Differentiation of murine B cell precursors in agar culture. Frequency, surface marker analysis and requirements for growth of clonable pre-B cells.

A semi-solid agar assay is described in which B cell progenitors, already present in day 12 fetal liver, generate colonies which contain antibody-secreting cells. Panning experiments, in which cells which initiate colony formation are depleted on plates coated with monoclonal antibodies, suggest that by the 13th day of gestation they express the antigen recognized by the monoclonal antibody AA4.1 and by day 14 they also express the B220 form of Ly-5 recognized by the monoclonal antibody 14.8. By similar criteria the precursor cells do not express mu, I-A, I-E or Lyb-2. Growth of cells in this assay is dependent upon soluble products provided by either fetal liver adherent cells, bone marrow adherent cells or colony-stimulating factor-containing conditioned media derived from placenta cells, L929 cells, WEHI-3 B(D-) cells, T helper cells or mouse lung cells. These experiments define two sets of growth conditions. In the first, when support is provided by fetal liver adherent cells, the limiting component appears to be the B cell precursor, allowing us to estimate the frequency of these cells during ontogeny. We find approximately 1 clonable pre-B cell in 300 000 fetal liver cells on day 12 of gestation and 1 in 6000 by day 16. Under the second set of growth conditions, when support is provided by bone marrow adherent cells or colony-stimulating factor-containing conditioned media, more than one cell, colony or cell product is limiting. Highly purified samples of granulocyte/macrophage colony-stimulating factor, colony-stimulating factor 1 and multilineage-hematopoietic growth factor are effective in this assay suggesting that the colony-stimulating factors are the active components under these conditions.