Production of human B cells from CD34+CD38− T− B− progenitors in organ culture by sequential cytokine stimulation

We investigated sequential cytokine addition on human hematopoietic stem cell (HSC) differentiation in murine fetal liver (FL), fetal spleen (FS) and bone marrow (BM) organ cultures (OC). Tissues were colonized with unpurified or FACS sorted CD34+CD38−CD10−CD19−CD3−CD8−CD4−(T− B−) cells from human cord blood (HUCB). CD19+ cell production and kinetics differed in each tissue. Fetal liver organ cultures (FLOC) inoculated with CD34+CD38−T−B− cells produced fewer CD19+ cells than fetal liver organ culture (FLOC) cultured with unpurified HUCB. CD19+ cell production was restored in the CD34+CD38−T−B− organ cultures by treating with SCF, LIF and IL-6 followed by IL-7 and removing all cytokines for the last 3 days of culture (a six-fold increase). FLOC also produced CD34+CD38−T−B− cells and monocyte-lineage CD33+CD14− cells, both of which increased after cytokine treatment. Re-colonization of secondary FLOC with CD34+CD38−T−B− cells generated in primary FLOC produced additional B-cells, monocytes and CD34+CD38− cells suggesting that the primary cells retained HSC activity. Expansion and differentiation of HSCs depended on the microenvironment of the recipient tissue as well as addition of cytokines in the appropriate order.