Basic Biology III
One of the most potentially powerful aspects of regenerative medicine is stem cell therapy. In this therapy, healthy tissues derived from stem cells will be implanted into patients with damaged tissue in order to restore function. However, there is currently a risk of immune rejection. Human induced pluripotent stem (hiPS) cells have the potential to revolutionize regenerative medicine. By reprogramming a patient's own cells into pluripotent stem cells, stem cell therapies can be performed with little to no risk of rejection. However, this nuclear reprogramming process is not well understood at a mechanistic level. Also, all procedures developed to date use cancer-related genes. This has raised fears that current hiPS cells could potentially have a high cancer risk if used therapeutically. In this proposed research, we intend to study the mutation load associated with reprogramming and the functional consequences of the mutations. We will use the obtained knowledge about transformation to develop safer methods of generating hiPS cells. We will perform large-scale screening of somatic mutations that have potential deleterious effects during the reprogramming of human primary cells into hiPS cells, and the differentiation of hiPS cells into somatic cell types. We will also characterize whether mutations occurred have any function, including the increase of cancer risk. These information will help us to understand how do the mutations occur and propagate. This proposed project will not only help us to gain additional mechanistic insights on nuclear reprogramming, but also allow great progress towards functional stem cell therapy, as safe hiPS cells will be available for therapeutic use.
Statement of Benefit to California:
Over the past decade, California has made great progress in stem cell research thanks to Proposition 71. Research into stem cell properties and applications has made the promise of regenerative cell therapies almost a reality. Human induced pluripotent stem (hiPS) cells offer the promise of treatments or cures for diseases that affect millions of people without a risk of immune rejection, including Alzheimer's disease, heart disease, organ failure, and spinal cord injury. However, before these cells can be used therapeutically in the clinic, a better understanding of the mechanisms of generating hiPS and the safety of the resultant cells must be gained. Our work will greatly improve understanding of the reprogramming process with respect to genomic mutations and integrity by determining the relative safety of a variety of available hiPS reprogramming techniques. We will work towards creating hiPS reprogramming methods that have been proven to be non-tumorigenic. Through this research, we hope to clear one of the biggest hurdles stopping hiPS cells: the risk of cancer. Due to the huge potential of stem cell therapies in regenerative medicine, this work has applications to a large number of diseases and genetic disorders that affect Californians, from infants to senior citizens.
Project Synopsis: The investigators seek to comprehensively identify genetic changes in protein coding sequences (exomes) by whole exome sequencing of human induced pluripotent stem cell (hiPSC) lines derived using different methods and different somatic donor cell types. The mutational load acquired during differentiation of hiPSC and human embryonic stem cells (hESC) will be determined in Aim 1. Moreover, for some of the genes found to be mutated in hiPSC, their functional role during reprogramming and their oncogenic potential in derived differentiated cells will be assessed in Aim 2. Significance and Innovation: - Several recent studies have demonstrated that the generation of hiPSC is accompanied by genetic alterations that may be selected for during the reprogramming process. Expanding on these data will be important for identifying safer sources of starting cells and safer reprogramming technologies that minimize mutational load. These studies should also shed light on whether mutations in specific genes tend to be selected for during reprogramming. - This study is highly significant, since evaluating the nature and impact of the hiPSC mutational load is important for understanding the process of reprogramming and for assessing potential safety issues of hiPSC. If mutations are selected for, and if some selected mutations are in cancer-promoting genes, this could have a serious impact on their potential therapeutic use. - Determining whether there is mutational pressure associated with differentiation is an interesting idea and has not yet been systematically analyzed. - This study is innovative in that it goes beyond just a description of the mutational load to important functional analyses, which are necessary to distinguish causative mutations from passenger mutations. - This proposal is limited to exome sequencing and is, therefore, less able to detect re-arrangements and other types of variation than alternative, more costly methodologies. - To achieve maximum benefit to science, the data (raw files and derived sequence files) should be deposited publicly. Human subjects protocols should be developed that allow this. Feasibility and Experimental Design: - The proposal is supported by strong preliminary data that hiPSCs contain mutations affecting protein coding regions. Some of these mutations were found to be present in the starting fibroblasts and others appear to have been acquired during the reprogramming process. - The proposed methodology is straightforward and logical; the experiments are clearly described. - In Aim 2, it is unclear how different genes will be prioritized for functional analyses, how the level of induced expression changes may influence results, or how changes in reprogramming efficiency will be quantified. Therefore, although technically feasible, it is unclear how informative these studies will be or what alternative strategies may be undertaken. - It is not clear if this study can be adequately powered using current technology and within the allowable budget for this RFA. - The proposal is to perform sequencing in-house, which is more expensive and error-prone than contract sequencing. It is therefore not a good choice for a project that requires exceptional accuracy and low error rate for detection of rare variants. - The key for identifying differences between two genomes lies in the quality of the statistical analysis; however, biostatistical methods are not discussed in this application. Principal Investigator (PI) and Research Team: - The PI is accomplished, has a strong track record in genome analysis and is well suited to carry out the proposed studies in Aim 1. The field will benefit from this PI applying his/her expertise to questions in stem cell biology. - The PI has excellent publications that are highly relevant to the proposed studies. - The PI has strong support from leaders in the iPSC field who will provide cell lines, DNA, and advisory support. - The personnel employed on the project have genome sequence analysis experience but it would have been beneficial to recruit someone with direct experience culturing pluripotent stem cells. It is unclear whether the primary team has any experience in evaluating stem cell function, although an appropriate collaborator will provide assistance. - There is no clear demonstration of bioinformatics expertise on the team. The identified person is a first year graduate student, and it is unclear that he/she will be in an environment with adequate support and expertise to handle cutting-edge bioinformatics. Responsiveness to the RFA: - This proposal is responsive to the RFA, as it addresses the genomic instability of human stem cells and the effects of such instability on tumorigenicity.
- Chad Cowan