The Development of T Lymphocytes in Fetal Thymus Organ Culture (original) (raw)

Abstract

Fetal thymus organ culture (FTOC) is a unique and powerful culture system that allows intrathymic T-lymphocyte development in vitro. T-cell development in FTOC well represents fetal thymocyte development in vivo. Here we describe the basic method for FTOC as well as several related techniques, including reconstitution of thymus lobes with T-lymphoid progenitor cells, high-oxygen submersion culture, reaggregation thymus organ culture, retrovirus-mediated gene transfer to developing thymocytes in FTOC, and coculture of progenitor cells with OP9-DL1 cells.

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References

  1. Owen JJT, Ritter MA (1969) Tissue interaction in the development of thymus lymphocytes. J Exp Med 129:431–442
    Article PubMed CAS Google Scholar
  2. Owen JJT (1974) Ontogeny of the immune system. Prog Immunol 2:163–173
    Google Scholar
  3. Mandel T, Russel PJ (1971) Differentiation of foetal mouse thymus. Ultrastructure of organ cultures and of subcapsular grafts. Immunology 21:659–674
    PubMed CAS Google Scholar
  4. Mandel TE, Kennedy MM (1978) The differentiation of murine thymocytes in vivo and in vitro. Immunology 35:317–331
    PubMed CAS Google Scholar
  5. Jenkinson EJ, van Ewijk W, Owen JJT (1981) Major histocompatibility complex antigen expression on the epithelium of the developing thymus in normal and nude mice. J Exp Med 153:280–292
    Article PubMed CAS Google Scholar
  6. Kingston R, Jenkinson EJ, Owen JJT (1985) A single stem cell can recolonize an embryonic thymus, producing phenotypically distinct T-cell populations. Nature 317:811–813
    Article PubMed CAS Google Scholar
  7. Takahama Y (2000) Differentiation of mouse thymocytes in fetal thymus organ culture. Methods Mol Biol 134:37–46
    PubMed CAS Google Scholar
  8. Takahama Y, Hasegawa T, Itohara S, Ball EL, Sheard MA, Hashimoto Y (1994) Entry of CD4-CD8- immature thymocytes into the CD4/CD8 developmental pathway is controlled by tyrosine kinase signals that can be provided through T cell receptor components. Int Immunol 6:1505–1514
    Article PubMed CAS Google Scholar
  9. Theiler K (1989) The house mouse. Springer-Verlag, New York, NY
    Google Scholar
  10. Kaufman MH (1992) The atlas of mouse development. Academic, San Diego, CA
    Google Scholar
  11. Butler H, Juurlink BH (1987) An atlas for staging mammalian and chick embryos. CRC, Boca Raton, FL
    Google Scholar
  12. Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7:1063–1066
    Article PubMed CAS Google Scholar
  13. Saitoh T, Nakano H, Yamamoto N, Yamaoka S (2002) Lymphotoxin-β receptor mediates NEMO-independent NF-kB activation. FEBS Lett 532:45–51
    Article PubMed CAS Google Scholar
  14. Schmitt MT, Zuniga-Pflucker JC (2002) Induction of T cell development from hematopoietic progenitor cells by Delta-like-1 in vitro. Immunity 17:749–756
    Article PubMed CAS Google Scholar
  15. Tsuda S, Rieke S, Hashimoto Y, Nakauchi H, Takahama Y (1996) IL-7 supports D-J but not V-DJ rearrangement of TCR-β gene in fetal liver progenitor cells. J Immunol 156:3233–3242
    PubMed CAS Google Scholar
  16. Watanabe Y, Katsura Y (1993) Development of T cell receptor αβ bearing T cells in the submersion organ culture of murine fetal thymus at high oxygen concentration. Eur J Immunol 23:200–205
    Article PubMed CAS Google Scholar
  17. Jenkinson EJ, Anderson G, Owen JJT (1992) Studies on T cell maturation on defined thymic stromal cell populations in vitro. J Exp Med 176:845–853
    Article PubMed CAS Google Scholar
  18. Hawley RG, Fong AZC, Burns BF, Hawley TS (1992) Transplantable myeloproliferative disease induced in mice by interleukin 6 retrovirus. J Exp Med 176:1149–1163
    Article PubMed CAS Google Scholar
  19. Nitta T, Nasreen M, Seike T, Goji A, Ohigashi I, Miyazaki T, Ohta T, Kanno M, Takahama Y (2006) IAN family critically regulates survival and development of T lymphocytes. PLoS Biol 4:e103
    Article PubMed Google Scholar
  20. Hozumi K, Mailhos C, Negishi N, Hirano K, Yahata T, Ando K, Zuklys S, Holländer GA, Shima DT, Habu S (2008) Delta-like 4 is indispensable in thymic environment specific for T cell development. J Exp Med 205:2507–2513
    Article PubMed CAS Google Scholar
  21. Koch U, Fiorini E, Benedito R, Besseyrias V, Schuster-Gossler K, Pierres M, Manley NR, Duarte A, Macdonald HR, Radtke F (2008) Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment. J Exp Med 205:2515–2523
    Article PubMed CAS Google Scholar
  22. Taghon TN, David ES, Zúñiga-Pflücker JC, Rothenberg EV (2005) Delayed, asynchronous, and reversible T-lineage specification induced by Notch/Delta signaling. Genes Dev 19:965–978
    Article PubMed CAS Google Scholar
  23. Ramsdell F, Zúñiga-Pflücker JC, Takahama Y (2006) In vitro systems for the study of T cell development: fetal thymus organ culture and OP9-DL1 cell coculture. Curr Protoc Immunol Chapter 3, Unit 3.18
    Google Scholar
  24. de Pooter R, Zúñiga-Pflücker JC (2007) T-cell potential and development in vitro: the OP9-DL1 approach. Curr Opin Immunol 19:163–168
    Article PubMed Google Scholar
  25. Takahama Y, Suzuki H, Katz KS, Grusby MJ, Singer A (1994) Positive selection of CD4+ T cells by TCR ligation without aggregation even in the absence of MHC. Nature 371:67–70
    Article PubMed CAS Google Scholar
  26. Takahama Y, Nakauchi H (1996) Phorbol ester and calcium ionophore can replace TCR signals that induce positive selection of CD4 T cells. J Immunol 157:1508–1513
    PubMed CAS Google Scholar
  27. Jenkinson EJ, Franchi LL, Kingston R, Owen JJT (1982) Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: application in the production of chimeric thymus rudiments. Eur J Immunol 12:583–587
    Article PubMed CAS Google Scholar
  28. Anderson G, Jenkinson EJ, Moore NC, Owen JJT (1993) MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. Nature 362:70–73
    Article PubMed CAS Google Scholar
  29. Jenkinson W, Jenkinson E, Anderson G (2008) Preparation of 2-dGuo-treated thymus organ cultures. J Vis Exp pii:906. doi:10.3791/906
    Google Scholar
  30. White A, Jenkinson E, Anderson G (2008) Reaggregate thymus cultures. J Vis Exp pii:905. doi:10.3791/905
    Google Scholar

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Authors and Affiliations

  1. Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Tokushima, Japan
    Takeshi Nitta & Izumi Ohigashi
  2. Division of Experimental Immunology, Institute for Genome Research, The University of Tokushima, Tokushima, Japan
    Yousuke Takahama

Authors

  1. Takeshi Nitta
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  2. Izumi Ohigashi
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  3. Yousuke Takahama
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Corresponding author

Correspondence toYousuke Takahama .

Editor information

Editors and Affiliations

  1. , Experimental Therapeutics, BC Cancer Agency, West 10th Ave. 675, Vancouver, V5Z 1L3, British Columbia, Canada
    Cheryl D. Helgason
  2. STEMCELL Technologies, Inc., W. Seventh Avenue 570, Vancouver, V5Z 1B3, British Columbia, Canada
    Cindy L. Miller

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Nitta, T., Ohigashi, I., Takahama, Y. (2013). The Development of T Lymphocytes in Fetal Thymus Organ Culture. In: Helgason, C., Miller, C. (eds) Basic Cell Culture Protocols. Methods in Molecular Biology, vol 946. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-128-8\_6

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