Basic Biology V
Human induced pluripotent stem cells (hIPSCs) could enable a paradigm shift in personalized medicine. However, a roadblock in advancing hIPSC-derived therapeutic cells to the clinic is that hIPSCs generated by current approaches are genetically unstable and, therefore, carry an elevated risk of cancer. What causes genomic instability in hIPSCs remains unknown. We propose a new mechanism to explain why hIPSCs demonstrate genomic instability and we present a simple, generally applicable and cost-effective method to prevent this deleterious process. The rationale for this project is based on our recent finding that hIPSCs have reduced levels of deoxyribonucleotide triphosphate (dNTP) precursors of DNA. The inability of cultured hIPSCs to produce sufficient dNTPs through a metabolic circuit known as the de novo pathway (DNP) triggers DNA replication stress, which is a well-established cause of genomic instability. To prevent the dNTP shortage and its consequences, we propose that, in addition to the DNP, a second dNTP biosynthetic mechanism known as the nucleoside salvage pathway (NSP) must be operational during hIPSC culture. We further propose that supplementing the hIPSC culture medium with deoxyribonucleoside substrates of the NSP will increase dNTP pools and significantly reduce genomic instability. If successful, the new hIPSC culture methods proposed here could be rapidly adopted in the field, with important consequences for the therapeutic potential of hIPSC-derivatives.
Statement of Benefit to California:
Human induced pluripotent stem cells (hIPSCs) may enable the development of autologous cell replacement therapies for many diseases that significantly impact human health in California. However, the genomic instability of hIPSCs obtained using current procedures has been linked with an increased risk of cancer, and therefore is a major challenge in the development of hIPSC-based therapies that can advance to the clinic. The technology we propose to develop utilizes optimization of the cell culture conditions, which should significantly increase the genomic stability of hIPSCs and their derivatives. Establishing the means to maintain genetically stable hIPSCs will be instrumental in unlocking the therapeutic promise of personalized pluripotent stem cell-based therapeutics. This research should also lead to economic benefits in California by creating new cell culture reagents, protocols and technologies with vast commercial potential.
The goals of this Exploratory Concepts Award proposal are to identify the main cause of genomic instability in human induced pluripotent stem cells (iPSCs) and to develop a method for preventing this instability, such that iPSCs will be safe for future therapeutic use. The hypothesis motivating this project is that the pool of deoxyribonucleotide triphosphates (dNTPs), the DNA building blocks, available in these cells is too small, which in turn leads to genomic instability. The aims of the project are to elucidate the link between dNTP pool size and genomic instability in iPSCs, and to identify optimum culture conditions that will increase dNTP pool size and thereby prevent genome instability in these cells. Novelty and Transformative Potential - The proposal lacks transformative potential. Although there may be some utility in pursuing the aims, reviewers viewed this proposal to be a poor fit for the Track 2 mechanism of this RFA. - The extent and the causes of genomic instability in iPSCs are under intense investigation in the scientific community. The proposal oversimplifies this problem by implying that limited dNTP pool size is the main cause of genomic instability, and that the cause is the same in different cell types. - If successful, this proposal may lead to improved culture conditions for human pluripotent stem cells; reviewers judged this to be a modest outcome. Feasibility and Experimental Design - The experimental design is clear and logical. The project uses well established, appropriate and complementary methods for tracking genomic instability. - The goals are achievable within the proposed time frame. - The research environment is suitable for success of the project. Principal Investigator (PI) and Research Team - The PI and co-PI have an established track record of successful research. -The co-PI has expertise critical for the success of the project. - The research team is of the appropriate size, and time commitment is appropriate for completion of the project. Responsiveness to the RFA - Although the project directly relates to stem cell biology, it does not meet the call for the Track 2 mechanism of this RFA to pursue highly novel, transformative research.