Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Generation of functional multipotent adult stem cells from GPR125+ germline progenitors

Abstract

Adult mammalian testis is a source of pluripotent stem cells1. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells2. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro1, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125–β-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125–LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125–LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Restricted GPR125 expression in adult mouse testis and derivation of multipotent cells from spermatogonial progenitor cells (SPCs).
Figure 2: Characterization and multipotent derivatives of Gpr125 lacZ/lacZ SPC lines
Figure 3: GPR125–LacZ MASCs exhibit multipotency and can form functional vessels.
Figure 4: Gpr125 lacZ/lacZ MASCs have an expression profile different from mouse embryonic stem cells.

References

  1. Guan, K. et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 440, 1199–1203 (2006)

    CAS  Article  ADS  Google Scholar 

  2. Kanatsu-Shinohara, M. & Shinohara, T. The germ of pluripotency. Nature Biotechnol. 24, 663–664 (2006)

    CAS  Article  Google Scholar 

  3. Valenzuela, D. M. et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nature Biotechnol. 21, 652–659 (2003)

    CAS  Article  Google Scholar 

  4. Oakberg, E. F. A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat. 99, 391–413 (1956)

    CAS  Article  Google Scholar 

  5. Fiering, S. N. et al. Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs. Cytometry 12, 291–301 (1991)

    CAS  Article  Google Scholar 

  6. Kuroda, N. et al. Distribution and role of CD34-positive stromal cells and myofibroblasts in human normal testicular stroma. Histol. Histopathol. 19, 743–751 (2004)

    CAS  PubMed  Google Scholar 

  7. Schaefer, B. C., Schaefer, M. L., Kappler, J. W., Marrack, P. & Kedl, R. M. Observation of antigen-dependent CD8+ T-cell/dendritic cell interactions in vivo. Cell. Immunol. 214, 110–122 (2001)

    CAS  Article  Google Scholar 

  8. Enders, G. C. & May, J. J. Developmentally regulated expression of a mouse germ cell nuclear antigen examined from embryonic day 11 to adult in male and female mice. Dev. Biol. 163, 331–340 (1994)

    CAS  Article  Google Scholar 

  9. Schrans-Stassen, B. H., Saunders, P. T., Cooke, H. J. & de Rooij, D. G. Nature of the spermatogenic arrest in Dazl-/- mice. Biol. Reprod. 65, 771–776 (2001)

    CAS  Article  Google Scholar 

  10. Tanaka, S. S. et al. The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev. 14, 841–853 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Costoya, J. A. et al. Essential role of Plzf in maintenance of spermatogonial stem cells. Nature Genet. 36, 653–659 (2004)

    CAS  Article  Google Scholar 

  12. Buaas, F. W. et al. Plzf is required in adult male germ cells for stem cell self-renewal. Nature Genet. 36, 647–652 (2004)

    CAS  Article  Google Scholar 

  13. Brinster, R. L. & Zimmermann, J. W. Spermatogenesis following male germ-cell transplantation. Proc. Natl Acad. Sci. USA 91, 11298–11302 (1994)

    CAS  Article  ADS  Google Scholar 

  14. Kanatsu-Shinohara, M. et al. Generation of pluripotent stem cells from neonatal mouse testis. Cell 119, 1001–1012 (2004)

    CAS  Article  Google Scholar 

  15. Schatten, G., Smith, J., Navara, C., Park, J. H. & Pedersen, R. Culture of human embryonic stem cells. Nature Methods 2, 455–463 (2005)

    CAS  Article  Google Scholar 

  16. Friedrich, G. & Soriano, P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5, 1513–1523 (1991)

    CAS  Article  Google Scholar 

  17. Reijo, R. A. et al. DAZ family proteins exist throughout male germ cell development and transit from nucleus to cytoplasm at meiosis in humans and mice. Biol. Reprod. 63, 1490–1496 (2000)

    CAS  Article  Google Scholar 

  18. Ehmcke, J., Hubner, K., Scholer, H. R. & Schlatt, S. Spermatogonia: origin, physiology and prospects for conservation and manipulation of the male germ line. Reprod. Fertil. Dev. 18, 7–12 (2006)

    Article  Google Scholar 

  19. Wang, P. J., Page, D. C. & McCarrey, J. R. Differential expression of sex-linked and autosomal germ-cell-specific genes during spermatogenesis in the mouse. Hum. Mol. Genet. 14, 2911–2918 (2005)

    CAS  Article  Google Scholar 

  20. Ryu, B. Y., Orwig, K. E., Kubota, H., Avarbock, M. R. & Brinster, R. L. Phenotypic and functional characteristics of spermatogonial stem cells in rats. Dev. Biol. 274, 158–170 (2004)

    CAS  Article  Google Scholar 

  21. Kanatsu-Shinohara, M., Toyokuni, S. & Shinohara, T. CD9 is a surface marker on mouse and rat male germline stem cells. Biol. Reprod. 70, 70–75 (2004)

    CAS  Article  Google Scholar 

  22. Shinohara, T., Avarbock, M. R. & Brinster, R. L. β1- and α6-integrin are surface markers on mouse spermatogonial stem cells. Proc. Natl Acad. Sci. USA 96, 5504–5509 (1999)

    CAS  Article  ADS  Google Scholar 

  23. Seydoux, G. & Braun, R. E. Pathway to totipotency: lessons from germ cells. Cell 127, 891–904 (2006)

    CAS  Article  Google Scholar 

  24. Keller, G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 19, 1129–1155 (2005)

    CAS  Article  Google Scholar 

  25. Sun, J. F. et al. Microvascular patterning is controlled by fine-tuning the Akt signal. Proc. Natl Acad. Sci. USA 102, 128–133 (2005)

    CAS  Article  ADS  Google Scholar 

  26. Simon, A. & Frisen, J. From stem cell to progenitor and back again. Cell 128, 825–826 (2007)

    CAS  Article  Google Scholar 

  27. Jiang, Y. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41–49 (2002)

    CAS  Article  ADS  Google Scholar 

  28. Baba, S. et al. Generation of cardiac and endothelial cells from neonatal mouse testis-derived multipotent germline stem cells. Stem Cells 25, 1375–1383 (2007)

    CAS  Article  Google Scholar 

  29. Kanatsu-Shinohara, M. et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 69, 612–616 (2003)

    CAS  Article  Google Scholar 

  30. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996)

    CAS  Article  ADS  Google Scholar 

  31. Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 22, 1567–1572 (2004)

    CAS  Article  Google Scholar 

  32. Ogawa, T., Arechaga, J. M., Avarbock, M. R. & Brinster, R. L. Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J. Dev. Biol. 41, 111–122 (1997)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Howard Hughes Medical Institute, Ansary Stem Cell Center for Regenerative Medicine and Memorial Sloan Kettering Cancer Center T32 grant (M.S.), an AACR–Genentech BioOncology Fellowship for Cancer Research on Angiogenesis (M.S.), the Heed Foundation (S.C.), the International Retinal Research Foundation (S.C.) and National Heart, Lung and Blood Institute grants (S.R.). We thank M. Hardy, P. Schlegel, Marc Goldstein, A. Brivanlou and S. Noggle for critical input. We are grateful to G. Enders for providing anti-GCNA antibody. We thank D. S. Johnston, G. Linkov and G. Zlotchenko for technical assistance.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Shahin Rafii.

Ethics declarations

Competing interests

S.R., M.S., S.V.S. and S.C. have filed a provisional patent application related to the use of GPR125.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-12 with Legends, Supplementary Tables 1 and 2, and the Legend for Supplementary Video 1. (PDF 1691 kb)

Supplementary Video

This file contains Supplementary Video 1. (MOV 1448 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Seandel, M., James, D., Shmelkov, S. et al. Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature 449, 346–350 (2007). https://doi.org/10.1038/nature06129

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature06129

This article is cited by

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.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing