title: In vitro reconstitution of human B-cell ontogeny: from CD34(+) multipotent progenitors to Ig-secreting cells.
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.
authors.affiliation: Department of Pediatrics, the Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, CA, USA.
authors.forename: A C
authors.initials: AC
authors.lastname: Fluckiger
authors.forename: E
authors.initials: E
authors.lastname: Sanz
authors.forename: M
authors.initials: M
authors.lastname: Garcia-Lloret
authors.forename: T
authors.initials: T
authors.lastname: Su
authors.forename: Q L
authors.initials: QL
authors.lastname: Hao
authors.forename: R
authors.initials: R
authors.lastname: Kato
authors.forename: S
authors.initials: S
authors.lastname: Quan
authors.forename: A
authors.initials: A
authors.lastname: de la Hera
authors.forename: G M
authors.initials: GM
authors.lastname: Crooks
authors.forename: O N
authors.initials: ON
authors.lastname: Witte
authors.forename: D J
authors.initials: DJ
authors.lastname: Rawlings
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000032:analysis / Q000096:biosynthesis*; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D023201:CD40 Ligand; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D019070:Cell Lineage / Q000276:immunology; D046508:Culture Techniques / Q000379:methods*; D016207:Cytokines / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D017578:Immunoglobulin Class Switching / Q000187:drug effects; D007136:Immunoglobulins / Q000032:analysis / Q000096:biosynthesis*; D016130:Immunophenotyping; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D005810:Multigene Family / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology; D013997:Time Factors
chemical_list: D015703:Antigens, CD; D016207:Cytokines; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D023201:CD40 Ligand