A SELECTIVE CULTURE SYSTEM FOR GENERATING TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE-POSITIVE LYMPHOID-CELLS IN-VITRO .5. DETECTION OF STAGE-SPECIFIC PRO-B-CELL STIMULATING ACTIVITY IN MEDIUM CONDITIONED BY MOUSE BONE-MARROW STROMAL CELLS

The selective in vitro generation of rat, mouse, and human terminal de oxynucleotidyl transferase-positive (TdT+) lymphoid cells in our long- term xenogeneic bone marrow (BM) culture system is characterized by ph ysical interaction between the developing lymphocytes and mouse BM-adh erent stromal cells and macrophages. In the present study, experiments in which microporous membrane culture inserts were inoculated with ra t BM cells demonstrated that although the generation of primitive B-li neage 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 s pleen) adherent cells and stromal-cell lines therefrom supported the p roliferation of early rat lymphoid cells in a dose-dependent manner. D ouble immunofluorescence for incorporated bromo-deoxyuridine (BrdU) an d early B-lineage markers of rat BM lymphoid cells maintained in cultu re inserts or conditioned medium (CM), and studies of their in vitro a nd in vivo developmental potentials, indicated that the lymphoprolifer ative response resulted from the selective stimulation of lymphoid ste m and/or progenitor cells. The most primitive of these target cells ha d a HIS24+ HIS50- TdT- cmu- sIg-, pre-pro-B-cell phenotype. Whereas th is subset normally constitutes less than 2% of B-lineage BM cells in v ivo, it comprises more than 25% of total lymphoid cells in vitro. In a ddition, the number of TdT+ cells, predominantly of the early pro-B-ce ll phenotype (HIS24+ HIS50- cmu- sIg-), was increased approximately te nfold above input levels. Based on these and previous findings, a sche matic model is proposed for the developmental pathway of early B-linea ge cells in rat BM from the level of the committed (possibly common) l ymphoid stem cell to that of the pre-B-cell.