In vitro reconstitution of human B-cell ontogeny: from CD34(+) multipotent progenitors to Ig-secreting cells

Blood. 1998 Dec 15;92(12):4509-20.

Abstract

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 stromal cell line, S17, leading to the sustained production of large numbers of CD10(+), CD19(+) early B progenitors. Reverse transcriptase-polymerase chain reaction (RT-PCR) and three-parameter flow cytometry for VpreB (surrogate light chain), cytoplasmic mu chain, and surface IgM expression were used to characterize the CD19(+) B progenitors present within these cultures. This analysis showed distinct B-lineage subpopulations, including pro-B cells, cycling pre-B cells, and IgM+, IgD-/+ immature B cells. The limited expansion of IgM+ B cells and the immature surface phenotype of this population (IgM+, IgD+, CD10(+), CD38(+)) suggested that HB-LTC conditions were unable to provide appropriate signals for further differentiation. A second culture stage was used to determine if these immature B cells were functionally 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 one consistent with an activated mature B cell, secretion of Ig, and isotype switching. Notably, IgM and IgG producing B cells were also generated using two-stage cultures established with highly purified multipotent CD34(+)CD38(-) hematopoietic stem cell progenitors. This culture model should permit detailed in vitro analysis and genetic manipulation of the major transition points in human B ontogeny, beginning with commitment to the B lineage and leading to development and activation of mature B cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antigens, CD / analysis
  • Antigens, CD / biosynthesis*
  • B-Lymphocytes / cytology*
  • B-Lymphocytes / immunology
  • CD40 Ligand
  • Cell Differentiation / drug effects
  • Cell Division / drug effects
  • Cell Line
  • Cell Lineage / immunology
  • Culture Techniques / methods*
  • Cytokines / pharmacology
  • Fetal Blood / cytology
  • Flow Cytometry
  • Hematopoietic Stem Cells / cytology*
  • Humans
  • Immunoglobulin Class Switching / drug effects
  • Immunoglobulins / analysis
  • Immunoglobulins / biosynthesis*
  • Immunophenotyping
  • Membrane Glycoproteins / metabolism
  • Mice
  • Multigene Family / immunology
  • Reverse Transcriptase Polymerase Chain Reaction
  • Stromal Cells / cytology
  • Time Factors

Substances

  • Antigens, CD
  • Cytokines
  • Immunoglobulins
  • Membrane Glycoproteins
  • CD40 Ligand