Genomic instability during culturing of human embryonic stem cells

The first year of our CIRM project was highly successful in that we met our goals of establishing the necessary genetically engineered human embryonic stem cells (hESC) clones and assay systems that are required to perform our proposed experiments on how culture conditions in the laboratory can influence the stability of hESCs chromosomes. The hESC clones that we developed will allow us to monitor how culture conditions influence the caps on the ends of chromosomes, called telomeres, which are essential for maintaining the stability of chromosomes. Our earlier studies in mouse embryonic stem (ES) cells and human cancer cells using this same system have shown that telomere loss can result in many of the chromosome rearrangements found to result in cancer. Therefore, having these genetically engineered hESC clones will help to determine culture conditions that prevent telomere loss and cancer by hESCs. We have also devoted a large amount of time adapting various assay systems to monitor how culture conditions influence oxidative stress, telomere loss, chromosome stability, and differentiation of hESCs. This initial phase of our study was very time consuming, because the unique characteristics and culture conditions of hESCs required that each assay system be tailored specifically for hESCs. However, these assay systems are now in hand and we have initiated our studies to monitor the status of these various endpoints periodically during culturing of the hESCs under different conditions in the laboratory. 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.

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.

The third and last year of our CIRM project was highly successful in some aspects and disappointing in others. We adapted a variety assay systems to monitor how culture conditions influence the well being of hESCs in culture to insure that they are in optimal condition to be introduced into humans. These assays monitored oxidative stress, telomere loss, chromosome stability, and differentiation. We found that most of these assays did not provide useful information on the status of hESCs in culture. However, one of the assays proved to be highly valuable and can now be used as a tool to optimize the way that hESCs are grown in culture. This assay is called Q-FISH, and is used to monitor the status of telomeres, which are the caps on the ends of chromosomes. Telomeres are very susceptible to cell stress, and Q-FISH provides a way of monitoring whether telomeres are dysfunctional. We showed that while adding some growth factors to hESC cultures can increase their growth rate, this is not always a good thing, in that it can promote replication stress and telomere dysfunction, which can lead to chromosome instability. Because chromosome instability can generate cancer cells, it should be limited whenever possible. We also found that adding the nucleoside building blocks for DNA to culture medium can limit replication stress in hESCs. Q-FISH is therefore an important tool for optimizing and limiting replication stress in hESCs grown in culture. The second part of our study proved to be much more frustrating. This part of the project involved the development of hESC clones in which we inserted specific genes adjacent to telomeres at the ends of chromosomes. This approach allows us to monitor when a telomere is lost and the consequences of telomere loss for chromosome instability and cancer. We have successfully used this approach to study the consequences of telomere loss in human tumor cells and mouse embryonic stem cells. The project started out well in that we were successful in generating three different hESC clones that have the necessary genes integrated adjacent to a telomere. However, despite considerable effort, we were unable to isolate cells in which the telomere had been lost. Therefore, we were unable to conduct the in depth studies that we have previously performed in human tumor cells and mouse embryonic stem cells. Based on our results, we now conclude that the reason for our frustrating results is that hESCs are very sensitive to telomere loss and that hESCs that lose a telomere fail to proliferate, either due to cell death or due to differentiation and cell cycle arrest. Importantly, while this was bad for our project, it is encouraging for the use of hESCs in humans, since it means that hESCs have very sensitive pathways for preventing chromosome instability due to telomere loss.