Short peer-review storytelling : One big technical problem of human embryonic stem cells (hESCs) (in contrast to mouse embryonic stem cells) that hESCs normally undergo high rates of spontaneous apoptosis and differentiation, making them difficult to maintain in culture. Now we are getting to know a bit more on the molecular background of these processes. In an article in the prestigious Journal of Biological Chemistry Qin et al.’s studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation.
J Biol Chem. 2007 Feb 23;282(8):5842-52
“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.”
Figure 2 for the pros:
p53 binds to mitochondria and induces apoptosis in hESCs. A, confocal immunofluorescent detection of p53 and a mitochondrial protein Hsp 75 before and 6 h after 20 J/m2 UV in hESCs. The nucleus is stained by DAPI (bar, 8 µm). Cells stained without primary antibodies were used as a negative control (data not shown). B, Western blot of p53, Cox IV (mitochondria), and histone H3 (nuclear) of crude cell lysates and mitochondrial fractions from hESCs, UV-irradiated hESCs, and UV-irradiated hESCs pretreated with 20 µM pifithrin-alpha for 12 h. Cells were harvested 4 h after 20 J/m2 UV irradiation. C, caspase-9 activity of hESCs, H1-null, and H1-p53si cells before and 14 h after 20 J/m2 UV. Results represent the average of triplicates. **, p < 0.01. D, establishment of p53 knockdown H1 cells using the lentiviral vector pLL3.7. H1 cells were infected by lentivirus encoding siRNA specific for p53 (H1-p53si) and a control without the siRNA sequence (H1-null). This lentiviral vector also contained a GFP marker under the control of the cytomegalovirus promoter. The GFP-positive cells were selected under fluorescence microscopy and were found to retain GFP expression after 25 passages. Immunofluorescence detection of p53 was found in H1-p53si and H1-null cells. Cells were irradiated with 20 J/m2 UV and were analyzed 6 h later. The nucleus is stained by DAPI (bar, 100 µm). Cells stained without primary antibodies were used as a negative control (data not shown). E, Western blot of p53 in H1, H1-null, H1-p53si cells before and 6 h after 20 J/m2 UV irradiation. F, percentage of non-viable cells of H1-null and H1-p53si cells at different time points after 20 J/m2 UV. Data represent the average of triplicates. G, percentage of viable cells 36 h after 20 J/m2 UV in H1-null and H1-p53si cells pre-treated with Me2SO (DMSO) or 20 µM pifithrin-µ (Calbiochem) for 2 h. ***, p < 0.001.