Year 3

The goal of our project is to improve methods for generating and banking human embryonic stem cells (hESCs), which are derived from human embryos. Since the federal government does not support work with human embryos, this research would be impossible without the funds we have received from the California Institute for Regenerative Medicine (CIRM). Additionally, our project has benefited greatly from CIRM Facilities Grants, which provided us with this specialized nonfederal laboratories that we need for experiments that employ human embryos.

Our project has three Specific Aims. The first is to derive hESC lines from intact human embryos that have been grown in the laboratory for approximately five days. This is the method that has been used to derive the overwhelming majority of the existing hESCs. In these experiments, cells that are destined to become hESCs emerge over several days. Thus, the precise origin and timing are unknown. Some investigators think that this element of randomness contributes to the significant differences that have been noted among hESCs. To date, we used this approach to derive one line, UCSF-4, which was approved by CIRM. Thus, other grantees of this agency can work with these cells. Since they were generated using standard techniques, UCSF-4 was approved by the National Institutes of Health. Therefore, researchers who have federal funding can also work with these cells.

The second Specific Aim proposed a new approach for deriving hESCs. We wanted to make lines from single cells that were removed from embryos at very precise stages of development. To date we have focused on the eight-cell stage, embryos that have been grown for three days in the laboratory. In these experiments, we used five embryos that were donated by a single couple. In all, we established 10 lines from individual cells. In other words, we derived multiple lines from the same embryo. Thus, this collection is unique because some of the lines are genetically identical and all have a high degree of genetic relatedness. This special property is important to stem cell biologists as we are trying to understand the changes that happened over time as the cells are maintained in the laboratory. Additionally, we have evidence that hESCs that are derived from embryos that have been grown for three rather than five days are more plastic in that they are better able to form the precursors of a broader range of cell types that comprise the human body.

Like UCSF-4, the ten lines that were derived from single cells removed from embryos were registered by CIRM. They were also submitted to the National Institutes of Health along with UCSF-4. However, we were notified that they do not fit the federal definition of an hESC line, which specifies derivation from an embryo that has been grown for five rather than three days in the laboratory. Accordingly, these lines are still pending approval, which will require changing the federal definition. Therefore, it is uncertain if they will ever be registered, a process that allows federally-funded scientists to work with hESC lines.

Our third Specific Aim was to derive a hESC line using Good Manufacturing Processes (GMP) as specified by the Food and Drug Administration. These guidelines have been established to make sure that cell-based therapies are safe for patients. For example, they are designed to prevent the spread of infectious agents that can contaminate animal cells to humans. One obstacle to producing GMP-grade cells is that hESC derivation requires a carpet of another cell type, which is thought to “feed” embryos the substances that are required for generation of hESC colonies. Thus these “feeder” cells must also be produced using GMP methods. To avoid this laborious step, we have developed methods for the “feeder-free” culture of hESCs. We plan to use the same approach to derive hESCs from intact embryos.

To date, with CIRM support, we have derived 11 hESC lines. Therefore, distributing these cells has become a significant part of this project. It is interesting to note that these lines are being used for a wide variety of experiments. For example, UCSF-4 is being studied by a Consortium that has been formed to understand how chemical modifications to genes control their expression. This is a particularly interesting question because the programs that specify the initial stages of human development are very poorly understood.

The other groups who are using our cells are focusing on a wide range of normal and disease processes. Several investigators are studying the mechanisms by which cells of the brain and spinal cord develop. One group hopes to use them as a therapeutic strategy for patients with amyotrophic lateral sclerosis or Lou Gehrig’s disease. Other investigators are studying formation of cardiac muscle, pancreatic beta cells and the liver. Finally, our lines are also being used for training purposes in our CIRM Shared Research and Teaching Facility.