Identifying sources of mutation in human induced pluripotent stem cells by whole genome sequencing
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.
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.
The goal of this study is to identify mutations in human iPSC lines and determine their likely origin. To accomplish this we previously used mouse models to develop and validate new methods to isolate iPSC lines from the same precursor donor cell and to perform comprehensive whole genome sequencing based mutation detection . In this grant period we have translated our iPSC tracing and lineage analysis methods to human iPSCs, established two methods to produce iPSCs using non integrating methods and improved our bioinformatic detection of mutations. We are now poised carry out the remainder of the proposed work within the timeframe allotted.
The use of induced pluripotent stem cells (iPSCs) in research and translational medicine depends on our ability to identify iPSC cell lines that do not contain potentially dangerous mutations that can lead to cancer, degeneration or uncontrolled variability in cell survival or function. Such mutations may arise from numerous sources including somatic mutations that originated in patient donor cells during development or aging, and as a consequence of particular reprogramming methods. In order to identify the cause of mutations in iPSCs we have developed and successfully optimized a lineage tracing approach to identify clonally related sister iPSCs that we produce from fibroblasts and blood cells, which are the two most promising sources for patient specific iPSCs. This advance now allows us to perform whole genome sequencing on these iPSCs and determine the major source and type of mutation that arise under different commonly used conditions for iPSC generation. Results of genome sequencing of these iPSCs will inform future efforts to identify the best means to produce iPSCs for research and therapeutic applications.
The impact of induced pluripotent stem cells on clinical and basic research depends on the extent to which they maintain the genomic background of the individual while avoiding changes to their DNA that can make them potentially dangerous or ineffective. The goal of this grant was to identify the extent to which different aspects of iPSC biology contribute to unwanted variation in these stem cell lines. To accomplish this goal, we adapted a method that we previously used to assess the role of mutations in mouse iPSC function, called the "sister line" approach. In this grant period we successfully adapted this approach to human iPSCs, generated a large number of "sister lines" and have applied sensitive whole genome sequencing to these lines to identify the source and scope of mutations that arise in the patient's original cells compared to events that occur during reprogramming into iPSCs. In addition we have compared the effects of different commonly used reprogramming methods which should serve as a useful guide to researchers generating iPSCs for research and clinical use. These results are being translated to researchers involved in other CIRM funded stem cell genomics and translational studies as well as to those funded by the NIH and other institutes.