Analyses of T-cell differentiation from hemopoietic stem cells in the G0 phase by an in vitro method

Junko Toki, Takayuki Kumamoto, Hajime Ogata, Masayo Kawamura, Manabu Fukumoto, Cherry, Yutaka Yamamoto, Soe Than, Muneo Inaba, Yasuo Himeno, Hiroo Imura, Robert A. Good, Susumu Ikehara

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4 Citations (Scopus)

Abstract

Using differential radiation sensitivity of components of mouse embryonal thymus, an in vitro method for studying T-cell differentiation from hemopoietic stem cells (HSCs) in the G0 phase was established. Intrathymic T-cell precursors present in embryonal thymus were found to be quite radioresistant (up to 20 Gy), and consequently 25-Gy-irradiated embryonal thymic lobes were used. Thymic lobes (25-Gy irradiated) taken from mouse fetuses (gestation day 15) were placed in Millipore-HA culture plates supported on squares of gelatin foam sponge in 24-well culture plates in which neonatal thymus stromal cells were cultured. HSCs (105 cells per well) in the G0 phase were added to these thymic lobes and cocultured at 37°C in a 5% CO2/95% air incubator. Half the culture medium was changed every week. After 3 weeks, a large number of colonies had formed. Immunohistochemical studies and fluorescence-activated cell sorter analyses revealed that the colonizing cells regularly develop and exhibit surface markers characteristic of T cells (Thy-1, IL-2R, L3T4, Lyt-2, etc.). In situ hybridization analyses revealed that mRNA expression for T-cell receptor (TCR) β chains occurred within colonizing cells. Using a monoclonal antibody (F23.1), expression of TCR β-chain variable domain (VβS) on the surface of these developing T cells was demonstrated. These cells responded to interleukin 2 and/or anti-CD3 monoclonal antibody, indicating functional T cells. This method will be useful in studying T-cell differentiation pathways from pluripotent HSCs and in clarifying the mechanisms involved in negative and positive selection of T cells within the thymus.

Original languageEnglish
Pages (from-to)7548-7551
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume88
Issue number17
DOIs
Publication statusPublished - 1991

Keywords

  • Embryonal thymus
  • In vitro T-cell differentiation
  • Organ culture

ASJC Scopus subject areas

  • General

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    Toki, J., Kumamoto, T., Ogata, H., Kawamura, M., Fukumoto, M., Cherry, Yamamoto, Y., Than, S., Inaba, M., Himeno, Y., Imura, H., Good, R. A., & Ikehara, S. (1991). Analyses of T-cell differentiation from hemopoietic stem cells in the G0 phase by an in vitro method. Proceedings of the National Academy of Sciences of the United States of America, 88(17), 7548-7551. https://doi.org/10.1073/pnas.88.17.7548