Year 3

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.