1 ) ssea-5 and l1cam would be immunoreactive and colocalized in human tumors ; ( 2 ) ssea-5 and l1cam immunoreactivity would persist in ipscs following retinal differentiating treatment . 
 ssea-5 immunofluorescence was most pronounced in primitive tumors , such as embryonal carcinoma . in tumor cell lines , ssea-5 was highly immunoreactive in capan-1 cells , while l1cam was highly immunoreactive in u87 mg cells . 
 ssea-5 and l1cam showed colocalization in undifferentiated ipscs , with immunopositive ipscs remaining after 20 days of retinal differentiating treatment . 
 this is the first demonstration of ssea-5 immunoreactivity in human tumors and the first indication of ssea-5 and l1cam colocalization . 
 ssea-5 and l1cam warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in ipsc populations prior to therapeutic use .