In vitro tracking of IL-7 responsiveness and gene expression during commitment of bipotent B-cell/macrophage...

The development of B lymphocytes from multipotent hematopoietic stem cells occurs through a series of intermediate cell types with increasingly restricted developmental potential. Despite intensive investigation, the underlying basis for commitment to a given lineage or the restriction in developmental potential of multipotent cells is unknown. To gain insight into this process we have developed an in vitro system that tracks a bipotent progenitor, which has the capacity to give rise to both B lymphocytes and macrophages, as it makes the transition to a B-lineage-committed precursor. The development of mature B lymphocytes from bipotent progenitors is dependent on interleukin 7 (IL-7), a pre-B-cell growth factor, in addition to other stromal-cell-derived factors such as IL-11 and mast cell growth factor (MGF). IL-7 acts on pre-B lymphocytes, but the stage of differentiation at which B-lineage cells become responsive to this factor, and its potential role in lineage commitment have not been investigated thoroughly. Here, we examine the requirements for IL-7 during the development of B lymphocytes from bipotent progenitors. Furthermore, we define onset of B-lineage-associated gene expression during the development of committed B-lineage cells under defined culture conditions.

We demonstrate that, under our experimental conditions, bipotent progenitors commit to differentiation through either the B or macrophage lineages within the first 3 days of culture. Cells that require IL-7 for survival first develop on day 3 of culture; however, commitment to the B lineage occurs at the same frequency in the presence or absence of this factor. After day 3 of culture, IL-7 is required both for the proliferation and survival of committed B-lineage progenitors and for the expression of several B-cell-associated genes, such as lambda5, VpreB, mb-1 and Rag1.

Our results demonstrate that the growth factor combination of IL-11 and MGF provides sufficient support for bipotent progenitors to commit to either the B or the macrophage lineage. Single-cell cloning assays revealed that IL-7 does not influence the decision to commit to the B lineage, despite the observation that the bipotent cells potentially respond to IL-7, as indicated by an increase in cell number, prior to the commitment event. Furthermore, the addition of IL-7 to cells developing along the B-cell pathway promotes the expression of mRNA transcripts which encode several B-cell-specific genes.