Skip to main content

In Vitro Generation of Lymphocytes From Embryonic Stem Cells

  • Protocol
Basic Cell Culture Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 290))

  • 4902 Accesses

Abstract

Lymphocytes arise during ontogeny via a series of increasingly restricted intermediates. Initially, the mesoderm gives rise to hemangioblasts, which can differentiate into endothelial precursors, or hematopoietic stem cells (HSCs). HSCs can either self-renew or differentiate into lineage-restricted progenitors and, ultimately, to mature effector cells. This complex process is only beginning to be understood, and the ability to generate lymphocytes from embryonic stem (ES) cells in vitro will facilitate further study by providing a model system in which the effects of genetic and environmental manipulations of ES-cell-derived progenitors can be examined. In this protocol, we describe procedures for generating either B- and NK- or T-lymphocytes from mouse ES cells in vitro.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Chen, U., Kosco, M., and Staerz, U. (1992) Establishment and characterization of lymphoid and myeloid mixed-cell populations from mouse late embryoid bodies, “embryonic-stem-cell fetuses.” Proc. Natl. Acad. Sci. USA 89, 2541–2545.

    Article  PubMed  CAS  Google Scholar 

  2. Gutierrez-Ramos, J. C. and Palacios, R. (1992) In vitro differentiation of embryonic stem cells into lymphocyte precursors able to generate T and B lymphocytes in vivo. Proc. Natl. Acad. Sci. USA 89, 9171–9175.

    Article  PubMed  CAS  Google Scholar 

  3. Potocnik, A. J., Nielsen, P. J., and Eichmann, K. (1994) In vitro generation of lymphoid precursors from embryonic stem cells. EMBO J. 13, 5274–5283.

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  6. Yoshida, H., Hayashi, S., Kunisada, T., et al. (1990) The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345,442–444.

    Article  PubMed  CAS  Google Scholar 

  7. Hirayama, F., Lyman, S. D., Clark, S. C., and Ogawa, M. (1995) The flt3 ligand supports proliferation of lymphohematopoietic progenitors and early B-lymphoid progenitors. Blood 85, 1762–1768.

    PubMed  CAS  Google Scholar 

  8. Hudak, S., Hunte, B., Culpepper, J., et al. (1995) FLT3/FLK2 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units. Blood 85, 2747–2755.

    PubMed  CAS  Google Scholar 

  9. Hunte, B. E., Hudak, S., Campbell, D., Xu, Y., and Rennick, D. (1996) flk2/flt3 ligand is a potent cofactor for the growth of primitive B cell progenitors. J. Immunol. 156,489–496.

    PubMed  CAS  Google Scholar 

  10. Jacobsen, S. E., Okkenhaug, C., Myklebust, J., Veiby, O. P., and Lyman, S. D. (1995) The FLT3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factors. J. Exp. Med. 181, 1357–1363.

    Article  PubMed  CAS  Google Scholar 

  11. Lyman, S. D. and Jacobsen, S. E. (1998) c-kit ligand and Flt3 ligand: stem/pro genitor cell factors with overlapping yet distinct activities. Blood 91,1101–1134.

    PubMed  CAS  Google Scholar 

  12. Veiby, O. P., Lyman, S. D., and Jacobsen, S. E. W. (1996) Combined signaling through interleukin-7 receptors and flt3 but not c-kit potently and selectively pro motes B-cell commitment and differentiation from uncommitted murine bone marrow progenitor cells. Blood 88, 1256–1265.

    PubMed  CAS  Google Scholar 

  13. Sitnicka, E., Bryder, D., Theilgaard-Monch, K., Buza-Vidas, N., Adolfsson, J., and Jacobsen, S. E. (2002) Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool. Immunity 17, 463–472.

    Article  PubMed  CAS  Google Scholar 

  14. Cho, S. K., Webber, T. D., Carlyle, J. R., Nakano, T., Lewis, S. M., and Zúñiga-Pflücker, J. C. (1999) Functional characterization of B lymphocytes generated in vitro from embryonic stem cells. Proc. Natl. Acad. Sci. USA 96, 9797–9802.

    Article  PubMed  CAS  Google Scholar 

  15. Kennedy, M. K., Glaccum, M., Brown, S. N., et al. (2000) Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J. Exp. Med. 191,771–780.

    Article  PubMed  CAS  Google Scholar 

  16. De Pooter, R. F., Cho, S. K., Carlyle, J. R., and Zuniga-Pflucker, J. C. (2003) In vitro generation of T lymphocytes from embryonic stem cell-derived prehemato-poietic progenitors. Blood 102, 1649–1653.

    Article  PubMed  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  18. Takahama, Y. (2000) Differentiation of mouse thymocytes in fetal thymus organ culture. Methods Mol. Biol. 134, 37–46.

    PubMed  CAS  Google Scholar 

  19. Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D., and Hayakawa, K. (1991) Resolution and characterization of pro-B and pre-pro-B cell stages in nor mal mouse bone marrow. J. Exp. Med. 173, 1213–1225.

    Article  PubMed  CAS  Google Scholar 

  20. Li, Y. S., Wasserman, R., Hayakawa, K., and Hardy, R. R. (1996) Identification of the earliest B lineage stage in mouse bone marrow. Immunity 5, 527–535.

    Article  PubMed  CAS  Google Scholar 

  21. Melchers, F., Rolink, A., Grawunder, U., et al. (1995) Positive and negative selection events during B lymphopoiesis. Curr. Opin. Immunol. 7, 214–227.

    Article  PubMed  CAS  Google Scholar 

  22. Carlyle, J. R., Michie, A. M., Cho, S. K., and Z úñiga-Pflücker, J. C. (1998) Natu ral killer cell development and function precede alpha beta T cell differentiation in mouse fetal thymic ontogeny. J. Immunol. 160, 744–753.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2005 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

de Pooter, R.F., Cho, S.K., Zúñiga-Pflücker, J.C. (2005). In Vitro Generation of Lymphocytes From Embryonic Stem Cells. In: Helgason, C.D., Miller, C.L. (eds) Basic Cell Culture Protocols. Methods in Molecular Biology™, vol 290. Humana Press. https://doi.org/10.1385/1-59259-838-2:135

Download citation

  • DOI: https://doi.org/10.1385/1-59259-838-2:135

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-284-1

  • Online ISBN: 978-1-59259-838-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics