Generation of ovarian follicles from mouse pluripotent stem cells

Abstract

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.