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Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells

Nature Protocols volume 8, pages 1513–1524 (2013)Cite this article

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Abstract

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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Figure 1: A schematic overview of the derivation of eggs from pluripotent stem cells.
Figure 2: Representative contour plots of FACS analyses.
Figure 3: Technical procedures of ovary reconstitution and transplantation.
Figure 4: Collection, maturation and fertilization of oocytes derived from PGCLCs.

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Katsuhiko Hayashi & Mitinori Saitou

  2. Department of Reprogramming Science, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan

    Katsuhiko Hayashi & Mitinori Saitou

  3. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama, Japan

    Katsuhiko Hayashi

  4. Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Kyoto, Japan

    Mitinori Saitou

  5. The World Premier International Research Center Initiative–Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan

    Mitinori Saitou

Authors
  1. Katsuhiko Hayashi
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  2. Mitinori Saitou
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Contributions

K.H. and M.S. conceived of the study; K.H. performed the experiments; K.H. and M.S. wrote the manuscript.

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Correspondence to Katsuhiko Hayashi or Mitinori Saitou.

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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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