Genetically Diverse Mouse Embryonic Stem Cells: A Platform for Cellular Systems Genetics

项目总结 这个应用程序目标是生成一个经过彻底验证的遗传多样性小鼠小组 胚胎干细胞(MESC)将使细胞系统遗传学得到广泛采用。表型 变异，表现为细胞状态的异质性，代表着实现完整的 承诺个体化、基于细胞的治疗和再生医学。但在遗传上的表型变异 多样化的干细胞也为大规模细胞基因筛选的发展提供了机会 环境相互作用(例如，药物基因组学、毒理基因组学)。各种各样的方法正在开始 确定驱动细胞状态转变的网络，但这些努力主要集中在批量分析上， 这不能提供足够的单元状态异质性的分辨率，并且掩盖了潜在的 稀有细胞类型的遗传变异。此外，使用人类多能干细胞的遗传学研究在很大程度上是 仅限于测试由于等位基因频率低和群体结构不平衡而需要的常见变异 样本大得令人望而却步，阻碍了对高功率和 决议。因此，我们目前缺乏对基础基因和机制的透彻了解。 多能干细胞的表型变异。杰克逊的多样性繁殖(DO)鼠种群 实验室是由基因定义的、多样化的，并提供了一个独特的、经济高效的机会来系统化 研究哺乳动物多能性的异质性。我们使用DO MESCs进行的试点研究建立了 在高功率和分辨率下识别调控基因座的可行性，以及在小鼠和 调节细胞状态转变的人类。在目标1中，我们将创建一个包含300个DO的参考映射面板 MESC系将成为细胞系统遗传学的黄金标准资源。这个小组将完全 通过杰克逊实验室/突变小鼠资源和 研究中心(MMRRC)。在目标2中，我们将在单细胞水平上确定转录网络 在体外通过向中胚层分化的早期阶段调节细胞状态的转变 144行的代表性子集。在目标3中，我们将定位导致小麦品种变异的数量性状基因座(QTL) 细胞状态特异的基因表达和细胞状态在种群中的分布。此外，我们还将建立 以及基于多基因得分的测试模型，该模型可以根据基因预测分化倾向。最后，一个 用于细胞可视化的基于Web的可搜索的表达表型数据库和交互工具 成分和eQTL将公开提供，以支持社区查询和假设 一代。总之，我们将为这项研究提供细胞系和基因表达数据的资源 这将促进再生医学、药物基因组学和毒素基因组学的新发现。

PROJECT SUMMARY 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.