Abstract
Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac1. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo2,3. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Lawson, K. A. & Hage, W. J. Clonal analysis of the origin of primordial germ cells in the mouse. Ciba Found. Symp. 182, 68–84, 84–91 (1994)
Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W. & Kemler, R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87, 27–45 (1985)
Leahy, A., Xiong, J. W., Kuhnert, F. & Stuhlmann, H. Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation. J. Exp. Zool. 284, 67–81 (1999)
Kyba, M., Perlingeiro, R. C. & Daley, G. Q. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 109, 29–37 (2002)
Saitou, M., Barton, S. C. & Surani, M. A. A molecular programme for the specification of germ cell fate in mice. Nature 418, 293–300 (2002)
Wang, P. J., McCarrey, J. R., Yang, F. & Page, D. C. An abundance of X-linked genes expressed in spermatogonia. Nature Genet. 27, 422–426 (2001)
Cooke, H. J., Lee, M., Kerr, S. & Ruggiu, M. A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum. Mol. Genet. 5, 513–516 (1996)
Seboun, E. et al. Gene sequence, localization, and evolutionary conservation of DAZLA, a candidate male sterility gene. Genomics 41, 227–235 (1997)
Moore, F. L. et al. Human Pumilio-2 is expressed in embryonic stem cells and germ cells and interacts with DAZ (Deleted in AZoospermia) and DAZ-like proteins. Proc. Natl Acad. Sci. USA 100, 538–543 (2003)
Zandstra, P. W., Le, H. V., Daley, G. Q., Griffith, L. G. & Lauffenburger, D. A. Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation. Biotechnol. Bioeng. 69, 607–617 (2000)
Bortvin, A. et al. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 130, 1673–1680 (2003)
Koshimizu, U., Watanabe, M. & Nakatsuji, N. Retinoic acid is a potent growth activator of mouse primordial germ cells in vitro. Dev. Biol. 168, 683–685 (1995)
Labosky, P. A., Barlow, D. P. & Hogan, B. L. Mouse embryonic germ (EG) cell lines: transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines. Development 120, 3197–3204 (1994)
Barlow, D. P., Stoger, R., Herrmann, B. G., Saito, K. & Schweifer, N. The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus. Nature 349, 84–87 (1991)
Stoger, R. et al. Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. Cell 73, 61–71 (1993)
Matsui, Y., Zsebo, K. & Hogan, B. L. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70, 841–847 (1992)
Resnick, J. L., Bixler, L. S., Cheng, L. & Donovan, P. J. Long-term proliferation of mouse primordial germ cells in culture. Nature 359, 550–551 (1992)
Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D. & Orkin, S. H. Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood 101, 508–516 (2003)
Fuhrmann, G. et al. Mouse germline restriction of Oct4 expression by germ cell nuclear factor. Dev. Cell 1, 377–387 (2001)
Wassarman, P. M. Mouse gamete adhesion molecules. Biol. Reprod. 46, 186–191 (1992)
Cao, T., Shannon, M., Handel, M. A. & Etkin, L. D. Mouse ret finger protein (rfp) proto-oncogene is expressed at specific stages of mouse spermatogenesis. Dev. Genet. 19, 309–320 (1996)
Ogawa, S. et al. Molecular cloning of a novel RING finger-B box-coiled coil (RBCC) protein, terf, expressed in the testis. Biochem. Biophys. Res. Commun. 251, 515–519 (1998)
Tezel, G., Nagasaka, T., Shimono, Y. & Takahashi, M. Differential expression of RET finger protein in testicular germ cell tumors. Pathol. Int. 52, 623–627 (2002)
Fenderson, B. A., O'Brien, D. A., Millette, C. F. & Eddy, E. M. Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies. Dev. Biol. 103, 117–128 (1984)
Hubner, K. et al. Derivation of oocytes from mouse embryonic stem cells. Science 300, 1251–1256 (2003)
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)
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997)
Selig, S., Okumura, K., Ward, D. C. & Cedar, H. Delineation of DNA replication time zones by fluorescence in situ hybridization. EMBO J. 11, 1217–1225 (1992)
Reijo, R. et al. Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans, is expressed in male germ cells before and after puberty. Genomics 35, 346–352 (1996)
Li, E., Bestor, T. H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992)
Acknowledgements
The authors thank S. L. Opitz, N. Watson and E. Dikovskaia for technical support; K. Hochedlinger and T. Holm for assistance with intracytoplasmic oocyte injection; and W. Lensch for critical revisions of the manuscript. This work was supported by grants from the National Institutes of Health, the National Science Foundation Biotechnology Process Engineering Center, and the Dutch Cancer Society ‘Koningin Wilhelmina fonds’. G.Q.D. is a Birnbaum Scholar of the Leukemia and Lymphoma Society of America. J.G. was sponsored by the Human Frontiers Science Foundation. K.E. is a Junior Fellow in the Harvard Society of Fellows.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare that they have no competing financial interests.
Rights and permissions
About this article
Cite this article
Geijsen, N., Horoschak, M., Kim, K. et al. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, 148–154 (2004). https://doi.org/10.1038/nature02247
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature02247
This article is cited by
-
Modelling in vitro gametogenesis using induced pluripotent stem cells: a review
Cell Regeneration (2023)
-
In vitro differentiation of primed human induced pluripotent stem cells into primordial germ cell-like cells
Molecular Biology Reports (2023)
-
Cell-Based Therapy Approaches in Treatment of Non-obstructive Azoospermia
Reproductive Sciences (2023)
-
Oocyte Arrested at Metaphase II Stage were Derived from Human Pluripotent Stem Cells in vitro
Stem Cell Reviews and Reports (2023)
-
Meiosis resumption in human primordial germ cells from induced pluripotent stem cells by in vitro activation and reconstruction of ovarian nests
Stem Cell Research & Therapy (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.