Stromal cell-free conditions favorable for human B lymphopoiesis in culture

Progress has been slow in defining molecular requirements for human B lymphopoiesis in part because of differences from experimental animals and also because of the lack of culture conditions that efficiently support the process. We recently found that human CD10+ lymphocytes were produced when CD34+ hematopoietic stem and progenitor cells were cultured in contact with human mesenchymal stem cells (hMSC). Further investigation revealed that it occurred even when progenitors were separated from hMSC by membrane filters. Experiments with neutralizing antibodies suggested that important heat labile factors produced by hMSC are unlikely to be IL-7, TSLP, CXCL12 or hemokinin-1. Further manipulation of culture conditions revealed that optimal lymphopoiesis required careful selection of fetal calf serum lots, maintenance of high cell densities, as well as recombinant cytokines (SCF, FL and G-CSF). G-CSF was particularly important when adult bone marrow rather than umbilical cord blood derived CD34+ cells were used to initiate the cultures. These improved methods should facilitate identification of molecules that can be used to speed regeneration of the humoral immune system following chemotherapy and might suggest ways to inhibit growth of B lineage malignancies.