In this study we show that, when compared to differentiated cells or cancer cells, hESCs express elevated levels of multiple pro-apoptotic BCL-2 family members. We took advantage of a microfluidics-based qPCR platform to measure all of the human BCL-2 family members across 48 samples simultaneously. The samples consisted of 12 cell lines each having 3 or, in some cases, 4 replicate cultures, which were established from separately thawed cryologically-preserved cell stocks. Additionally, hESC lines (BG01 and TE06) were each grown under both MEF feeder and feeder-free conditions. Owing to the disparate cell types evaluated for gene expression, we first measured expression levels of 15 reference genes, and identified two genes (PGK1/PPIA) that best reflected total mRNA levels. As a result of this experimental design, we are confident that the gene expression levels calculated from our qPCR experiments accurately reflect differences in abundance of mRNA transcripts among the dozen cell types studied. We hypothesized that one, or perhaps two BH3-only BCL-2 family members would be dominantly expressed in hESCs, and that these pro-apoptotic transcripts would be balanced by select pro-survival BCL-2 family members. From these patterns of expression, we hypothesized that the principal pathways governing apoptosis in hESCs could be revealed.
Contrary to our expectations, we found that hESCs express the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF, and PUMA at levels far greater than that seen in the seven human primary cells, hESC-derived neural stem cells, or cancer lines (). However, only one of the pro-survival BCL-2 family members, BCL-2, was found to be moderately elevated in hESCs compared to that seen in other cell types. Transcript abundance of two other pro-survival BCL-2 family members, BCL-x and MCL-1, was merely comparable to that seen in the other ten cell lines, and expression levels of BCL-w, and A1 were minimal in hESCs (our assays did not distinguish between transcripts for BCL-xL and BCL-xS). What is more, unsupervised hierarchical clustering of these gene expression profiles resulted in the clustering of NOXA, BIK, BIM, BMF, and PUMA together with hESC pluripotency markers like NANOG and SOX2. We interpreted these findings as further indication that elevated expression of these BH3-only BCL-2 family members is a genuine feature of hESCs when they are either co-cultured with MEFs or grown on Matrigel with conditioned media.
Despite the fact that well over a decade has passed since the first human embryonic stem cell lines were established, we have yet to uncover the complete list of MEF-derived molecular determinants of hESC pluripotency and proliferation 
. It is formally possible that a deficient trophic environment explains the ubiquitous observation that hESCs have elevated rates of apoptosis 
. From this, it would logically follow that death of hESCs in culture might require certain pro-apoptotic BH3-only BCL-2 family members. However, it was only recently reported that BCL-2 family members play a role in governing basal rates of apoptosis in hESCs 
. In one study by Ardehali et al., BCL-2 overexpression in hESCs was found to improve overall growth rates, eliminate reliance on knock-out serum replacement, increase clonogenicity after single cell dissociation, and reduce apoptosis associated with newly formed embryoid bodies 
. In another study by Ohgushi et al., apoptosis in hESCs resulting from single cell dissociation was shown to be inhibited by overexpression of the pro-survival BCL-2 family member BCL-xL 
. That a pro-survival BCL-2 family member could reduce dissociation-induced apoptosis and increase the overall growth rate of hESCs suggests that basal rates of apoptosis are most likely mediated by constitutive expression of pro-apoptotic BH3-only family members. Our data suggest that the pro-apoptotic BH3-only family members responsible for basal rates of apoptosis include NOXA, BIK, BIM, BMF, and PUMA, or a subset thereof.
Despite the constitutive overexpression of the pro-apoptotic BCL-2 family members NOXA, BIK, BIM, BMF and PUMA in hESCs, it is conceivable that the activities of these molecules would be inhibited via post-translational modifications, and that apoptosis in hESCs may be controlled by other novel signaling pathways. Accumulating evidence in the literature indicates that many BH3-only BCL-2 family members are regulated by post-translational modifications 
. Whereas nothing is currently known about what, if any, post-translational modifications of pro-apoptotic BH3-only BCL-2 family members might occur in hESCs, limiting the apoptotic activity of these molecules to some extent would be essential for net proliferation to occur.
For many of the pro-apoptotic BCL-2 family members, including BAD, BIM, BMF, BIK and NOXA, apoptosis-promoting activity can be regulated by phosphorylation. Perhaps the most well known example of this is in the case of BAD, wherein phosphorlyation of serine residues by multiple kinases, including AKT, can promote complex formation with 14-3-3, resulting in cytosolic sequestration 
. Similarly, ERK1/2 or RSK1/2-mediated phosphorylation of BIM blunts the pro-apoptotic activity of this molecule 
. Instead of being sequestered in the cytosol by 14-3-3, as in the case of BAD, BMF associates with DLC2, thereby limiting BMF's pro-apoptotic activity 
. BIK is also under post-translational control, but in this case, casein kinase-mediated phosphorylation of BIK stimulates its apoptotic function 
. More recently, NOXA was also discovered to be the target of phosphorylation 
. The atypical cyclin-dependent kinase CDK5 phosphorylates NOXA when cells experience glucose replete conditions. Phosphorylation of NOXA causes cytosolic sequestration via an unknown mechanism and blunted apoptotic function, in addition to inducing a novel NOXA-mediated stimulation of the pentose phosphate pathway 
. To date, no evidence of post-translational modifications of PUMA have been identified, but based on the growing list of post-translational modifications that regulate BCL-2 family member function, post-translational modifications of PUMA remain a distinct possibility.
The picture that emerges is one in which hESCs may express pro-apoptotic BH3-only BCL-2 family members in a constitutive fashion, but that the apoptotic function of these molecules could be held in check either by complex formation with pro-survival BCL-2 family members (likely BCL-xL or MCL-1) or by apoptotic-inhibitory phosphorylation of NOXA, BIK, BIM, BMF and possibly PUMA, or complex formation with other inhibitory molecules (e.g., 14-3-3 for BAD, DLC2 for BMF). The advantage of such a scenario might be that apoptotic responses could be engaged more rapidly and robustly, requiring only kinase cascades to control activation of apoptosis. The danger to the cell, and a possible explanation of the challenge of growing hESCs, is that seemingly minor changes in the environment could potentially result in cell death.
In conclusion, we have shown that, compared with differentiated cells, hESCs express an unusual repertoire of BCL-2 family members. hESCs cultured under standard growth conditions contain elevated levels of transcripts encoding pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF, and PUMA, when compared to differentiated cells. This, in light of the fact that complementary increases in pro-survival BCL-2 family members are not seen in hESCs, suggests that this expression profile could help to explain the elevated rates of apoptosis seen in hESCs. Recent reports demonstrating that overexpression of BCL-2 
or BCL-xL 
limits dissociation-induced apoptosis and increases overall growth rates is compatible with our finding that hESCs express elevated levels of pro-apoptotic BH3-only BCL-2 family members, and thus may be “primed” for apoptosis in a way that differentiated cells are not, and supports the notion that improvements in hESC culture methods could reduce the basal rates of apoptosis. With the large-scale propagation of hESCs remaining a major hurdle to the development of hESC-based therapies, our ability to limit the basal rate of apoptosis will be an important step toward achieving this goal.
Furthermore, given that, even in the face of elevated expression of pro-apoptotic BH3-only BCL-2 family members, hESCs are able to proliferate under standard growth conditions, hESCs might utilize novel regulatory mechanisms to control initiation of apoptosis. The importance of such a possibility is highlighted by the fact that one of the greatest obstacles to overcome in regenerative medicine is the potential of introducing teratoma-forming cells during transplantation of cells with desired phenotypes. Therefore, our ability to exploit apoptotic pathways unique to hESCs could improve the clinical outcome of therapies relying on hESC-derived cells.