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

Abstract

The selectivein vitrogeneration of rat, mouse, and human terminal deoxynucleotidyl transferase-positive (TdT+lymphoid cells in our long-term xenogeneic bone marrow (BM) culture system is characterized by physical interaction between the developing lymphocytes and mouse BM-adherent stromal cells and macrophages. In the present study, experiments in which micropor)us membrane culture inserts were inoculated with rat BM cells demonstrated that although the generation of primitive B-lineage lymphoid cells requires the 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 thymus or spleen) adherent cells and stromal-cell lines therefrom supported the proliferation of early rat lymphoid cells in a dose-dependent manner. Double immunofluorescence for incorporated bromo-deoxyuridine (BrdU) and early B-lineage markers of rat BM lymphoid cells maintained in culture inserts or conditioned medium (CM), and studies of their in vitro andin vivodevelopmental potentials, indicated that the lymphoproliferative response resulted from the selective stimulation of lymphoid stem and/or progenitor cells. The most primitive 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-), was increased 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 rat BM from the level of the committed (possibly common) lymphoid stem cell to that of the pre-B-cell.