It was found that human pluripotent stem cells are exquisitely sensitive to this genotoxic agent compared to differentiated cells, with DNA damage occurring as a result of stem cell exposure to only natural products drug discovery 0.5 ug/mL concentration of etoposide[44]. Incubation of hESCs with 0.2 umol/L etoposide for 16 h resulted in 80% hESC death[45]. The minor surviving fraction of hESC that recovered after etoposide treatment displayed undifferentiated morphology, even though the ability of these cells to differentiate into derivatives of all three germ layers was not directly examined[45]. The altered expression of key apoptosis regulators such as TP53 and BBC3 can at least

partly explain a rapid and extensive induction of apoptosis in etoposide-treated hESC cultures[46].

A high degree of sensitivity of hESC cultures to camptothecin, an inhibitor of DNA topoisomerase I, was recently observed[47]. Camptothecin exerts its cytotoxic effects by inducing DNA double-strand breaks (DSBs) in S-phase cells[48]. Even though CDKN1A mRNA was induced almost 5-fold compared to sham-treated H9 hESC cultures (1 µmol/L camptothecin, 3 h post exposure), the level of p21 protein remained undetectable[47]. This report also supports the prevailing view that P53 signaling pathway is crucial in execution of apoptosis and in preventing the propagation of DNA damage in genotoxic stress-exposed hESC cultures. Very recently, the adriamycin-induced DNA damage response in hESCs was characterized with ChIP-seq and microarray analysis[49]. About 1,326 genes were responsive to adriamycin in H9 line of hESCs, with TP53-target genes being implicated mostly in cell death, cell cycle (P < 10-6), and cell motility and migration (P < 10-4). TP53 was found to target highly distinct subsets of genes during genotoxic stress exposures compared to induced differentiation in hESCs, resulting in specific outcomes that partly overlap, but largely differ[49]. Importantly, genotoxic stress

– induced targets of TP53 in hESCs, human colon cancer cells, and human normal cells, such as fibroblasts and keratinocytes, are surprisingly different[50-52]. Therefore, changes in DNA damage-elicited gene expression are governed not only by stimulus-specific upstream signaling, but cellular GSK-3 milieu as well. Induction of apoptosis was observed as a default response to moderate and high levels of genotoxic stress in hESCs in many studies[20,53-55]. One of the radiomimetic drugs, neocarzinostatin, was shown to elicit a robust programmed cell death at concentrations as low as 0.1 µg/mL in H1 line of hESCs[20]. There were dramatic differences in how pro-apoptotic gene expression alterations manifest; for example, the levels of BAX remained unchanged, whereas BBC3, FAS, APAF1, and NOXA changed more than 2-fold[20].