The objective of this application is to generate a thoroughly-validated panel of genetically diverse mouse embryonic stem cells (mESC) that will enable widespread adoption of cellular systems genetics. Phenotypic variation, manifesting as heterogeneity in cell state, represents a significant challenge for realizing the full promise of individualized, cell-based therapies, regenerative medicine. But phenotypic variation in genetically diverse stem cells also presents an opportunity for the advancement of large scale, cellular screens of gene by environment interactions (e.g. pharmacogenomics, toxicogenomics). A variety of approaches are beginning to identify the networks that drive cell state transitions, but these efforts have largely focused on bulk assays, which do not provide sufficient resolution of cell state heterogeneity, and mask the contribution of underlying genetic variation on rare cell types. Moreover, genetic studies using human pluripotent stem cells are largely limited to testing common variants due to low allele frequencies and imbalanced population structure requiring prohibitively large samples and impeding identification of core regulatory networks with high power and resolution. Therefore, we currently lack a thorough understanding of the genes and mechanisms that underlie phenotypic variation in pluripotent stem cells. The Diversity Outbred (DO) mouse population at The Jackson Laboratory is genetically defined, diverse, and presents a singular, cost-effective opportunity to systematically investigate heterogeneity in mammalian pluripotency. Our pilot studies using DO mESCs establish the feasibility of identifying regulatory loci at high power and resolution, as well as networks conserved in mice and humans that regulate cell state transitions.
In Aim 1, we will create a reference mapping panel of 300 DO mESC lines that will serve as a gold standard resource for cellular systems genetics. This panel will be fully credentialed and banked for broad availability through The Jackson Laboratory / Mutant Mouse Resource and Research Centers (MMRRC).
In Aim 2, we will determine at the single cell level the transcriptional networks that regulate cell state transitions in vitro through the early stages of differentiation to mesoderm in a representative subset of 144 lines.
In Aim 3, we will map quantitative trait loci (QTL) that underlie variation in cell state-specific gene expression and in the distribution of cell states in a population. In addition, we will build and test models based on polygenic scores that can predict differentiation propensity from genotype. Finally, a web-based searchable database of expression phenotypes and interactive tools for visualization of cell composition and eQTL will be made publicly available to support community queries and hypothesis generation. In sum, we will produce a resource of cell lines and gene expression data for the research community that will spur new discoveries in regenerative medicine, pharmacogenomics, and toxicogenomics.
Patient-derived pluripotent stem cells are essential tools for studying human disease biology and for the development of individualized regenerative medicine, however, stem cells from genetically diverse patients behave in unpredictable ways. One potential route to overcome this confounding variable and help realize the promise of precision medicine is accurate prediction of the diverse cellular states that are dictated by an individual's genome. This project will generate key resources to help achieve this goal by providing experimental tools for the identification and validation of critical genes and pathways governing cell state transitions and differentiation propensity, and the information needed to build predictive models based on an individual's genetic background.
