The second year of our CIRM project was very productive in that we were able to successfully establish and utilize a variety of assays for monitoring deleterious changes that could occur in human embryonic stem cells (hESCs) during growth in culture. These studies are important in that they provide valuable information on how to avoid alterations in hESCs during growth in culture that could make them cause cancer when introduced into humans. These assays are designed to detect alterations in hESCs brought on by the stress of unlimited growth in culture, including changes in chromosome number, dysfunction of the caps on the ends of chromosomes, called telomeres, and oxidative stress. Telomeres are particularly important for these studies, because they have been shown to be highly sensitive to cell stress brought on by continuous cell division and damage due to oxygen, which can occur in cultured cells. Although most of the assays showed little changes in hESCs during growth in culture, the Q-FISH assay for monitoring telomeres demonstrated significant amounts of telomere dysfunction in the cultured hESCs. This telomere function has detrimental consequences for the stability of hESC chromosomes, as demonstrated by the presence of anaphase bridges, which occur when chromosomes fuse together following the loss of a telomere. We are currently using the Q-FISH assay to address the reason for the telomere dysfunction in the cultured hESCs. Our results thus far have shown that telomere dysfunction is not affected by changes in the level of oxygen, suggesting that oxidative damage is not responsible. However, the addition of a growth factor, Activin A, which increases the growth rate of hESCs, caused a 50% increase in telomere dysfunction. This result suggests that the telomere dysfunction in hESCs in culture is a result of replication stress, which occurs when cells are continuously dividing under adverse conditions. We are therefore now using the Q-FISH assay to explore alterations in growth conditions that can ameliorate this stress. The second part of our project is to investigate the consequences of telomere loss in hESCs. Our previous studies with mouse embryonic stem cells demonstrated that telomere loss resulting from a DNA break adjacent to a telomere resulted in chromosome fusion leading to extensive chromosome instability. Other laboratories have shown that similar chromosome rearrangements resulting from telomere loss can result in cancer in mice. Importantly, we also found that mouse embryonic stem cells were capable of preventing this chromosome instability by adding a new telomere to the site of the break, a process called chromosome healing. We have shown that chromosome healing serves a vital role in preventing chromosome instability due to a deficiency in other mechanisms for repair of DNA near telomeres. Our studies in mouse embryonic stem cells required introducing DNA sequences at locations near telomeres so that we could introduce breaks in DNA at specific locations and select cells that had lost a telomere. This method allows us to simulate normal processes at a defined telomere, which allows us to study the consequences of telomere loss. To perform similar studies in hESCs, we have now generated hESC clones that contain these same DNA sequences integrated adjacent to a telomere. We are now in the process of generating DNA breaks at these locations near telomeres in the hESCs clones to determine whether telomere loss in hESCs also results in chromosome instability, and whether chromosome healing can prevent this chromosome instability. These studies will allow us to determine whether telomere loss and chromosome instability in hESCs can result in chromosome changes leading to cancer, and will provide critical information on how to culture hESCs to avoid specific alterations that could lead to cancer during hESC therapy.