A selective culture system for generating terminal deoxynucleotidyl transferase-positive lymphoid precursor cells in vitro. IV. 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 deoxynucleotidyl 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 sequentially appearing subsets of B cell progenitors in the culture system are characterized. The first lymphoid subsets to appear, and presumably 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 nonadherent phase, suggesting the release of a constant proportion of the adherent lymphoid cells from the feeder layer. Lastly, a wave of TdT- lymphoid cells is observed in the nonadherent fraction. All of the lymphoid cells generated in the cultures expressed the rat equivalent of the B220 antigen, whereas only one-half of the lymphoid cells (and one-half of the TdT+ cells) expressed the heat stable antigen (HSA), while none expressed surface or cytoplasmic immunoglobulin (Ig). This phenotypic profile identifies one-half of the generated lymphoid cells as pre-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 demonstrate that virtually all the precursors of TdT+ lymphoid cells (pre-TdT cells) 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 activity 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, it was observed that a decrease in the concentration of fetal bovine serum (FBS) in the culture medium resulted in the selective release of the adherent TdT+, but not TdT-, lymphoid cells into the nonadherent compartment. These studies indicated that a stepwise progression of the earliest detectable stages in lymphoid development was associated with changes in stromal cell/lymphoid cell interactions that were partly regulated by serum-dependent adhesion mechanisms.