A Selective Culture System for Generating Terminal Deoxynucleotidyl Transferase-Positive Lymphoid Cells In Vitro. V. Detection of Stage-Specific Pro-B-Cell Stimulating Activity in Medium Conditioned by Mouse Bone Marrow Stromal Cells
A Selective Culture System for Generating Terminal Deoxynucleotidyl Transferase-Positive Lymphoid Cells In Vitro. V. Detection of Stage-Specific Pro-B-Cell Stimulating Activity in Medium Conditioned by Mouse Bone Marrow Stromal Cells
SEAN D. McKENNA"
IRVING GOLDSCHNEIDER
The selective in vitro generation of rat, mouse, and human terminal deoxynucleotidyl transferase-positive (TdT lymphoid cells in our long-term xenogeneic bone marrow (BM) culture system is characterized by physical interaction between the developing lymphocytes and mouse BM-adherent stromal cells and macrophages. In the present study, experiments in which micropor)us membrane culture inserts were inoculated with rat BM cells demonstrated that although the generation of primitive B-lineage lymphoid cells requires the presence of a mouse BM feeder layer, cognitive recognition events are not necessary. Similarly, cell-free (and serum-free) medium conditioned with mouse BM (but not thymus or spleen) adherent cells and stromal-cell lines therefrom supported the proliferation of early rat lymphoid cells in a dose-dependent manner. Double immunofluorescence for incorporated bromo-deoxyuridine (BrdU) and early B-lineage markers of rat BM lymphoid cells maintained in culture inserts or conditioned medium (CM), and studies of their in vitro and in vivo developmental potentials, indicated that the lymphoproliferative response resulted from the selective stimulation of lymphoid stem and/or progenitor cells. The most primitive of these target cells had a HIS24 HIS50- TdT- c/- sIg-, pre-pro-B-cell phenotype. Whereas this subset normally constitutes less than 2% of B-lineage BM cells in vivo, it comprises more than 25% of total lymphoid cells in vitro. In addition, the number of TdT cells, predominantly of the early pro-B-cell phenotype (HIS24 HIS50-c/-sIg-), was increased approximately tenfold above input levels. Based on these and previous findings, a schematic model is proposed for the developmental pathway of early B-lineage cells in rat BM from the level of the committed (possibly common) lymphoid stem cell to that of the pre-B-cell.
Terminal transferase (TdT); pro-B cell stimulating factor; in vitro culture system; bone marrow stromal cells
INTRODUCTION
The growth and differentiation of lymphoid cells
from stem and progenitor cells are thought to be
regulated by cell-cell contact as well as by the
release of soluble factors
(reviewed in Kincade et al.,
1989; Dorshkind, 1990)
. A major technical advance
in the study of these cellular and biochemical events
in lymphopoiesis has been the development of
long-term, feeder layer-depetdent, in vitro bone
marrow (BM) culture systems, in which
manipula"Corresponding author.
tion of culture conditions profoundly affects the
lineages and/or stages of development that are
generated. For example, Dexter-type cultures
selectively produce myeloid lineage cells and
multipotential stem cells, but few lymphoid cells
(Dexter et al.,
1977)
, whereas the Whitlock-Witte culture system
selectively produces large numbers of pre-B cells
(c/+, sIg-), but not myeloid or multipotent stem
cells
(Whitlock and Witte, 1982; Whitlock et al.,
1984)
. Recently, modifications to the
WhitlockWitte culture system have been described that
permit the generation of B-lineage cells more primitive
than those observed in standard cultures. These
modifications include seeding the BM feeder layers
with mouse fetal liver cells
(Denis et al., 1984,
1987)
, transfecting the cultured ells with the BCR/
ABL chimeric oncogene (Scherele et al., 1990), and
initiating the culture in the presence of interleukin-4
(Peschel et al., 1989)
.
The role of oluble factors in the generation of
pre-B-cells has been extensively studied using bone
marrow stromal cell lines established from the
foregoing cultures. In particular, interleukin-7 (IL-7), a
cytokine first purified from mouse BM stromal cells,
has been found to stimulate the proliferation of
pre-B (B220 /) and possibly pro-B (B220-) cells from
mouse BM
(Namen et al., 1988a,1988b; Henney,
1989; Lee et al., 1989)
. However, despite the effect
of IL-7, the long-term survival of early B-lineage
cells in primary cultures generally is not maintained
in the absence of direct contact with BM stromal
cells
(Kierney and Dorshkind, 1987; Sudo et al.,
1989)
. This is consistent with the development of
foci of proliferating lymphoid precursor cells on and
within the adherent BM stromal-cell layer
(Whitlock
and Witte, 1982; Whitlock et al., 1984; Hayashi et
al., 1984; Medlock et al., 1993a, 1993b)
.
Hardy et al. (1991)
have reported that the earliest
phenotypically distinct mouse B-lineage cell
population, called "pre-pro-" B-cells (B220 $7/ BP-I-,
HSA-, Ig genes in germline configuration), requires
only contact with BM stromal cells to survive in a
4-day coculture system. However, later stages of
lymphopoiesis, marked by expression of HSA and
progressive Ig gene rearrangements, were
increasingly (...truncated)