Production of human B cells from CD34 super(+)CD38 super(-) T super(-) B super(-) 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 super(+)CD38 super(-)CD10 super(-)CD19 super(-)CD3 super(-)CD8 super(-)CD4 super(-)(T super(-) B super(-)) cells from human cord blood (HUCB). CD19 super(+) cell production and kinetics differed in each tissue. Fetal liver organ cultures (FLOC) inoculated with CD34 super(+)CD38 super(-)T super(-)B super(-) cells produced fewer CD19 super(+) cells than fetal liver organ culture (FLOC) cultured with unpurified HUCB. CD19 super(+) cell production was restored in the CD34 super(+)CD38 super(-)T super(-)B super(-) 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 super(+)CD38 super(-)T super(-)B super(-) cells and monocyte-lineage CD33 super(+)CD14 super(-) cells, both of which increased after cytokine treatment. Re-colonization of secondary FLOC with CD34 super(+)CD38 super(-)T super(-)B super(-) cells generated in primary FLOC produced additional B-cells, monocytes and CD34 super(+)CD38 super(-) 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.