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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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References

  1. Sasaki, H. & Matsui, Y. Epigenetic events in mammalian germ-cell development: reprogramming and beyond. Nat. Rev. Genet. 9, 129–140 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Ewen, K.A. & Koopman, P. Mouse germ cell development: from specification to sex determination. Mol. Cell Endocrinol. 323, 76–93 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Saitou, M. & Yamaji, M. Germ cell specification in mice: signaling, transcription regulation, and epigenetic consequences. Reproduction 139, 931–942 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. Lawson, K.A. et al. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 13, 424–436 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hilscher, B. et al. Kinetics of gametogenesis. I. Comparative histological and autoradiographic studies of oocytes and transitional prospermatogonia during oogenesis and prespermatogenesis. Cell Tissue Res. 154, 443–470 (1974).

    Article  CAS  PubMed  Google Scholar 

  6. Speed, R.M. Meiosis in the foetal mouse ovary. I. An analysis at the light microscope level using surface-spreading. Chromosoma 85, 427–437 (1982).

    Article  CAS  PubMed  Google Scholar 

  7. Edson, M.A., Nagaraja, A.K. & Matzuk, M.M. The mammalian ovary from genesis to revelation. Endocr. Rev. 30, 624–712 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Campbell, K.H., McWhir, J., Ritchie, W.A. & Wilmut, I. Sheep cloned by nuclear transfer from a cultured cell line. Nature 380, 64–66 (1996).

    Article  CAS  PubMed  Google Scholar 

  9. Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R. & Yanagimachi, R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374 (1998).

    Article  CAS  PubMed  Google Scholar 

  10. Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Hayashi, K. et al. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 338, 971–975 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Hubner, K. et al. Derivation of oocytes from mouse embryonic stem cells. Science 300, 1251–1256 (2003).

    Article  PubMed  Google Scholar 

  13. Toyooka, Y., Tsunekawa, N., Akasu, R. & Noce, T. Embryonic stem cells can form germ cells in vitro. Proc. Natl. Acad. Sci. USA 100, 11457–11462 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Geijsen, N. et al. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, 148–154 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Nayernia, K. et al. In vitro–differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev. Cell 11, 125–132 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Tesar, P.J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Hayashi, K. & Surani, M.A. Self-renewing epiblast stem cells exhibit continual delineation of germ cells with epigenetic reprogramming in vitro. Development 136, 3549–3556 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Saitou, M., Kagiwada, S. & Kurimoto, K. Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells. Development 139, 15–31 (2012).

    Article  CAS  PubMed  Google Scholar 

  19. Chuma, S. et al. Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis. Development 132, 117–122 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Matoba, S. & Ogura, A. Generation of functional oocytes and spermatids from fetal primordial germ cells after ectopic transplantation in adult mice. Biol. Reprod. 84, 631–638 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Hashimoto, K., Noguchi, M. & Nakatsuji, N. Mouse offspring derived from fetal ovaries or reaggregates which were cultured and transplanted into adult females. Dev. Growth Differ. 34, 233–238 (1992).

    Article  Google Scholar 

  22. Ying, Q.L., Nichols, J., Chambers, I. & Smith, A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281–292 (2003).

    CAS  PubMed  Google Scholar 

  23. Brons, I.G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Guo, G. et al. Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136, 1063–1069 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ohinata, Y. et al. A signaling principle for the specification of the germ cell lineage in mice. Cell 137, 571–584 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Ohinata, Y., Sano, M., Shigeta, M., Yamanaka, K. & Saitou, M. A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter. Reproduction 136, 503–514 (2008).

    Article  CAS  PubMed  Google Scholar 

  27. Ying, Q.L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo 3rd edn. (Cold Spring Harbor Laboratory Press, 2003).

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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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