Advertisement

Conversion of Primordial Germ Cells to Pluripotent Stem Cells: Methods for Cell Tracking and Culture Conditions

  • Go Nagamatsu
  • Toshio Suda
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1052)

Abstract

Primordial germ cells (PGCs) are unipotent cells committed to germ lineage: PGCs can only differentiate into gametes in vivo. However, upon fertilization, germ cells acquire the capacity to differentiate into all cell types in the body, including germ cells. Therefore, germ cells are thought to have the potential for pluripotency. PGCs can convert to pluripotent stem cells in vitro when cultured under specific conditions that include bFGF, LIF, and the membrane-bound form of SCF (mSCF). Here, the culture conditions which efficiently convert PGCs to pluripotent embryonic germ (EG) cells are described, as well as methods used for identifying pluripotent candidate cells during culture.

Keywords

Primordial germ cell Acquisition of pluripotency Tracking Purification 

Notes

Acknowledgements

We thank Dr. Takeo Kosaka for contributing to the establishment of 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; and by the Takeda Science Foundation.

Supplementary material

Movie 1

 Time-laps bio-imaging of EG cell formation. (AVI 71045 kb).

References

  1. 1.
    Sasaki H, Matsui Y (2008) Epigenetic events in mammalian germ-cell development: reprogramming and beyond. Nat Rev Genet 9:129–140PubMedCrossRefGoogle Scholar
  2. 2.
    Saitou M, Kagiwada S, Kurimoto K (2012) Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells. Development 139:15–31PubMedCrossRefGoogle Scholar
  3. 3.
    Yoshimizu T, Sugiyama N, De Felice M, Yeom YI, Ohbo K, Masuko K et al (1999) Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev Growth Differ 41:675–684PubMedCrossRefGoogle Scholar
  4. 4.
    Yamaji M, Seki Y, Kurimoto K, Yabuta Y, Yuasa M, Shigeta M et al (2008) Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nat Genet 40:1016–1022PubMedCrossRefGoogle Scholar
  5. 5.
    Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448:313–317PubMedCrossRefGoogle Scholar
  6. 6.
    Matsui Y, Zsebo K, Hogan BL (1992) Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70:841–847PubMedCrossRefGoogle Scholar
  7. 7.
    Nagamatsu G, Kosaka T, Kawasumi M, Kinoshita T, Takubo K, Akiyama H et al (2011) A germ cell-specific gene, Prmt5, works in somatic cell reprogramming. J Biol Chem 286:10641–10648PubMedCrossRefGoogle Scholar
  8. 8.
    Nagamatsu G, Kosaka T, Saito S, Takubo K, Akiyama H, Sudo T et al (2012) Tracing the conversion process from primordial germ cells to pluripotent stem cells in mice. Biol Reprod 86(182):1–11Google Scholar
  9. 9.
    Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J et al (2008) The ground state of embryonic stem cell self-renewal. Nature 453:519–523PubMedCrossRefGoogle Scholar
  10. 10.
    Silva J, Barrandon O, Nichols J, Kawaguchi J, Theunissen TW, Smith A (2008) Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol 6:e253. 10.1371/journal.pbio.0060253PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Go Nagamatsu
    • 1
    • 2
  • Toshio Suda
    • 1
  1. 1.The Sakaguchi Laboratory, Department of Cell Differentiation, School of MedicineKeio UniversityTokyoJapan
  2. 2.Precursory Research for Embryonic Science and Technology, Japan Science and Technology AgencySaitamaJapan

Personalised recommendations