Functional characterization of lymphoid cells generated in serum-deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice.
We have investigated the phenotypic and functional characteristics of murine pre-B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL-7). Both serum-supplemented and serum-deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/lpr and its congenic strain MRL/+. SCF (100 ng/ml) and IL-7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum-supplemented and serum-deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum-supplemented cultures but were either ineffective or had modest activity in serum-deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL-7 in the absence of serum had almost exclusively a pre-B cell phenotype (BP-1+, B220+, slg-, CD4-, CD8-, Mac-1-, RB-6-). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12-14. At this time, the pre-B cells failed to differentiate further and started to die. Pre-B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 x 10(6) pre-B cells had readily detectable serum levels of IgM (54 +/- 26 micrograms/ml) and IgG (60 +/- 95 micrograms/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (> 80% BP-1+) populations of functional pre-B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum-deprived cultures stimulated with SCF and IL-7. These cultures, therefore, provide a highly enriched source of pre-B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice.
Ashany
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Hospital for Special Surgery, Cornell University Medical Center, New York, New York, USA.
Elkon
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Migliaccio
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Migliaccio
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D000818:Animals; D001327:Autoimmune Diseases / Q000276:immunology*; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D006410:Hematopoiesis / Q000187:drug effects; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology; D016130:Immunophenotyping; D066298:In Vitro Techniques; D015851:Interleukin-7 / Q000494:pharmacology*; D051379:Mice; D008809:Mice, Inbred C3H; D008817:Mice, Mutant Strains; D016513:Mice, SCID; D013154:Spleen / Q000166:cytology; D019089:Stem Cell Factor; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology
D003470:Culture Media; D016298:Hematopoietic Cell Growth Factors; D015851:Interleukin-7; D019089:Stem Cell Factor