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.
Key Words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
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.
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.
Nakano, T. (1995) Lymphohematopoietic development from embryonic stem cells in vitro. Semin. Immunol. 7, 197–203.
Nakano, T., Kodama, H., and Honjo, T. (1994) Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Robertson, E. J. (1997) Derivation and maintenance of embryonic stem cell cultures. Methods Mol. Biol. 75, 173–184.
Takahama, Y. (2000) Differentiation of mouse thymocytes in fetal thymus organ culture. Methods Mol. Biol. 134, 37–46.
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.
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.
Melchers, F., Rolink, A., Grawunder, U., et al. (1995) Positive and negative selection events during B lymphopoiesis. Curr. Opin. Immunol. 7, 214–227.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights 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