A Selective Culture System for Generating TerminalDeoxynucleotidyl Transferase-Positive Lymphoid CellsIn Vitro. V. Detection of Stage-Specific Pro-B-CellStimulating Activity in Medium Conditioned by Mouse Bone Marrow Stromal Cells

摘要

The selectivein vitrogeneration of rat, mouse, and human terminal deoxynucleotidyltransferase-positive (TdT+lymphoid cells in our long-term xenogeneic bone marrow (BM)culture system is characterized by physical interaction between the developing lymphocytesand mouse BM-adherent stromal cells and macrophages. In the present study, experimentsin which micropor)us membrane culture inserts were inoculated with rat BM cellsdemonstrated that although the generation of primitive B-lineage lymphoid cells requiresthe presence of a mouse BM feeder layer, cognitive recognition events are not necessary.Similarly, cell-free (and serum-free) medium conditioned with mouse BM (but not thymusor spleen) adherent cells and stromal-cell lines therefrom supported the proliferation ofearly rat lymphoid cells in a dose-dependent manner. Double immunofluorescence forincorporated bromo-deoxyuridine (BrdU) and early B-lineage markers of rat BM lymphoidcells maintained in culture inserts or conditioned medium (CM), and studies of their in vitroandin vivodevelopmental potentials, indicated that the lymphoproliferative responseresulted from the selective stimulation of lymphoid stem and/or progenitor cells. The mostprimitive of these target cells had a HIS24+HIS50-TdT-cμ-sIg-, pre-pro-B-cell phenotype.Whereas this subset normally constitutes less than 2% of B-lineage BM cellsin vivo,it comprises more than 25% of total lymphoid cellsin vitro. In addition, the number of TdT+cells, predominantly of the early pro-B-cell phenotype (HIS24+HIS50-TdT-cμ-sIg-), wasincreased approximately tenfold above input levels. Based on these and previous findings,a schematic model is proposed for the developmental pathway of early B-lineage cells in ratBM from the level of the committed (possibly common) lymphoid stem cell to that of thepre-B-cell.