Regulation of Apoptosis and Differentiation by p53 in Human Embryonic Stem Cells

Abstract

The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs), hESCs normally undergo high rates of spontaneous apoptosis and differentiation, making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However, despite the accumulation of p53, it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types, including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation.Human embryonic stem cells (hESCs) 3 are capable of essentially unlimited self-renewal and retain the developmental potential to differentiate into almost any cell type. These characteristics of hESCs make them attractive for tissue and cellbased therapies (1, 2). Previously, basic fibroblast growth factor and activin A were identified as self-renewal factors (3-6). However, for reasons that are not clear, hESCs often display high rates of spontaneous apoptosis and differentiation in culture, thus making the process of expanding these cells highly inefficient (3, 7-10). For example, Dravid et al. (8) reported that, under routine culture conditions, Ͼ30% of hESCs undergo spontaneous apoptosis. Furthermore, Ezashi et al. (12) showed that nearly 40% of hESCs undergo spontaneous differentiation after 12 days of culture in normoxic conditions. Finally, Maitra et al. (13) reported that multiple passages of hESCs can cause genomic alterations, which may limit the therapeutic application of hESCs. In contrast to hESCs, mouse embryonic stem cells (mESCs) undergo lower rates of spontaneous apoptosis and differentiation (14). Moreover, they maintain their pluripotency and genomic stability longer than hESCs (15). The reason for these different species-specific phenotypes in embryonic stem cells is currently unknown.The p53 tumor suppressor gene is a strong candidate for playing a role in the observed phenotypes of hESCs, because it regulates various cellular processes, including apoptosis, differentiation, and genomic integrity (16). In many cell types p53 plays a crucial role in controlling apoptosis and cell cycle arrest when these cells are exposed to stress-inducing conditions (17). In response to stress, p53 accumulates and transactivates downstream target genes such as mdm2 (responsible for the feedback degradation circuitry of p53), p21 (responsible for cell cycle control), bax, noxa, and puma (responsible for DNA damage-induced ...

Activation of p53 by nutlin leads to rapid differentiation of human embryonic stem cells

“…Our model clearly and principally differs from that of Qin et al (2007) and this may be a reason for this discrepancy. Qin et al induced p53 with UV, which caused within 6 h a significant increase of transcriptionally inactive p53 (and therefore there was no upregulation of p21 or hdm2) and downregulation of both OCT4 and NANOG at the transcriptional level.…”

“…Recently, Qin et al (2007) suggested that p53 could directly downregulate the expression of OCT4 transcription, which is important for retaining human stem cell pluripotency. Similarly, it has been shown that in mouse ESCs, p53 may bind to Nanog promoter and therefore inhibit its transcription (Lin et al, 2005).…”

Human Embryonic Stem Cells Are Capable of Executing G1/S Checkpoint Activation

“…Curiously, no such cell cycle block was noted in a previous study of UVC-irradiated hESCs; rather, massive cell death under DNA-damaging conditions was observed [29]. However, as this study did not use synchronized hESCs and did not focus on cell cycle regulation, rigorous comparisons to our data are difficult.…”

“…The function of p53-p21 axis has been studied by several groups in both mouse and human ESCs and in pluripotent human embryonal carcinoma cells using various strategies [10,[29][30][31][32]. Surprisingly, there have been many discrepancies between their findings, including differences in the ability of cells to transactivate the p21 gene or to produce increased levels of p21 protein when p21 transcripts are upregulated [10,[29][30][31]33]. When we examined p53 expression in hESCs with UVC-induced G1 delay, we found that at 3 hours after UVC irradiation, p53 protein is already dramatically increased, is phosphorylated on serines 15, 33, and 392, and localizes completely to cell nuclei.…”

Regulation of Mammary Progenitor Cells by p53 and Parity

“…We also used the p53 staining protocol that worked in regular tissue to stain irradiated mammospheres and we did not get typical nucleus staining. It is reported that ES cells could not activate p53-dependent stress response and that the p53 protein in ES cells were cytoplasmic and translocated ineffectively into nucleus 21,22 . It is possible that similar mechanism may exist in the mammary stem/progenitor cells to protect the body from losing the cells to DNA damage.…”