Dissect formative pluripotency using cultured pluripotent stem cells

项目摘要/摘要 多能干细胞(PSCs)的产生彻底改变了发育生物学和再生 医药。稳定维持培养中的PSCs，并引导其高效、准确地分化为 对于各种类型的细胞，了解支配多能性(能力)的分子机制是很重要的 在体内产生任何组织的细胞)。已经定义了多能性的两个阶段，即幼稚和初级 由于成功地获得了小鼠胚胎干细胞(ESCs)和外胚层，对其进行了详细的研究 干细胞(EpiSCs)。小鼠胚胎干细胞与出生4天的小鼠的外胚层最为相似 胚泡(~胚胎第4天，或E4)，而“启动”的EpiSCs表现出类似的全球基因表达特征 到植入后小鼠胚胎的E7外胚层。然而，尽管有这些进步，还是有 缺乏成熟的PSC模型，类似于E5-6植入后早期的外胚层，这与 到了多能性的形成阶段。形成性多能性存在于一个天真的时间窗口内 多能性被重新配置，以准备多谱系能力，包括生殖细胞。在功能上，形成性 直接原始生殖细胞的嵌合体能力和迁移性是多能性的特征。 (PGC)诱导。最近的几项研究试图通过短暂的上胚样细胞来定义这种状态 (EpiLCs)从ESCs分化而来。然而，到目前为止，还没有产生稳定的形成PSCs。通过 通过调节成纤维细胞生长因子、转化生长因子-β和WNT通路，我们最近从小鼠和人身上获得了PSCs(参考文献 作为FTW-PSC)，允许在体外直接诱导PGC样细胞，并能够促进 活体内或种间嵌合体。FTW-PSCs具有分子、细胞和表型特征 具有形成多能性的特点。这项提议总体目标是使用这些新的 建立了细胞系，全面剖析了不同物种的形成状态。建议数 研究将阐明几种转录因子在调节小鼠和人类形成中的作用 多能性，以及证明FTW-PSCs是一个强大的分子解剖平台 人类和小鼠PGC规范背后的机制。此外，我们还将建立一个体外平台 用于基于形成FTW-PSCs的小鼠功能卵母细胞和人卵原细胞的生成，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。我们的建议已经 给再生医学和生殖生物学带来革命性变化的巨大潜力。

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.