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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4509-4520
RAPID COMMUNICATION
In Vitro Reconstitution of Human B-Cell Ontogeny: From
CD34+ Multipotent Progenitors to Ig-Secreting Cells
Anne-Catherine Fluckiger,
Eva Sanz,
Maria Garcia-Lloret,
Thomas Su,
Qian-Lin Hao,
Roberta Kato,
Shirley Quan,
Antonio de la Hera,
Gay M. Crooks,
Owen N. Witte, and
David J. Rawlings
From the Department of Pediatrics, the Jonsson Comprehensive Cancer
Center, the Department of Microbiology and Molecular Genetics, the
Howard Hughes Medical Institute, and the Molecular Biology Institute,
University of California, Los Angeles, Los Angeles, CA; the Department
of Medicine, Universidad de Acala, Acala de Henares, Madrid, Spain; the
Centro de Investigaciones Biologicas, Valazquez, Madrid, Spain; and the
Division of Research Immunology and Bone Marrow Transplantation,
Childrens Hospital Los Angeles, Los Angeles, CA.
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 µ 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.
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