Multiple mechanisms have been described that sensitize ESCs

Multiple mechanisms have been described that sensitize ESCs to DNA damage-induced apoptosis. First, human ESCs possess unique ROCK-dependent mechanisms in singularized molar weight calculator that lead to a myosin-mediated form of cell blebbing, which rapidly triggers apoptosis upon cell detachment (Ohgushi et al., 2010). Second, unlike differentiated cells, certain human ESC lines have been found to express a constitutively pre-activated form of the proapoptotic BCL-2 protein BAX at the Golgi apparatus, which may quickly translocate to the outer mitochondrial membrane and initiate execution of the intrinsic apoptosis pathway upon DNA damage (Dumitru et al., 2012). Notably, the basal level of pre-activated BAX varies among different human ESC lines and, for example, is not detectable in the H1 cell line. Nonetheless, H1 cells show the typical sensitivity to DNA damage, suggesting that additional mechanisms might be involved in priming ESCs for rapid cell death (Liu et al., 2014).
Results
Discussion Pluripotent stem cells must tightly control the balance between cell survival and death to prevent unfavorable mutations and to ensure genomic integrity. First, maintenance of genomic stability must be particularly stringent, because any genetic alterations in pluripotent stem cells can impair the functionality of their progeny and compromise tissue renewal. Pluripotent stem cells bear an enhanced tumorigenic potential and share several characteristics with cancer cells, such as replicative immortality (Ben-David and Benvenisty, 2011). These properties require a quick and strong apoptotic response in DNA-damaged cells to prevent an accumulation of mutations that could facilitate deregulated proliferation or predispose cells to acquire further mutations associated with cancer development. It has been shown that ESCs are hypersensitive to DNA damage and readily undergo apoptosis (Qin et al., 2007; Madden et al., 2011; Liu et al., 2013), although much less is known about iPSCs. DNA damage sensitivity in human ESCs was shown to correlate with a property, termed mitochondrial priming, that is determined by the balance between pro- and antiapoptotic BCL-2 proteins (Liu et al., 2013). In the present study, we found that, compared to differentiated fibroblasts, iPSCs exhibit a low apoptosis threshold. Interestingly, the high apoptosis sensitivity was restricted to stimuli activating the mitochondrial pathway, whereas iPSCs were strongly resistant to the extrinsic apoptosis pathway, presumably by the downregulation of several death receptors. Unlike human ESCs (Dumitru et al., 2012), however, the increased sensitivity of iPSCs was not confined to DNA-damaging stimuli, but also observed after treatment with ER stress- or Golgi disassembly-inducing agents that also trigger the mitochondrial pathway. Human ESCs have been reported to maintain BAX in an active conformation at the Golgi apparatus under basal conditions (Dumitru et al., 2012), but this mechanism is unlikely the sole reason of their increased apoptosis sensitivity. In support of this notion is the finding that the H1 ESC line displays the typical apoptosis hypersensitivity, but lacks expression of active BAX at the Golgi (Dumitru et al., 2012). Moreover, we found that, also in human iPSCs, BAX was evenly distributed in the cytosol, but was not localized at the Golgi (Figure S2). Nevertheless, our results showed that, even under basal conditions, iPSCs reveal a strong accumulation of p53, a tumor suppressor, which also acts as a barrier to somatic cell reprogramming (Tapia and Schöler, 2010). p53 activates the transcription of multiple genes involved in apoptosis, in particular proapoptotic BCL-2 proteins. Indeed, compared to fibroblasts, expression of several p53 target genes including BAK, BIM, and NOXA was strongly upregulated, indicating that iPSCs exhibit high mitochondrial priming compared to differentiated cells. Fibroblasts, however, could be sensitized to DNA damage-induced apoptosis using ABT-737, an inhibitor of antiapoptotic BCL-2 proteins.