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.