TY - JOUR
T1 - Analyses of T-cell differentiation from hemopoietic stem cells in the G0 phase by an in vitro method
AU - Toki, Junko
AU - Kumamoto, Takayuki
AU - Ogata, Hajime
AU - Kawamura, Masayo
AU - Fukumoto, Manabu
AU - Cherry,
AU - Yamamoto, Yutaka
AU - Than, Soe
AU - Inaba, Muneo
AU - Himeno, Yasuo
AU - Imura, Hiroo
AU - Good, Robert A.
AU - Ikehara, Susumu
PY - 1991
Y1 - 1991
N2 - 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.
AB - 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.
KW - Embryonal thymus
KW - In vitro T-cell differentiation
KW - Organ culture
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U2 - 10.1073/pnas.88.17.7548
DO - 10.1073/pnas.88.17.7548
M3 - Article
C2 - 1881895
AN - SCOPUS:12044252603
VL - 88
SP - 7548
EP - 7551
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 17
ER -