Evaluation of ABCG2 Expression in Human Embryonic Stem Cells: Crossing the Same River Twice?   (2009)

A recent publication in Stem Cells states that human embryonicstem (ES) cells do not express ABCG2 and ‘‘…absence ofABCG2 isa novelfeatureof humanpluripotentstem cells,whichdistinguishes them from many other stem cells including mouseES cells’’ [1]. This is in sharp contrast to our observations [2]and the report of several other investigators who detectedABCG2 mRNA in various human ES cells [3–6]. The presenceof multidrug resistance ABC (MDR-ABC) transporters may sig-niﬁcantly contribute to stem cell defense mechanisms; thus, thisis an important question that should be addressed properly.Our interest in ABC transporters dates back to the discov-ery of their role in cancer drug resistance over two decadesago. Since then, we have had ample opportunity to experiencehow insufﬁcient methodology and a simplifying approachmay obscure the assessment of the impact of MDR-ABCtransporters on cancer patient survival. Measuring the func-tional expression of ABC transporters proved challengingbecause of the heterogeneity of tumors, the varying levels ofexpression, and the unreliability of the assay systems usedthroughout the trials. As a result, most reports were consid-ered controversial, and the true contribution of MDR-ABCtransporters to therapy failure could only be established onceassay conditions were standardized [7]. The key teaching ofthese extensive studies have immediate relevance to exploringtransporter expression in stem cells. First, MDR-ABC trans-porters are active extrusion pumps that may signiﬁcantly mod-ify cellular homeostasis or endobiotic and xenobiotic resist-ance even at low levels. Therefore, the assays measuring theirimpact should be sensitive, quantitative, and should preferablytarget the function of the MDR-ABC transporters. Second,samples are often heterogeneous for MDR-ABC expression,as these proteins are rapidly regulated by numerous mecha-nisms, both at the transcriptional and processing levels. How-ever, this initial heterogeneity may be relevant in circumstan-ces of stress, survival, or proliferation. Third, in many cases,the cell type, the mechanism of cell transformation, or differ-entiation does not determine the expression or function ofMDR-ABC transporters. Rather, ABC transporters are modu-lated by numerous environmental conditions [7, 8].In the case of the paper by Zeng et al. [1], the appre-ciation of these features is not possible as there are manyexperimental ﬂaws that are reminiscent of the limitationsthat our ﬁeld had to overcome to evaluate the MDR ofcancer. First, the reverse transcription polymerase chainreaction (RT-PCR) results are not quantitated, and there isno effort to perform quantitative PCR studies for thedetection of the relevant messages. Second, the Hoechstdye efﬂux studies lack the essential negative control. Third,instead of using a highly speciﬁc ABCG2 inhibitor, theauthors make their case on the basis of the effect of vera-pamil, which is a weak and nonspeciﬁc inhibitor ofABCG2. Fourth, the immunostaining studies are not con-vincing, the antibody used requires cell permeabilization,and the membrane localization of ABCG2 is not examined.Fifth, detection of subpopulations is contradictory and isnot evaluated in the context of co-expression of stem cellmarkers. Therefore, this study does not allow conclusionsto be drawn regarding the presence or up- and downregu-lation of ABCG2 in human ES cells.In contrast, we emphasize again that with appropriateexperimental tools, the functional although heterogeneousexpression of membrane ABCG2 is detectable in undifferenti-ated human stem cells. Detailed documentation is not possi-ble here, but the key features of ABCG2 expression in fourdifferent ES cell lines are depicted in Figure 1 and in thesupporting information video. Here we used properly quanti-tated real-time PCR measurements, ﬂow cytometry, and con-focal microscopy with costaining of relevant surface markers.Furthermore, we compare ES cells grown on MEF or Matri-gel, and we also evaluate the expression pattern of a mesen-chymal-like cell line (Figure 1C (F2)). We also document amicroscopic measurement of Hoechst dye uptake in undiffer-entiated stem cells, which is modulated by a speciﬁc ABCG2inhibitor. All these measurements suggest that ABCG2 ispresent at relatively high levels in the undifferentiatedhuman ES cells, highlighting its role in the protection of thisvaluable sanctuary against the damage by toxins, drugs, orhypoxia [8, 9].