Functional maturation of murine B lymphocyte precursors. I. Selection of adherent cell-dependent precursors from bone marrow and fetal liver.
A cell culture assay is described which is suitable to explore interactions between cells of the bone marrow (BM) microenvironment on one side and B lymphocyte progenitors on the other. First, a heterogeneous adherent BM (aBM) cell population was established on Cytodex 1 microcarriers. Then, adherent cell and surface IgM+(sIgM+) cell-depleted BM precursors or adherent cell-depleted day 12 fetal liver cells were added. The generation of B cells in these cultures was monitored by staining with fluorochrome-labeled anti-mu-chain antibody and by lipopolysaccharide (LPS) induction of protein A plaque-forming cells at limiting dilution. In the absence of aBM cells, some B cells arose after 24 hr from BM precursors but not from day 12 fetal liver cells. With aBM cells, BM precursors gave rise to a distinct second wave of B cells starting after 5 days of culture. When fetal liver cells were cultured on aBM cells, B cells appeared after a delay of 4 to 5 days. By using Ig allotype-congenic mouse strains (C.AL 20, BALB/c) and an allotype-specific plaque assay, we established that mature B cells originate from the putative progenitors and not from the aBM cell population. In an attempt to eliminate the aBM cell-independent progenitor subset, mice were pretreated with 5-fluorouracil 5 days before BM cells were collected. The remaining cells still contained B cells, but the frequency of c mu+ sIgM- pre-B cells was less than 10(-5). Remaining B cells were removed by anti-mu panning. In cultures of this precursor cell population, LPS-responsive B cells appeared after a delay of about 1 wk, and their generation was totally aBM cell-dependent and was maintained for more than 2 wk.
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D001853:Bone Marrow / Q000196:embryology; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D005260:Female; D005472:Fluorouracil / Q000494:pharmacology; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D007075:Immunoglobulin M / Q000032:analysis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000166:cytology* / Q000196:embryology; D008212:Lymphocyte Depletion; D051379:Mice; D008807:Mice, Inbred BALB C; D011247:Pregnancy; D051381:Rats; D011917:Rats, Inbred Lew; D011947:Receptors, Antigen, B-Cell / Q000032:analysis
D007075:Immunoglobulin M; D008070:Lipopolysaccharides; D011947:Receptors, Antigen, B-Cell; D005472:Fluorouracil