Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell–like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in ∼1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro.
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We would like to thank S. Ogushi, K. Kurimoto, H. Ohta, S. Shimamoto, S. Aramaki and Y. Ishikura for their technical assistance, and T. Mori for encouragement. We also thank S. Matoba and A. Ogura for their advice on ovary reconstitution. This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan; by JST-PRESTO/Core Research for Evolutionary Science and Technology (CREST)/ERATO; and by the Takeda Science Foundation.
The authors declare no competing financial interests.
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Hayashi, K., Saitou, M. Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. Nat Protoc 8, 1513–1524 (2013). https://doi.org/10.1038/nprot.2013.090
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