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.

  • Article
  • Published:

Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro

Nature Immunology volume 5, pages 410–417 (2004)Cite this article

Abstract

Embryonic stem cells (ESCs) have the potential to serve as a renewable source of transplantable tissue-specific stem cells. However, the molecular cues necessary to direct the differentiation of ESCs toward specific cell lineages remain obscure. Here we report the successful induction of ESC differentiation into mature functional T lymphocytes with a simple in vitro coculture system. The directed differentiation of ESCs into T cells required the engagement of Notch receptors by Delta-like 1 ligand (DL1) expressed on the OP9-DL1 stromal cell line. We found a normal program of T cell differentiation in ESC–OP9-DL1 cell cocultures. ESC-derived T cell progenitors effectively reconstituted the T cell compartment of immunodeficient mice, enabling an effective response to a viral infection. These findings provide a powerful tool for the molecular analysis of T cell development and open new avenues for the development of immunotherapeutic approaches using defined sources of stem cells.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: T cell development from ESCs in vitro.
Figure 2: ESC-derived T cell development recapitulates thymic development in vitro.
Figure 3: Gene expression analysis of ESCs cultured in vitro.
Figure 4: Functionally mature CD8+ T cells develop from ESCs cultured on OP9-DL1 cells.
Figure 5: ESC-derived T cells can reconstitute immune function in immunodeficient hosts.

Similar content being viewed by others

References

  1. 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).

    CAS  Google Scholar 

  2. Kennedy, M. et al. A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature 386, 488–493 (1997).

    Article  CAS  Google Scholar 

  3. Nakano, T., Kodama, H. & Honjo, T. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101 (1994).

    Article  CAS  Google Scholar 

  4. Nakano, T. Lymphohematopoietic development from embryonic stem cells in vitro . Semin. Immunol. 7, 197–203 (1995).

    Article  CAS  Google Scholar 

  5. Cho, S.K. et al. Functional characterization of B lymphocytes generated in vitro from embryonic stem cells. Proc. Natl. Acad. Sci. USA 96, 9797–9802 (1999).

    Article  CAS  Google Scholar 

  6. Keller, G.M. In vitro differentiation of embryonic stem cells. Curr. Opin. Cell Biol. 7, 862–869 (1995).

    Article  CAS  Google Scholar 

  7. Kaufman, M.H., Robertson, E.J., Handyside, A.H. & Evans, M.J. Establishment of pluripotential cell lines from haploid mouse embryos. J. Embryol. Exp. Morphol. 73, 249–261 (1983).

    CAS  Google Scholar 

  8. Orkin, S.H. & Morrison, S.J. Stem-cell competition. Nature 418, 25–27 (2002).

    Article  CAS  Google Scholar 

  9. de Pooter, R.F., Cho, S.K., Carlyle, J.R. & Zúñiga-Pflücker, J.C. In vitro generation of T lymphocytes from embryonic stem cell-derived prehematopoietic progenitors. Blood 102, 1649–1653 (2003).

    Article  CAS  Google Scholar 

  10. Schmitt, T.M. & Zúñiga-Pflücker, J.C. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro . Immunity 17, 749–756 (2002).

    Article  CAS  Google Scholar 

  11. Pui, J.C. et al. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 11, 299–308 (1999).

    Article  CAS  Google Scholar 

  12. Radtke, F. et al. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10, 547–558 (1999).

    Article  CAS  Google Scholar 

  13. Wolfer, A., Wilson, A., Nemir, M., MacDonald, H.R. & Radtke, F. Inactivation of Notch1 impairs VDJb rearrangement and allows pre-TCR-independent survival of early ab lineage thymocytes. Immunity 16, 869–879 (2002).

    Article  CAS  Google Scholar 

  14. Washburn, T. et al. Notch activity influences the ab versus gd T cell lineage decision. Cell 88, 833–843 (1997).

    Article  CAS  Google Scholar 

  15. Robey, E. et al. An activated form of Notch influences the choice between CD4 and CD8 T cell lineages. Cell 87, 483–492 (1996).

    Article  CAS  Google Scholar 

  16. Izon, D.J. et al. Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength. Immunity 14, 253–264 (2001).

    Article  CAS  Google Scholar 

  17. Fowlkes, B.J. & Robey, E.A. A reassessment of the effect of activated Notch1 on CD4 and CD8 T cell development. J. Immunol. 169, 1817–1821 (2002).

    Article  CAS  Google Scholar 

  18. Deftos, M.L., Huang, E., Ojala, E.W., Forbush, K.A. & Bevan, M.J. Notch1 signaling promotes the maturation of CD4 and CD8 SP thymocytes. Immunity 13, 73–84 (2000).

    Article  CAS  Google Scholar 

  19. Engel, I., Johns, C., Bain, G., Rivera, R.R. & Murre, C. Early thymocyte development is regulated by modulation of E2A protein activity. J. Exp. Med. 194, 733–745 (2001).

    Article  CAS  Google Scholar 

  20. Barndt, R.J., Dai, M. & Zhuang, Y. Functions of E2A-HEB heterodimers in T-cell development revealed by a dominant negative mutation of HEB. Mol. Cell. Biol. 20, 6677–6685 (2000).

    Article  CAS  Google Scholar 

  21. Scott, E.W., Simon, M.C., Anastasi, J. & Singh, H. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265, 1573–1577 (1994).

    Article  CAS  Google Scholar 

  22. Ting, C.N., Olson, M.C., Barton, K.P. & Leiden, J.M. Transcription factor GATA3 is required for development of the T-cell lineage. Nature 384, 474–478 (1996).

    Article  CAS  Google Scholar 

  23. Williams, R.L. et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684–687 (1988).

    Article  CAS  Google Scholar 

  24. Shortman, K. & Wu, L. Early T lymphocyte progenitors. Annu. Rev. Immunol. 14, 29–47 (1996).

    Article  CAS  Google Scholar 

  25. Rothenberg, E.V., Telfer, J.C. & Anderson, M.K. Transcriptional regulation of lymphocyte lineage commitment. Bioessays 21, 726–742 (1999).

    Article  CAS  Google Scholar 

  26. Anderson, M.K., Weiss, A.H., Hernandez-Hoyos, G., Dionne, C.J. & Rothenberg, E.V. Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T cell development at the pro-T cell stage. Immunity 16, 285–296 (2002).

    Article  CAS  Google Scholar 

  27. Zhuang, Y., Soriano, P. & Weintraub, H. The helix-loop-helix gene E2A is required for B cell formation. Cell 79, 875–884 (1994).

    Article  CAS  Google Scholar 

  28. Bain, G. et al. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 79, 885–892 (1994).

    Article  CAS  Google Scholar 

  29. Peschon, J.J. et al. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180, 1955–1960 (1994).

    Article  CAS  Google Scholar 

  30. Mombaerts, P. et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 (1992).

    Article  CAS  Google Scholar 

  31. Tomita, K. et al. The bHLH gene Hes1 is essential for expansion of early T cell precursors. Genes Dev. 13, 1203–1210 (1999).

    Article  CAS  Google Scholar 

  32. Anderson, M.K. et al. Definition of regulatory network elements for T cell development by perturbation analysis with PU.1 and GATA3. Dev. Biol. 246, 103–121 (2002).

    Article  CAS  Google Scholar 

  33. 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).

    Article  CAS  Google Scholar 

  34. Robertson, E.J. Derivation and maintenance of embryonic stem cell cultures. Methods Mol. Biol. 75, 173–184 (1997).

    CAS  Google Scholar 

  35. Cho, S.K. & Zúñiga-Pflücker, J.C. Development of lymphoid lineages from embryonic stem cells in vitro . Methods Enzymol. 365, 158–169 (2003).

    Article  Google Scholar 

  36. Rodewald, H.-R., Kretzschmar, K., Takeda, S., Hohl, C. & Dessing, M. Identification of pro-thymocytes in murine fetal blood: T lineage commitment can precede thymus colonization. EMBO J. 13, 4229–4240 (1994).

    Article  CAS  Google Scholar 

  37. Carlyle, J.R. & Zúñiga-Pflücker, J.C. Requirement for the thymus in ab T lymphocyte lineage commitment. Immunity 9, 187–197 (1998).

    Article  CAS  Google Scholar 

  38. Godfrey, D.I., Kennedy, J., Suda, T. & Zlotnik, A. A developmental pathway involving four phenotypically and functionally distinct subsets of CD3−CD4−CD8− triple negative adult mouse thymocytes defined by CD44 and CD25 expression. J. Immunol. 150, 4244–4252 (1993).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank P. Poussier for critical review of the manuscript and G. Knowles for assistance in cell sorting. Supported by Canadian Institutes of Health Research (R.F.d.P. and J.C.Z.-P.), the National Cancer Institute of Canada and the Canadian Cancer Society.

Author information

Author notes

  1. Sarah K Cho

    Present address: Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, California, 92093, USA

Authors and Affiliations

  1. Department of Immunology, University of Toronto, Sunnybrook and Women's College Health Sciences Centre, 2075 Bayview Avenue, Toronto, M4N 3M5, Ontario, Canada

    Thomas M Schmitt, Renée F de Pooter, Sarah K Cho & Juan Carlos Zúñiga-Pflücker

  2. Departments of Medical Biophysics and Immunology, Ontario Cancer Institute, Toronto, M5G 2M9, Ontario, Canada

    Matthew A Gronski & Pamela S Ohashi

Authors
  1. Thomas M Schmitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Renée F de Pooter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthew A Gronski
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sarah K Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pamela S Ohashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Juan Carlos Zúñiga-Pflücker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Juan Carlos Zúñiga-Pflücker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schmitt, T., de Pooter, R., Gronski, M. et al. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat Immunol 5, 410–417 (2004). https://doi.org/10.1038/ni1055

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni1055

This article is cited by

Search

Advanced 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