An innovative tool for high throughput, cost-effective monitoring of chromosomal abnormalities in stem cells
Tools and Technologies I
Most normal, human cells have two copies of each chromosome – one inherited from one’s Mom and the other from Dad. Though normal stem cells have two copies of each chromosome, they can lose or gain chromosomes as they are grown in the laboratory. These abnormal stem cells are not like their normal counterparts and there is evidence that such cells might cause cancer when transplanted into patients. The development of chromosomal abnormalities in stem cells is one of the major barriers preventing the clinical application of stem cell therapies. This was demonstrated recently when the FDA suspended a clinical trial of a stem cell based treatment for spinal cord injury over concerns that the cells might have chromosomal abnormalities. Here we propose to generate stem cells that will emit a fluorescent signal when they develop chromosomal abnormalities. This way, scientists can study how the laboratory conditions in which the cells are grown influence whether or not the cells develop chromosomal abnormalities. Once we understand what causes the cells to develop chromosomal abnormalities we can grow stem cells in a manner that will prevent this, thus hastening the progress of stem cell therapies from the laboratory into the clinic.
Statement of Benefit to California:
The State of California faces immense challenges to its health care system, with soaring medical costs and an aging population. At the same time, investors are becoming more wary of funding high-risk technology development that has fueled California’s high-tech and biotech booms. The taxpayers of California have made a substantial investment in scientists who are dedicated to development of stem cell therapies that may revolutionize medicine and health care by providing new treatments for incurable conditions such as diabetes, Parkinson's disease, and spinal cord injuries. Stem cell therapies, however, are in an early stage and it is critical that research over the next few years be focused on development of cells that will be both safe and effective. A significant roadblock to progress toward the clinic is the fact that stem cells become chromosomally unstable with prolonged time in culture and such cells, when injected into patients, may cause cancer. Our current understanding of the factors that cause cells to develop chromosomal abnormalities is inadequate. We propose to develop tools that will allow scientists to monitor chromosomal abnormalities in live stem cells in a high throughput, cost effective manner. This information will be used to optimize the large-scale culture of chromosomally stable human embryonic stem cells (hESCs) for therapeutic purposes. The technology and data developed for this proposal will help to ensure that stem cells used for therapy are normal and free of chromosomal abnormalities that can cause devastating effects like cancer. We propose to develop this technology so that it can be integrated into the repertory of methods that can be used to ensure quality control and safety of cell therapies. We will make these tools available to stem cell researchers and clinicians throughout California. Ultimately, this technology will benefit California by attracting highly skilled jobs and tax revenues, and by making the State a leader in a field that is poised to be the economic engine of the future.
This application focuses on the development of human embryonic stem cell (hESC) reporter lines for detecting aneuploidy and using these lines to identify culture conditions that lead to chromosomal instability. In the first aim, the Principal Investigator (PI) will identify unstable chromosomal regions that will be targeted for analysis. Aim 2 will focus on the design and introduction of fluorescent reporter constructs into the loci identified in Aim 1. To detect the presence of aneuploidy, the investigators will evaluate fluorescence intensity, which should be proportional to copy number of the region in which it is inserted. For the final aim, the applicants will use the engineered reporter lines to screen for conditions that alter reporter construct fluorescence, and therefore presumably contribute to aneuploidy or other forms of genomic instability. The reviewers were enthusiastic about the concept of the proposed technology but were unconvinced of its utility and feasibility, largely due to weaknesses in the underlying premise. The scope of the final aim seemed overly ambitious and the budget was inadequately justified. Finally, while impressed with the qualifications of the primary investigators, the reviewers expressed concern that the many substantial responsibilities were fragmented and dispersed amongst too many personnel. If successful, the impact of the proposed technology would be great. The ability to identify and eliminate culture conditions that cause chromosomal instability would be of enormous importance in the field, as one of they key roadblocks in stem cell technology are concerns about the maintenance of genomic stability as cells are maintained and propagated. The feasibility of this proposal was in doubt for several reasons. Most significantly, reviewers were not convinced that reporter fluorescence would reliably report aneuploidy, largely due to unanticipated cellular events that might affect the relationship between gene and protein expression. Furthermore, one reviewer noted that only one copy of the target would express the reporter, and the applicant did not provide explanation for how loss of heterozygosity would impact the detection scheme. Additional concerns were raised about the strategy for choosing chromosomal regions to study. One reviewer questioned the premise that it would be straightforward to identify single loci that are predictive of relevant chromosomal aberrations, as preliminary analysis has demonstrated substantial variability in chromosomal regions that show autosomal copy number variation among individual hESC lines. Additional doubts were raised about the feasibility of Aim 3, where the proposed numbers of strains, conditions and variables to be assessed amount to a seemingly impossible task. Finally, the reviewers were disappointed that the applicants did not propose to validate their screen for aneuploid-promoting culture conditions by testing hESC with nonengineered genomes. The reviewers found the applicant team to exceptionally well qualified. The PI has extensive experience developing tools for genotyping and haplomapping, and the senior collaborators are pioneers in the fields of stem cell genotyping and modification. Some reviewers were concerned with the nontrivial number of tasks that were assigned to each member of the team, and questioned whether the overall responsibility for bringing the project together might be too fragmented. Some reviewers expressed uncertainty about the appropriateness of the budget. The applicants did not supply details of the subcontractor expenses and did not justify how the supply budget was determined. The general feeling was that the requests were excessive. In summary, the technologies and information derived from the proposed effort would be powerful and useful, but the applicants failed to convince the reviewers of the project’s feasibility, especially given the distributed responsibilities of the PI and his/her collaborators.