Dissect formative pluripotency using cultured pluripotent stem cells

使用培养的多能干细胞剖析形成性多能性

• 批准号: 10455585
• 负责人: Jun Wu
• 金额: $ 34.06万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-22 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10455585
• 关键词: Adult; Affinity Chromatography; Cell Differentiation process; Cell Lineage; Cells; ChIP-seq; Characteristics; Chemicals; Chimera organism; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; Derivation procedure; Development; Developmental Biology; Embryo; Epiblast; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Foundations; Gastrula; Gene Expression Profile; Generations; Genetic; Germ Cells; Goals; Homeodomain Proteins; Human; In Situ; In Vitro; Infertility; Laboratories; Maintenance; Methods; Modeling; Molecular; Mus; Names; Oocytes; Oogonia; Pathway interactions; Phase; Phenotype; Play; Pluripotent Stem Cells; Proteins; Proteomics; Regenerative Medicine; Regulation; Regulatory Element; Reproductive Biology; Resources; Role; Signal Transduction; Somatic Cell; Structure of primordial sex cell; Study models; Testing; Time; Tissues; Transforming Growth Factor beta; Transgenes; Validation; austin; base; blastocyst; cell type; embryonic stem cell; established cell line; experimental study; implantation; in vivo; induced pluripotent stem cell; insight; novel; permissiveness; pluripotency; regeneration potential; stem cell model; stem cells; tool; transcription factor; transcriptome

PROJECT SUMMARY/ABSTRACT Derivation of pluripotent stem cells (PSCs) has revolutionized developmental biology and regenerative medicine. To stably maintain PSCs in culture and guide them to differentiate with high efficiency and fidelity into a variety of cell types, it is important to understand the molecular mechanisms governing pluripotency (the ability of a cell to generate any tissues in the body). Two phases of pluripotency, naïve and primed, have been defined and studied in detail thanks to the successful derivation of mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), respectively. Mouse ESCs most closely resemble epiblast from a 4-day-old mouse blastocyst (~embryonic day 4, or E4), while “primed” EpiSCs display a global gene expression signature similar to the E7 epiblast of a post-implantation mouse embryo. Despite these advances, however, however, there is lack of a well-established PSC model that resembles E5-6 early post-implantation epiblast, which corresponds to the formative phase of pluripotency. Formative pluripotency exists within a time window during which naïve pluripotency is reconfigured to prepare for multilineage competency, including germ cells. Functionally, formative pluripotency is characterized by both chimera competency and permissiveness for direct primordial germ cell (PGC) induction. Several recent studies have attempted to define this state by transient epiblast-like cells (EpiLCs) differentiated from ESCs. To date, however, stable formative PSCs have not yet been generated. By modulating the FGF, TGF-β and WNT pathways, we recently derived PSCs from both mice and humans (referred to as FTW-PSCs) that are permissive for direct PGC-like cell induction in vitro and are capable of contributing to intra- or inter-species chimeras in vivo. FTW-PSCs harbor molecular, cellular and phenotypic features characteristic of formative pluripotency. The overall objective of this proposal is to use these newly established cell lines to comprehensively dissect the formative state across species. The proposed studies will elucidate the roles of several transcription factors in regulating mouse and human formative pluripotency, as well as demonstrate that FTW-PSCs are a robust platform for dissecting the molecular mechanisms underlying human and mouse PGC specification. In addition, we will establish an in vitro platform for the generation of functional mouse oocytes and human oogonia based on formative FTW-PSCs, thereby providing an invaluable resource for studying germ cell development and human infertility. Our proposal has tremendous potential to revolutionize regenerative medicine and reproductive biology.

项目摘要/摘要 多能干细胞（PSC）的推导已彻底改变了发育生物学和再生 药品。在文化中稳定地维持PSC，并指导它们以高效率和忠诚为差异化 多种细胞类型，重要的是要了解多能性的分子机制（能力 细胞生成体内任何组织）。已定义了两个幼稚和底漆的多能阶段 并详细研究了小鼠胚胎干细胞（ESC）和层细胞的成功推导 干细胞（EPISC）分别。鼠标最类似于4天老鼠的epeblast 胚泡（〜胚胎第4天或E4），而“启动” episcs显示出全局基因表达签名相似 到植入后小鼠胚胎的E7纤维细胞。但是，尽管有这些进步，但是 缺乏类似于E5-6早期植入后的E5-6的PSC模型，该模型对应于E5-6 到多能的形成阶段。在一个天真的时间窗口内存在形成性多能 重新配置多功能以准备多ineage的能力，包括生殖细胞。在功能上，形成性 多能性具有嵌合体能力和直接原始生殖细胞的允许性 （PGC）诱导。最近的一些研究试图通过短暂的epiblast样细胞来定义这种状态 （EPILC）与ESC区分开。但是，迄今为止，尚未生成稳定的形成性PSC。经过 调节FGF，TGF-β和WNT途径，我们最近从小鼠和人类中得出PSC（引用 对于允许在体外直接PGC样细胞诱导的ftw-PSCs），能够有助于 体内种内或种间嵌合体。 FTW-PSC携带分子，细胞和表型特征 形成性多能的特征。该提案的总体目的是使用这些新的 建立的细胞系以全面剖析跨物种的形成状态。提议 研究将阐明几种转录因子在控制小鼠和人类格式中的作用 多能性，并证明FTW-PSC是剖析分子的强大平台 人和小鼠PGC规范的机制。此外，我们将建立一个体外平台 为基于形成性FTW-PSC的功能性小鼠卵母细胞和人类Oogonia的产生，从而 为研究生殖细胞发育和人类不育症提供了宝贵的资源。我们的建议有 彻底改变再生医学和生殖生物学的巨大潜力。