Funding opportunities

Identifying sources of mutation in human induced pluripotent stem cells by whole genome sequencing

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02186
Principle Investigator: 
Funds requested: 
$1 755 864
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Stem cell research offers new tools to help treat and cure diseases that affect diverse cells types in the body such as neurological diseases, heart disease and diabetes by producing human cells for transplantation or to enable drug discovery . Recent advances have allowed researchers to generate patient-matched cell types from the skin or other tissues of patients. These patient-matched cells are important because they are unlikely to cause immune rejection upon transplantation and they may help to model diseases caused by gene variations found only in rare individuals. Despite their promise, patient-matched cells differ from traditional stem cells in ways that may cause them to be less stable or increase their potential to cause tumors. This is because patient-matched cells are generated from tissues taken from adult patients using methods that dramatically alter the chromosomes of these cells. These factors could endow these reprogrammed cells with mutations that would not be present in cells derived from embryonic sources. To ensure the safety and clinical utility of reprogrammed cells, it is critical to establish methods to identify potentially oncogenic or detrimental mutations. This proposal is designed to identify the source and scope of mutations in reprogrammed pluripotent cell lines using cutting edge whole genome sequencing methods. Results of these studies will establish the relative safety of current methods to produce patient-matched reprogrammed cells and help to improve methods to speed the translation of these advances into therapies.
Statement of Benefit to California: 
California has become an epicenter of stem cell advancement in part due to the funding of innovative collaborative research by the CIRM. We have established new methods that will improve the safety and effectiveness of regenerative medicine in cases where cell therapies are generated by converting adult cells into other cell types, including pluripotent cells and differentiated cells such as neurons and heart cells. This will help to reduce the costs of ongoing research funded by CIRM and by other California entities. Results of these studies will help to accelerate the translational application of basic biomedical advances being achieved across the state.
Review Summary: 
Project Synopsis: Using whole genome sequencing, the applicant proposes to uncover mutations present in human induced pluripotent stem cells (hiPSC) and in the somatic cells from which they were derived. The overall goal is to identify the optimal reprogramming method and source of somatic cells leading to hiPSC with the lowest mutational burden. In the first aim, the applicant will derive hiPSC from fibroblasts using a lentiviral vector and use lineage tracing to determine to what extent mutations were present in the original somatic cell or arose early during the reprogramming process. In addition, two components of the reprogramming method will be evaluated for their contribution to mutational load. The remaining goals include assessing the mutational load acquired when using a non-integrating reprogramming method (Aim 2) and determining the extent of somatic mutations present in donor cells of distinct lineage or age (Aim 3). Significance and Innovation: - This is a very significant project that addresses an important problem and could have a major impact, since understanding the extent of mutations in hiPSC is key to assessing their biomedical utility. - The use of a lineage tracing system to identify sister clones was considered innovative and invaluable in determining whether the source of mutations in a given hiPSC line can be traced to the somatic cell from which it was derived. - The project is almost purely descriptive, but may identify the source of mutations in hiPSC, a first step towards understanding the molecular basis of genetic change. - The rationale is logical and scientifically sound. - To achieve maximum benefit to science, the data (raw files and derived sequence files) should be deposited publicly. Human subjects protocols should be developed to ensure that this happens. Feasibility and Experimental Design: - Overall, this project appears feasible and the preliminary data are compelling and supportive of the proposed experiments, although it would have been reassuring if data using hiPSC had been included. - The preliminary data do not help establish the efficiency for generating sister clones; this is critical for the success of the lineage tracing approach. - Although the applicant will use lineage tracing to track the source of an identified mutation, direct whole genome sequencing of bulk somatic cell populations is proposed as well. Unlike hiPSC lines, which are clonally derived, these cells represent a potentially heterogeneous population, with individual subpopulations possibly carrying different mutations. Whole genome sequencing will not easily resolve those differences, and a comparison to hiPSC lines may be misleading. A reviewer suggested analyzing somatic subclones rather than DNA extracted from bulk somatic cell cultures. - The applicant plans to utilize a contract organization for sequencing; this is a strength, as the best quality data can likely be obtained through outsourcing to a commercial organization that operates at high-throughput and performs extensive process quality control, something that is unlikely to be possible in a research or core facility at an academic institution. - The key to identifying differences between two genomes lies in the quality of the biostatistical analyses. This application provides little detail in this regard, but confidence in the track record of the relevant team members alleviates this concern. - Suggestion by a reviewer: more modern databases than the one proposed should be utilized for the validation of putative point mutations. - There is little if any mention of alternative plans or potential difficulties; this is especially pertinent for the non-integrating reprogramming method, as an entire aim depends on its success. - It is not clear if this study can be adequately powered using current technology and costs. Principal Investigator (PI) and Research Team: - The PI has an outstanding track record and impeccable training. - The PI’s recent productivity has been relatively low, with the notable exception of one recent high impact publication. - The research team has much of the necessary expertise to conduct the proposed research. - The proposed collaboration with two computational biology and bioinformatics experts provides critical technological expertise in whole genome sequencing and analysis, both of which are integral to the success of the project. However, funding allocated to their effort is somewhat constraining. - Expertise working with or generating hiPSC is missing from the team, but extensive expertise working with mouse iPSC is reassuring. Responsiveness to the RFA: - This proposal is responsive to the RFA, as it addresses the genomic instability of human stem cells.
Conflicts: 

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