Abstract
BackgroundUnder appropriate culture conditions, undifferentiated embryonic stem (ES) cells can undergo multiple self-renewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. However, whether it plays an important role in stem cell maintenance remains to be defined.Methodology/Principal FindingsHere we provide evidence to show that an increase in the level of ABCG2 was observed accompanied by ES colony expansion and then were followed by decreases in the level of protoporphyrin IX (PPIX) indicating that ABCG2 plays a role in maintaining porphyrin homoeostasis. RNA-interference mediated inhibition of ABCG2 as well as functional blockage of ABCG2 transporter with fumitremorgin C (FTC), a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle and reduced expression of the pluripotent gene Nanog. Overexpression of ABCG2 in ES cells was able to counteract the increase of endogenous PPIX induced by treatment with 5-Aminolevulinic acid suggesting ABCG2 played a direct role in removal of PPIX from ES cells. We also found that excess PPIX in ES cells led to elevated levels of reactive oxygen species which in turn triggered DNA damage signals as indicated by increased levels of γH2AX and phosphorylated p53. The increased level of p53 reduced Nanog expression because RNA- interference mediated inhibition of p53 was able to prevent the downregulation of Nanog induced by FTC treatment.Conclusions/SignificanceThe present work demonstrated that ABCG2 protects ES cells from PPIX accumulation during colony expansion, and that p53 and γH2AX acts as a downstream checkpoint of ABCG2-dependent defense machinery in order to maintain the self-renewal of ES cells.
Highlights
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts
The ABCG2 multidrug-transporter is widely expressed in a variety of tissue stem cells and cancer stem cells, suggesting that it plays an important role in self-renewal maintenance
We have identified an endogenous role of ABCG2 in maintaining the selfrenewal of embryonic stem (ES) cells during self-renewal cycles
Summary
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. ES cells are unique in that unlike differentiated cells they do not accumulate DNA damage during multiple self-renewal cycles. It has been shown that the mutation frequency in ES cells is low because ES cells are sensitive to DNA damage and readily undergo apoptosis or differentiation in order to remove damaged cells from the self-renewal pool [5,6,7]. Under appropriate culture conditions, undifferentiated embryonic stem (ES) cells can undergo multiple selfrenewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. Whether it plays an important role in stem cell maintenance remains to be defined
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