Stromal cell-independent differentiation of human cord blood CD34(+)CD38(-) lymphohematopoietic progenitors toward B cell lineage

To study the cytokine regulation of early stages of human B-lymphopoiesis, we developed a stroma-free two-step culture system. Single human cord blood CD34(+)CD38(-) cells were individually cultured by micromanipulation with interleukin (IL)-3, stem cell factor (SCF), flt3 ligand (FL), IL-6 and gran ulocyte colony-stimulating factor (G-CSF). About 10% of the cells formed pr imary colonies, which were individually tested for myeloid and B-lymphoid p otentials by reculturing aliquots of the primary colony cells into secondar y myeloid and B-lymphoid cultures. One third of the primary colonies proved capable of differentiation into CD19(+)IgM(+) cells, as well as into myelo id lineage cells. RT-PCR analyses revealed that some cells in the primary c ulture had already matured to express B cell-specific transcripts. Thus, th e combination of IL-3, SCF, FL, IL-6 and G-CSF supported the differentiatio n of CD34+CD38- lymphohematopoietic progenitors toward B cell lineage in ad dition to myeloid lineages. Screening of cytokines to identify the minimum requirement of cytokines in the primary culture revealed that IL-3 and SCF were essential and that the addition of FL, and to a lesser extent IL-6 or G-CSF, to the combination of IL-3 and SCF remarkably enhanced the primary c olony formation and the generation of CD19(+) cells in the secondary B-lymp hoid culture.