Generation of ovarian follicles from mouse pluripotent stem cells
Generation of ovarian follicles from mouse pluripotent stem cells
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DOI：
10.1126/science.abe0237
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发表时间：
2021-07
期刊：
影响因子：
56.9
通讯作者：
Takashi Yoshino;Takahiro Suzuki;Go Nagamatsu;Haruka Yabukami;Mika Ikegaya;Mami Kishima;Haruka Kita;T. Imamura;K. Nakashima;R. Nishinakamura;M. Tachibana;Miki Inoue;Y. Shima;K. Morohashi;K. Hayashi
中科院分区：
文献类型：
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作者：
Takashi Yoshino;Takahiro Suzuki;Go Nagamatsu;Haruka Yabukami;Mika Ikegaya;Mami Kishima;Haruka Kita;T. Imamura;K. Nakashima;R. Nishinakamura;M. Tachibana;Miki Inoue;Y. Shima;K. Morohashi;K. Hayashi
重建卵泡最近的进展使得多能干细胞在体外生成卵母细胞成为可能。然而，这些细胞需要体细胞环境才能充分发育为生殖细胞。吉野等人。应用体内分化过程的已知信息来确定将胚胎干细胞分化为性腺体细胞样细胞的培养条件（参见 Yang 和 Ng 的观点）。当胚胎干细胞产生的卵巢性腺组织与早期原始生殖细胞或体外衍生的原始生殖细胞样细胞结合时，生殖细胞在重建的卵泡内发育成可存活的卵母细胞，可以受精并产生可存活的后代。该系统为小鼠配子生产提供了一种替代方法，并增进了我们对哺乳动物繁殖和发育的理解。 《科学》，abe0237，本期第 11 页。 eabe0237;另见 abj8347，第 17 页。在由多能干细胞重建的小鼠卵巢环境中成功产生了 282 个功能性卵子。简介 生殖细胞在生殖器官的特定环境中发育。在整个卵子发生过程中，卵母细胞被卵泡结构中的体细胞包裹，为卵母细胞发育中的关键事件（例如减数分裂和生长）提供大量必需的信号和成分。卵母细胞和滤泡体细胞之间的相互作用以阶段依赖性方式受到调节。最近，体外配子发生，即使用多能干细胞在培养物中重建生殖细胞发育，已在哺乳动物物种（包括小鼠和人类）中得到开发。在小鼠中，功能性卵母细胞可以通过多能干细胞衍生的原始生殖细胞样细胞 (PGCLC) 在胚胎第 12.5 天与胚胎卵巢体细胞重新聚集而产生。因此，体外配子发生有望成为在培养物中产生大量卵母细胞的创新方法。这对于人类和濒临灭绝的动物的应用特别有用。然而，生殖细胞发育的体外重建高度依赖于胚胎卵巢组织提供的体细胞环境，而这种环境很难从哺乳动物物种中获得。在这里，我们提供了一个使用小鼠多能干细胞重建卵巢体细胞环境的模型系统。基本原理 在小鼠发育过程中，胚胎卵巢起源于新生中胚层，然后是生殖脊区域的中间中胚层和体腔上皮。为了从小鼠多能干细胞形成胚胎卵巢体细胞，需要在培养物中提供适当的信号来模拟这些胚胎事件。使用带有报告构建体的小鼠胚胎干细胞（mESC）来监测每个步骤关键基因的表达，我们着手探索重现分化过程的培养条件。在适当的条件下，应在诱导胚胎卵巢体细胞中赋予忠实的基因表达和功能。诱导细胞的功能应通过支持能够受精和随后发育的功能性卵母细胞的生成的能力来验证。结果基于报告基因表达，我们确定了一系列培养条件，以逐步方式重现从多能细胞到性腺体细胞的分化过程。在这些条件下，mESC通过新生中胚层、中间中胚层和体腔上皮状态分化为表达性腺体细胞代表性标记基因Nr5a1的胎儿卵巢体细胞样细胞（FOSLC）。 FOSLC 在胚胎第 12.5 天表现出与胚胎卵巢体细胞相似的转录谱和细胞组成。当 FOSLC 与源自 mESC 的 PGCLC 聚集时，PGCLC 进入减数分裂，随后的卵母细胞生长伴随着培养中 FOSLC 衍生卵泡的发育。 PGCLC 衍生的卵母细胞在 FOSLC 衍生的卵泡中发育，能够受精并发育成活的后代。这些结果证明功能性卵泡结构的重建完全能够支持卵母细胞的产生。结论 我们的结果表明，功能性性腺体细胞可以通过培养中的忠实分化过程从 mESC 中诱导出来。产生的材料可以作为替代胚胎卵巢组织进行体外配子发生的有用来源。此外，该系统有助于更好地了解性腺体细胞分化以及卵母细胞和滤泡体细胞之间的相互作用。由于它不需要胚胎性腺，因此该方法为其他哺乳动物物种的应用提供了可能性，而伦理和技术问题较少。该系统将加速我们对性腺发育的理解，并为研究和繁殖提供配子的替代来源。完全由小鼠多能干细胞重建卵泡结构，包括卵母细胞。左图显示了从 mESC 重建 FOSLC 和 PGCLC 的示意图。重建环境中的卵母细胞受精后产生后代。右图代表源自 FOSLC（红色）和 PGCLC（蓝色）的完全生长的卵丘-卵母细胞复合物。卵母细胞在一个特殊的充满液体的囊（即卵泡）中成熟，它提供减数分裂和生殖细胞生长所需的信号。已经开发出将多能干细胞衍生的原始生殖细胞样细胞（PGCLC）与胚胎卵巢体细胞一起培养时产生功能性卵母细胞的方法。在这项研究中，我们开发了培养条件来重建从多能细胞到胎儿卵巢体细胞样细胞（FOSLC）的逐步分化过程。当 FOSLC 与源自小鼠胚胎干细胞的 PGCLC 聚集时，PGLCC 进入减数分裂，产生能够受精并发育成活后代的功能性卵母细胞。在重建的卵巢环境中生成功能性小鼠卵母细胞提供了一种体外卵母细胞生成和卵泡生成的方法，以更好地了解哺乳动物的繁殖。
Reconstituting the ovarian follicle Recent advances have enabled the generation of oocytes from pluripotent stem cells in vitro. However, these cells require a somatic environment to develop fully as reproductive cells. Yoshino et al. applied what is known about differentiation processes in vivo to determine a culture condition to differentiate embryonic stem cells into gonadal somatic cell–like cells (see the Perspective by Yang and Ng). When the embryonic stem cell–generated ovarian gonadal tissue was combined with early primordial germ cells or in vitro–derived primordial germ cell–like cells, germ cells developed into viable oocytes within the reconstituted follicles that could be fertilized and result in viable offspring. This system enables an alternative method for mouse gamete production and advances our understanding of mammalian reproduction and development. Science, abe0237, this issue p. eabe0237; see also abj8347, p. 282 Functional eggs were successfully produced in a mouse ovarian environment reconstituted from pluripotent stem cells. INTRODUCTION Germ cells develop in a specific environment in the reproductive organs. Throughout oogenesis, oocytes are encapsulated by somatic cells in follicle structures that provide numerous signals and components essential for key events in oocyte development, such as meiosis and growth. The interaction between the oocyte and the somatic follicular cells is regulated in a stage-dependent manner. Recently, in vitro gametogenesis, reconstitution of germ cell development in culture using pluripotent stem cells, has been developed in mammalian species, including mice and humans. In mice, functional oocytes can be produced from pluripotent stem cell–derived primordial germ cell–like cells (PGCLCs) by reaggregation with embryonic ovarian somatic cells at embryonic day 12.5. Therefore, in vitro gametogenesis is expected to be an innovative means of producing a robust number of oocytes in culture. This should be particularly useful for application to humans and endangered animals. However, the in vitro reconstitution of germ cell development is highly dependent on the somatic cell environment provided by embryonic ovarian tissue, which is difficult to obtain from mammalian species. Here, we provide a model system that reconstitutes the ovarian somatic cell environment using mouse pluripotent stem cells. RATIONALE During mouse development, the embryonic ovaries originate from the nascent mesoderm, followed by the intermediate mesoderm and coelomic epithelium at the genital ridge region. For the formation of embryonic ovarian somatic cells from mouse pluripotent stem cells, appropriate signals need to be provided in culture to mimic those embryonic events. Using mouse embryonic stem cells (mESCs) harboring reporter constructs that monitor the expression of key genes for each step, we set out to explore culture conditions for the recreation of the differentiation process. Faithful gene expression and functionality should be conferred in induced embryonic ovarian somatic cells under the appropriate conditions. The functionality of the induced cells should be verified by the ability to support the generation of functional oocytes capable of fertilization and subsequent development. RESULTS Based on reporter gene expression, we determined a series of culture conditions that recreate the differentiation process from pluripotent cells to gonadal somatic cells in a stepwise manner. Under these conditions, mESCs differentiated into fetal ovarian somatic cell–like cells (FOSLCs) expressing Nr5a1, a representative marker gene of gonadal somatic cells, through the nascent mesoderm, intermediate mesoderm, and coelomic epithelium states. FOSLCs exhibited a transcriptional profile and cellular composition similar to those in embryonic ovarian somatic cells at embryonic day 12.5. When FOSLCs were aggregated with PGCLCs derived from mESCs, the PGCLCs entered meiosis, and subsequent oocyte growth accompanied the development of FOSLC-derived follicles in culture. PGCLC-derived oocytes developing in the FOSLC-derived follicles were capable of fertilization and developed to live offspring. These results demonstrate the reconstitution of functional follicle structures that are fully capable of supporting oocyte production. CONCLUSION Our results demonstrate that functional gonadal somatic cells can be induced from mESCs through a faithful differentiation process in culture. The generated material may serve as a useful source to replace embryonic ovarian tissue for in vitro gametogenesis. Furthermore, this system contributes to a better understanding of gonadal somatic cell differentiation and the interactions between oocytes and follicular somatic cells. Because it does not require embryonic gonads, the methodology opens the possibility for application in other mammalian species with fewer ethical and technical concerns. This system will accelerate our understanding of gonadal development and provide an alternative source of gametes for research and reproduction. Reconstitution of follicle structures, including oocytes, entirely from mouse pluripotent stem cells. Illustrations on the left show a schematic overview of reconstitution of both FOSLCs and PGCLCs from mESCs. Oocytes in the reconstituted environment gave rise to offspring after fertilization. The right image represents fully grown cumulus-oocyte complexes derived from FOSLCs (red) and PGCLCs (blue). Oocytes mature in a specialized fluid-filled sac, the ovarian follicle, which provides signals needed for meiosis and germ cell growth. Methods have been developed to generate functional oocytes from pluripotent stem cell–derived primordial germ cell–like cells (PGCLCs) when placed in culture with embryonic ovarian somatic cells. In this study, we developed culture conditions to recreate the stepwise differentiation process from pluripotent cells to fetal ovarian somatic cell–like cells (FOSLCs). When FOSLCs were aggregated with PGCLCs derived from mouse embryonic stem cells, the PGCLCs entered meiosis to generate functional oocytes capable of fertilization and development to live offspring. Generating functional mouse oocytes in a reconstituted ovarian environment provides a method for in vitro oocyte production and follicle generation for a better understanding of mammalian reproduction.