Dissect formative pluripotency using cultured pluripotent stem cells

项目概要/摘要 多能干细胞（PSC）的衍生已经彻底改变了发育生物学和再生生物学。 药为了稳定地维持PSC在培养物中并引导其高效且保真地分化为 多种细胞类型，重要的是要了解管理多能性的分子机制（能力 细胞生成体内任何组织的能力）。多能性的两个阶段，幼稚和引发，已被定义 并详细研究了由于小鼠胚胎干细胞（ESC）和外胚层的成功衍生， 干细胞（EpiSC）。小鼠胚胎干细胞与4日龄小鼠的上胚层最相似 胚泡（~胚胎第4天，或E4），而“致敏的”EpiSC显示出与胚胎第4天相似的全局基因表达特征。 植入后小鼠胚胎的E7外胚层。尽管有这些进步，但是， 缺乏类似于E5-6早期植入后上胚层的良好建立的PSC模型，其对应于 多能性的形成阶段。形成性多能性存在于一个时间窗口内，在此期间， 多能性被重新配置以准备多谱系能力，包括生殖细胞。功能性，形成性 多能性的特征是嵌合体能力和对直接原始生殖细胞的容许性 (PGC)诱导最近的几项研究试图通过短暂的外胚层样细胞来定义这种状态 （EpiLCs）从ESC分化。然而，迄今为止，尚未产生稳定形成的PSC。通过 通过调节FGF、TGF-β和WNT通路，我们最近从小鼠和人获得PSC（参考文献100）。 作为FTW-PSC），其允许体外直接的PGC样细胞诱导，并且能够有助于 种内或种间嵌合体。FTW-PSC具有分子、细胞和表型特征 具有形成性多能性的特征。这项建议的总体目标是利用这些新的 建立了细胞系，以全面剖析不同物种的形成状态。拟议 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-PSC是一个强大的平台，用于解剖分子 人类和小鼠PGC特化的潜在机制。此外，我们将建立一个体外平台， 用于基于形成性FTW-PSC产生功能性小鼠卵母细胞和人卵原细胞，从而 为研究生殖细胞发育和人类不育提供了宝贵的资源。我们的建议 巨大的潜力来彻底改变再生医学和生殖生物学。

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.