In vitro reconstitution of human B-cell ontogeny: From CD34(+) multipotentprogenitors to Ig-secreting cells

We describe a long-term, in vitro culture system initiated with CD34(+) or CD34(+)CD38(-) umbilical cord blood hematopoietic progenitors that supports normal human B-lineage development, including the production of mature Ig- secreting B cells. In the first stage (human B-progenitor long-term culture [HB-LTC]), CD34(+) hematopoietic progenitors are cultured on the murine st romal cell line, S17, leading to the sustained production of large numbers of CD10(+), CD19(+) early B progenitors. Reverse transcriptase-polymerase c hain reaction (RT-PCR) and three-parameter flow cytometry for VpreB (surrog ate light chain), cytoplasmic mu chain, and surface IgM expression were use d to characterize the CD19+ B progenitors present within these cultures. Th is analysis showed distinct B-lineage subpopulations, including pro-B cells , cycling pre-B cells, and IgM(+), IgD(-/+) immature B cells. The limited e xpansion of IgM(+) B cells and the immature surface phenotype of this popul ation (IgM(+), IgD(+), CD10(+), CD38(+)) suggested that HB-LTC conditions w ere unable to provide appropriate signals for further differentiation. A se cond culture stage was used to determine if these immature B cells were fun ctionally competent. Purified CD19+ cells were transferred onto fibroblasts expressing human CD40-ligand in the presence of IL-10 and IL-4. This lead to cell proliferation, modulation of the IgM(+) cell surface phenotype to o ne consistent with an activated mature B cell, secretion of Ig, and isotype switching. Notably, IgM and IgG producing B cells were also generated usin g two-stage cultures established with highly purified multipotent CD34(+)CD 38(-) hematopoietic stem cell progenitors. This culture model should permit detailed in vitro analysis and genetic manipulation of the major transitio n points in human B ontogeny, beginning with commitment to the B lineage an d leading to development and activation of mature B cells. (C) 1998 by The American Society of Hematology.