Stepwise Differentiation from Naïve State Pluripotent Stem Cells to Functional Primordial Germ Cells Through an Epiblast-Like State

  • Katsuhiko Hayashi
  • Mitinori Saitou
Part of the Methods in Molecular Biology book series (MIMB, volume 1074)


A group of pluripotent cells appearing during mammalian embryogenesis is the source for all the cell lineages that compose the embryo proper. In mice, pluripotent cells are first established in the inner cell mass (ICM) of the preimplantation blastocyst. After implantation, the ICM soon transforms into a cup-shaped epithelium, called the postimplantation epiblast. The two types of pluripotent cells, the ICM and postimplantation epiblast cells, are distinct, based on the differences in their gene expression profiles, epigenetic status, and differentiation capacity. During gastrulation, some of the postimplantation epiblast cells adjacent to the extraembryonic ectoderm are specified as primordial germ cells (PGCs), precursors of the germ cell lineage, in response to bone morphogenetic protein 4 (BMP4). Recently, we succeeded in reconstituting epiblast differentiation and PGC specification in vitro using pluripotent stem cells. Here, we describe the culture method of a stepwise differentiation from pluripotent stem cells to functional PGCs.

Key words

Embryonic stem cells Inner cell mass Postimplantation epiblast Primordial germ cells BMP4 



We would like to thank Drs. Hiroshi Ohta and Kazuki Kurimoto for their contribution to establishing this culture system. This study was supported, in part, by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; by PRESTO; by JST-CREST/ERATO; and by the Takeda Science Foundation. We apologize to researchers whose work has not been cited due to space limitations.


  1. 1.
    Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV et al (1999) Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev 13:424–436PubMedCrossRefGoogle Scholar
  2. 2.
    Yabuta Y, Kurimoto K, Ohinata Y, Seki Y, Saitou M (2006) Gene expression dynamics during germline specification in mice identified by quantitative single-cell gene expression profiling. Biol Reprod 75:705–716PubMedCrossRefGoogle Scholar
  3. 3.
    Kurimoto K, Yabuta Y, Ohinata Y, Shigeta M, Yamanaka K, Saitou M (2008) Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice. Genes Dev 22:1617–1635PubMedCrossRefGoogle Scholar
  4. 4.
    Seki Y, Hayashi K, Itoh K, Mizugaki M, Saitou M, Matsui Y (2005) Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice. Dev Biol 278:440–458PubMedCrossRefGoogle Scholar
  5. 5.
    Hajkova P, Ancelin K, Waldmann T, Lacoste N, Lange UC, Cesari F et al (2008) Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature 452:877–881PubMedCrossRefGoogle Scholar
  6. 6.
    Saitou M, Kagiwada S, Kurimoto K (2012) Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells. Development 139:15–31PubMedCrossRefGoogle Scholar
  7. 7.
    Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156PubMedCrossRefGoogle Scholar
  8. 8.
    Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78:7634–7638PubMedCrossRefGoogle Scholar
  9. 9.
    Brons IG, Smithers LE, Trotter MW, Rugg-Gunn P, Sun B, de Sousa C, Lopes SM et al (2007) Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448:191–195PubMedCrossRefGoogle Scholar
  10. 10.
    Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, Mack DL et al (2007) New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448:196–199PubMedCrossRefGoogle Scholar
  11. 11.
    Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R et al (2003) Derivation of oocytes from mouse embryonic stem cells. Science 300:1251–1256PubMedCrossRefGoogle Scholar
  12. 12.
    Toyooka Y, Tsunekawa N, Akasu R, Noce T (2003) Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci U S A 100:11457–11462PubMedCrossRefGoogle Scholar
  13. 13.
    Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ (2004) Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427:148–154PubMedCrossRefGoogle Scholar
  14. 14.
    Nayernia K, Nolte J, Michelmann HW, Lee JH, Rathsack K, Drusenheimer N et al (2006) In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev Cell 11:125–132PubMedCrossRefGoogle Scholar
  15. 15.
    Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M (2011) Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146:519–532PubMedCrossRefGoogle Scholar
  16. 16.
    Nichols J, Ying QL (2006) Derivation and propagation of embryonic stem cells in serum- and feeder-free culture. Methods Mol Biol 329:91–98PubMedGoogle Scholar
  17. 17.
    Ohinata Y, Sano M, Shigeta M, Yamanaka K, Saitou M (2008) A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter. Reproduction 136:503–514PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Katsuhiko Hayashi
    • 1
  • Mitinori Saitou
    • 2
  1. 1.Department of Anatomy and Cell Biology, Graduate School of MedicineKyoto UniversityKyotoJapan
  2. 2.Department of Anatomy and Cell biology, Graduate School of MedicineKyoto UniversityKyotoJapan

Personalised recommendations