This protocol is an extension to: Nat. Protoc. 8, 1513-1524 (2013); doi: 10.1038/nprot.2013.090; published online 11 July 2013Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol. This Protocol Extension describes an adaptation of this existing Protocol in which oogenesis also occurs in vitro, thus substantially modifying the technique. Oocytes generated from embryonic stem cells (ESCs) or induced pluripotent stem cells give rise to healthy pups. Here, we describe the protocol for oocyte generation in culture. The protocol is mainly composed of three different culture stages: in vitro differentiation (IVDi), in vitro growth (IVG), and in vitro maturation (IVM), which in total take ∼5 weeks. In each culture period, there are several checkpoints that enable the number of oocytes being produced in the culture to be monitored. The basic structure of the culture system should provide a useful tool for clarifying the complicated sequence of oogenesis in mammals.

中文翻译：

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用多能干细胞在培养皿中重建小鼠卵子形成。

该协议是对以下内容的扩展：Nat。协议。8，1513-1524（2013）; doi：10.1038 / nprot.2013.090；2013年7月11日在线发布，从多能干细胞中培养功能性卵母细胞应该为提高我们对卵子发生的基本机制的理解提供有用的模型系统。此外，它作为卵母细胞繁殖的替代来源具有潜在的应用。使用有关生殖工程和干细胞生物学的最先进的小鼠模型，我们先前开发了一种从培养中的多能细胞开始产生功能性原代生殖细胞的培养方法，并在先前的规程中对其进行了描述。该方案扩展描述了该现有方案的改编，其中卵子发生也发生在体外，从而大大改进了该技术。由胚胎干细胞（ESC）或诱导多能干细胞产生的卵母细胞会产生健康的幼仔。在这里，我们描述了培养中卵母细胞生成的协议。该方案主要由三个不同的培养阶段组成：体外分化（IVDi），体外生长（IVG）和体外成熟（IVM），总共需要约5周的时间。在每个培养阶段，都有几个检查点可以监控培养物中卵母细胞的数量。培养系统的基本结构应为阐明哺乳动物卵子形成的复杂序列提供有用的工具。体外分化（IVDi），体外生长（IVG）和体外成熟（IVM），总共需要大约5周的时间。在每个培养阶段，都有几个检查点可以监控培养物中卵母细胞的数量。培养系统的基本结构应为阐明哺乳动物卵子形成的复杂序列提供有用的工具。体外分化（IVDi），体外生长（IVG）和体外成熟（IVM），总共需要大约5周的时间。在每个培养阶段，都有几个检查点可以监控培养物中卵母细胞的数量。培养系统的基本结构应为阐明哺乳动物卵子形成的复杂序列提供有用的工具。