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Another Chapter in the Story of ESCs and ABCG2

Original article from STEM CELLS

“Regulation and Expression of the ATP-Binding Cassette Transporter ABCG2 in Human Embryonic Stem Cells”

ATP-binding cassette sub-family G member 2 (ABCG2) is known to function as a multidrug efflux pump and plays a role in resistance to chemotherapeutic agents in cancer cells. While it has been implicated as the cause of the “side population” in adult stem cells, its role in human embryonic stem cells (hESCs) remains unclear. Two competing groups have reported seemingly contradictory findings; one reports that ABCG2 mRNA and protein are present at a high level in two hESC lines (HUES1 and HUES9) (Apati et al and Sarkadi et al), while another has suggested that ABCG2 mRNA and protein is absent in three hESC lines (HUES1, H9 and CT2) (Zeng et al). These results therefore suggest that the role ABCG2 in hESCs merits another detailed investigation, which has now been attempted in a recent report in Stem Cells from the laboratory of Michael M. Gottesman at the National Cancer Institute at the National Institutes of Health. Their analysis found ABCG2 mRNA to be expressed in all hESC lines while protein levels were low or undetectable; suggesting a putative role for miRNA-mediated regulation of ABCG2 expression (Padmanabhan and Chen et al).

mRNA analysis of 9 hESC lines (BG01, BG02, BG03, ES02, SA01, TE03, UC01, UC06, and WA01) by RT-PCR and/or Northern blotting found abundant ABCG2 mRNA.   By RT-PCR, all lines showed a similar level of expression, while Northern blot analysis (8 day exposure) found mRNA expression at around 2.8–3.8 kb, consistent with the sizes of the full-length ABCG2 mRNAs in the 5 cell lines tested (TE06, WA01, UC06, BG03 and BG02).   Additionally, no splicing events were found for ABCG2.   Further mRNA analysis by whole genome analysis allowed the segregation of total gene expression levels into high, intermediate, and low to no mRNA expression categories.   From three samples from the BGO1 hESC line, it was determined that ABCG2 mRNA expression was at an intermediate level, alongside other important genes such as POUF51, KLF4 and TERT.

Subsequent protein analysis by immunostaining using an anti-ABCG2 that specifically recognizes an extracellular epitope of ABCG2 in the BG01 and WA01 hESC lines and control cells (mitoxantrone-resistant H460 cancer cell line H460/MX, which expresses high levels of ABCG2 protein, and its parental line H460 which does not express ABCG2 protein), found high levels of surface ABCG2 expression in the H460/MX cells, but no expression was observed for the hESC lines similar to the H460 cells. However, analysis of two additional hESC lines (TE03 and TE06), which are known for their high level of spontaneous differentiation, found a few isolated ABCG2+ colonies.

Western analysis of BG01 and WA01 hESC lines found that while ABCG2 was expressed highly in H460/MX cells, H460 cells express ABCG2 very weakly while the undifferentiated hESCs had undetectable ABCG2 protein despite having a similar mRNA level to the H460 cells. Additionally, the hESCs grown as embryoid bodies for 8-10 days failed to induce ABCG2 protein, suggesting that expression of ABCG2 is not specifically linked to differentiation but may be induced by specific signals. This theory was examined through the exposure of hESCs to BMP4, which efficiently differentiated WA01 and H9 hESCs by day 6 of exposure, and was accompanied by an increase in SSEA-1 expression and a decrease in SSEA-4 expression. In response to BMP4, ABCG2 levels increased slowly to a 7-fold increase at day 6 compared to untreated controls.

The intermediate levels of mRNA and the low or no expression of protein, suggests that ABCG2 expression in hESCs may be regulated by miRNAs. Previous studies have shown that two miRNAs (miRNA-519c and miRNA-520h) regulate ABCG2 protein translation in human cancer cells (To et al 2008 and To et al 2009) but this phenomenon has not been observed in hESCs. Encouragingly, expression of both miRNAs was found in WA01 cells, and levels of both miRNAs decreased around 9-fold by 144 hours of BMP4 treatment, concomitant with the increase in ABCG2 protein levels. miRNA control of ABCG2 was confirmed through the expression of specific miRNA hairpin inhibitors of miRNA-519c and miRNA-520h in WA01 cells which led to a 2.6-fold and 1.9-fold increase in ABCG2 protein after BMP4 treatment. However, co-expression of both inhibitors in WA01 cells abolished the increase in ABCG2, while H9 cells only showed a moderate increase in ABCG2 protein levels in response to miR-519 inhibitor only.   Conversely, expression of miR-519 and miR-520 mimics (chemically modified double-stranded oligonucleotides designed to mimic the function of endogenous mature miRNAs) decreased the levels of ABCG2 protein in H9 cells by 32% and 40% respectively when exposed to BMP4, however no obvious downregulation of surface ABCG2 was found after the expression of miR-519m or miR-520m or both in WA01 cells.

This paper suggests that all hESC lines express ABCG2 mRNA and that the protein is generally absent under self-renewing conditions; however the dynamics of ABCG2 appear to be cellular context-dependent. It must also be noted that the failure to analyse all aspects of ABCG2 dynamics in all hESC lines does not allow for concrete generalisations, with a side-by-side analysis of all hESC lines within this study and in the initial two studies likely to uncover more fine details. BMP4 treatment is however seen to be implicated as an inducer of ABCG2 protein expression while the miRNAs miRNA-519c and miRNA-520h appear to have a direct role in the repression of ABCG2 protein expression. These findings are a welcome addition to the ABCG2 story, and demonstrate the requirement for analysis of such biological concepts in multiple hESC lines. Further work will surely uncover finer detail regarding ABCG2 expression in hESCs and perhaps in induced pluripotent stem cells (iPSCs), its role in self-renewal/pluripotency/differentiation, and its control, which hopefully will remove the controversy so far generated.

 

References

Apati A et al.
High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells.
Biochim Biophys Acta 2008; 1778:2700–2709.

Padmanabhan and Chen et al.
Regulation and Expression of the ATP-Binding Cassette Transporter ABCG2 in Human Embryonic Stem Cells.
Stem Cells. 2012; 30(10):2175-87.

Sarkadi B et al.
Evaluation of ABCG2 expression in human embryonic stem cells: Crossing the same river twice?
Stem Cells 2010; 28:174–176.

To KK et al.
Escape from hsa-miR-519c enables drug-resistant cells to maintain high expression of ABCG2.
Mol Cancer Ther 2009; 8:2959–2968.

To KK et al.
Regulation of ABCG2 expression at the 30 untranslated region of its mRNA through modulation of transcript stability and protein translation by a putative microRNA in the S1 colon cancer cell line.
Mol Cell Biol 2008; 28:5147–5161.

Zeng H et al.
Lack of ABCG2 expression and side population properties in human pluripotent stem cells.
Stem Cells 2009; 27:2435–2445.

 

STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.