A SELECTIVE CULTURE SYSTEM FOR GENERATING TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE-POSITIVE LYMPHOID PRECURSOR CELLS IN-VITRO .4. PROPERTIES AND DEVELOPMENTAL RELATIONSHIPS OF THE LYMPHOID-CELLS IN THE ADHERENT AND NONADHERENT COMPARTMENTS OF THE CULTURE

In a selective culture system, the in vitro generation of terminal deo xynucleotidyl transferase positive (TdT) B cell progenitors from rat, mouse, and human bone marrow (BM) is characterized by initial lymphoid colonization of a mouse BM feeder layer. In the present study, four s equentially appearing subsets of B cell progenitors in the culture sys tem are characterized. The first lymphoid subsets to appear, and presu mably the least mature, are the adherent TdT(-) and TdT(+) cells that reach plateau numbers on days 3 and 7, respectively. The appearance of TdT(+) cells in the adherent phase of the culture is closely followed by a parallel increase in the number of TdT(+) cells in the nonadhere nt phase, suggesting the release of a constant proportion of the adher ent lymphoid cells from the feeder layer. Lastly, a wave of TdT(-) lym phoid cells is observed in the nonadherent fraction. All of the lympho id cells generated in the cultures expressed the rat equivalent of the B220 antigen, whereas only one-half of the lymphoid cells (and one-ha lf of the TdT(+) cells) expressed the heat stable antigen (HSA), while none expressed surface or cytoplasmic immunoglobulin (Ig). This pheno typic profile identifies one-half of the generated lymphoid cells as p re-pro-B cells and the remainder as early to late pro-B cells. It also demonstrates that TdT is initially expressed prior to HSA expression in rat B cell differentiation. In vitro transfer experiments demonstra te that virtually all the precursors of TdT lymphoid cells (pre-TdT ce lls) in rat BM adhere to the mouse BM feeder layer during the first 24 hours of culture, and by day 7 of culture, the total pre-TdT cell act ivity in the adherent cell compartment increases more than 20-fold on a per cell basis and more than 70-fold on a per well basis. Finally, i t was observed that a decrease in the concentration of fetal bovine se rum (FBS) in the culture medium resulted in the selective release of t he adherent TdT, but not TdT, lymphoid cells into the nonadherent comp artment. These studies indicated that a stepwise progression of the ea rliest detectable stages in lymphoid development was associated with c hanges in stromal cell/lymphoid cell interactions that were partly reg ulated by serum-dependent adhesion mechanisms.