Basic Biology IV
Stem cell biology is expected to bring great progress both in basic biomedical research and also eventually by providing personalized treatments and cures for many diseases, including some of the most severe ones. However, there is evidence accumulating that perhaps the genome structure of stem cells in culture is not always completely stable. If this is a widespread phenomenon then it could confuse the results coming out of basic research that is making use of stem cells. And also it could have severe consequences for the patient, if a stem cell line is used in a treatment that has some instability in its genome which could render the treatment useless - or even dangerous to the patient, since genome instability can under certain conditions make cell lines become cancerous. There are new and powerful DNA analysis instruments available that allow for the first time to capture all genome variation and potential instability in a cell and we propose to make use of those instruments to study the genomes in a variety of different stem cell lines while they are kept in culture over extended periods as time and also while they are turned into neuronal and heart muscle tissue. We will be able to detect and measure genomic instability or variability if it exists and we will also be able to determine what, if any, the effects of such variability could be on the molecular mechanisms of control inside the cells.
Statement of Benefit to California:
Both stem cell biology and genomics technology are developing at a very fast pace and the breakthroughs in each field can be called revolutionary. Furthermore it is to be expected that it will be at the confluence of the two fields where some of the largest benefits for both basic research and translational medicine - and in particular for personalized medicine - can and will be found. California is in a particularly strong position, like no other region in the world, to be a leader in precisely this emerging field, stem cell genomics. Both sides of the equation are already in place and each field in its own right is a strong as only in a few other places. In particular genomics should be as strong as nowhere else in the world since all the components are concentrated in California, for example in Silicon Valley. However, for historic reasons there is for example no very large genome sequencing center in California. Personalized stem cell genomics could quite possibly be the key ingredient that makes personalized medicine become a full reality and move into profitable translational applications and into the clinic and it is important for California to make sure that both halves of the emerging field are strongly represented and supported so that the state can be at the very forefront of this development.
The applicant proposes a comprehensive analysis of genomic sequence variation in cultured human pluripotent stem cells at various passage numbers, in differentiated derivatives, and in original fibroblast cells that were used to generate the human induced pluripotent stem cell (hiPSC) lines included in this study. Some of these hiPSC lines were derived from patients with various disorders. Whole genome sequencing will be performed on all cell samples and will be correlated with data on genome-wide RNA expression, DNA methylation, distribution of several chromatin marks and higher level chromosome organization, all also obtained in this project. Significance and Innovation - Although understanding genomic and epigenomic instability in pluripotent stem cells and their derivatives is of importance, as it directly impacts the safety of therapeutic cell products, the applicant failed to explain how the planned work would advance the field and how it differs notably from what has been reported previously. - The applicant did not provide a strong rationale for the choice of the particular cell lines or culture conditions included in this project. - The project applies standard sequencing and related analytic tools to a set of samples derived using established methodologies; it is not innovative or creative. - The proposal to pursue the analysis of copy number variations (CNVs) is of significance, as CNVs tend to be more susceptible to instability than many other types of variants and, therefore, may serve as excellent indicators for instability in general. Feasibility and Experimental Design - Although a good amount of preliminary data is presented, the information is unfocused and largely descriptive in nature; the descriptions are difficult to follow and their specific relevance in the context of the application is very unclear. - The applicant does not acknowledge the extraordinary complexity of the proposed cell culture endeavors. There is no mention of basic cell culture parameters; neglect of such important issues greatly reduced reviewer enthusiasm for the proposal. - The experimental design was not well presented; it was unclear how many total samples, replicates and conditions will be analyzed. The proposal lacked a clear hypothesis and description of expected outcomes. - Without a clear hypothesis, the sample size required to arrive at a reliable answer cannot be calculated. If the sample sizes for the proposed studies are too small, and there was concern that might be the case, the studies can be subject to statistical traps that, if undetected or inappropriately reported, could lead to erroneous conclusions. - If provided with quality sequence data from quality cells, the applicant appeared capable of using state-of-the art technology and analysis to detect genome variation and instability. Principal Investigator (PI) and Research Team - The applicant is a junior investigator with expertise in the computational biology of genome variation but with little track record in terms of leadership or completion of large-scale projects. - The PI is co-author on several high impact publications that are collaborative genome reports dealing principally with mapping of structural genome variants. It is not clear how the applicant contributed to these studies. - It was not clear what experiments or data analysis would be conducted in laboratory of the Partner-PI, who is a junior investigator. Responsiveness to the RFA - The proposal is responsive to the RFA; it addresses basic molecular mechanisms operating in human stem cells.