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Laboratory of Developmental Biology, University of Wisconsin, Madison, WI 53706
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R, but did not express cytoplasmic
µ-chain, surface IgM (sIgM), or MHC class II molecules. PCR analysis
showed that the cells expressed TdT, 
5, and
RAG-1 genes, but that their Ig genes were still in
germline configuration. The cell line was dependent on direct contact
with S17 stromal cells for growth, but, in contrast to bone marrow stem
cells, required no additional growth factors for maintenance and
expansion. When stimulated with IL-7 and LPS, YS-PPB cells and cells
from all tested clonal sublines differentiated into sIgM+ B
cells in vitro. Irradiated mice reconstituted with YS-PPB cells yielded
spleens containing 38% sIgM+ donor-derived B cells,
demonstrating that YS-PPB cells, although stably arrested in
development at the boundary between pre-pro-B and pro-B stages of B
cell differentiation, still retain their competence to differentiate
into mature, Ig-producing B cells when transferred to a normal host
environment. Thus, this new cell line can provide a reproducible source
of B cell precursors arrested at that critical time in B cell
differentiation when the machinery for Ig gene rearrangement is in
place but rearrangement has not yet occurred. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the first hemopoietic stem cells can be identified both intraembryonically and in the yolk sac as early as day 7.5, the first B cell lineage commitment appears not to occur until days 11 to 12, when pro-B cells can be detected in the fetal liver (5, 6). The primary source of these cells has not been established, but it is believed that they originate extrinsically from the intraembryonic aorta/gonad/mesonephros or AGM region, from the blood islands of the extra-embryonic yolk sac, or from both (2, 7, 8).
B cell development in vitro, and presumably in vivo, is dependent on direct physical association between hemopoietic stem cells and the surrounding stromal cells that may include fibroblasts, adventitial reticular cells, epithelial cells, and endothelial cells (9). Cell-cell interactions, largely as yet undefined but partly mediated by released growth factors, generate the growth and differentiation signals that promote progression along B cell developmental pathways. The progressive stages of B cell development that result from these interactions can be defined by sequential rearrangements of Ig loci, surface expression of various stage-specific markers, distinctive growth factor requirements, and the acquisition of functional specializations (10-12).
The complexity of B cell development makes difficult a detailed in vivo analysis of these developmental events. Our knowledge of these processes has been appreciably expanded through the study of established, long term, stable B lymphocyte culture systems. These early stage B-lineage cells have been obtained exclusively from fetal liver and bone marrow. Our purpose has been to study B cell development in the mouse embryonic yolk sac at a stage preceding the time that B-lineage cells can be detected in the fetal liver, with the promising possibility that we might be able to detect new and possibly unique early stages in B cell development.
During the course of our studies, we detected an aberrant culture in which early yolk sac hemopoietic stem cells (AA4.1+; nonadherent; density, 1.077), explanted onto a bone marrow stromal cell (S17) feeder layer, resulted in the formation of a large number of uniform, small lymphocytes, and that these cells proliferated readily while maintaining their appearance on transfer to new S17 feeder layers. We show that these cells, which we describe in terms of their cell surface Ags, Ig gene status, and response to growth factors, appeared to be arrested at the boundary between pre-pro-B cells and pro-B cells, and they could subsequently be induced to give rise to mature B cell lineages both in vitro and in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/cAu (Ly5.1) and C57BL/Au (Ly5.2; originally from E. A. Boyse) mice were generated in our own colony. The appearance of the vaginal plug was designated day 0 of gestation. Four- to 6-wk-old C57BL/Au (Ly5.2) mice were used in restitution experiments.
Antibodies
The following Abs specific for surface markers were used: CD43, CD34, FITC-CD44, phycoerythrin-heat stable antigen (HSA),3 FITC-CD3, FITC-H-2Kd/H-2Iad, FITC-sIgM (µ chain), FITC-CD5, FITC-CD19, and goat anti-rat or mouse IgG-FITC from PharMingen (San Diego, CA); FITC-CD4, phycoerythrin-CD8, and FITC-Thy1.2 from Becton Dickinson (Mountain View, CA); anti-murine c-Kit (ACK-2) from Life Technologies (Gaithersburg, MD); AA4.1 Ab from J. P. McKearn (Searle/Monsanto, St. Louis, MO); Biotin-Sca-1 from I. L. Weissman (Stanford University, Stanford, CA); FITC-avidin from Tago (Burlingame, CA); Joro37.5 from R. Palacios (M. D. Anderson Cancer Center, Houston, TX), B220 (14.8) from P. Kincade (Oklahoma Medical Research Institute), anti-Mac-1 from the American Type Culture Collection (Manassas, VA); FITC-anti-mIL-7R from M. Sandor (University of Wisconsin-Madison); anti-Ly5.1 and anti-Ly5.2 from R. R. Hardy (University of Pennsylvania School of Medicine); goat anti-mouse polyclonal Ab from PharMingen (San Diego, CA); and anti-mouse polyvalent Ig (IgG, IgA, IgM) conjugated with alkaline phosphatase from Sigma (St. Louis, MO).
Isolation of AA4.1+ yolk sac cells
Single cell suspensions were prepared from yolk sacs isolated from day 10 BALB/c (Ly5.1) mouse embryos. AA4.1+ yolk sac cells were isolated according to previously published methods (2). In brief, yolk sac cells were separated using a discontinuous gradient of Percoll (Sigma; 1.054, 1.066, and 1.077 g/cm3). Gradient-separated (<1.077 g/cm3) yolk sac cells were incubated in tissue culture dishes in 37°C in 5% CO2 for 1 h, after which nonadherent cells were collected. Polystyrene dishes (Falcon 1001, Falcon Plastics, Oxnard, CA) were precoated with protein G-purified mAb AA4.1 (10 µg/ml) in 5 ml of 0.05 M Tris-HCl/0.15 M NaCl, pH 9.5, at room temperature for 1 h. After washing and blocking with PBS and 1% FBS, cells were layered onto Ab-coated plates in 5 ml of RPMI 1640/5% FBS and incubated at 4°C for 1 h. Nonadherent cells were removed, and plates were washed 8 to 10 times with PBS/5% FBS. The remaining adherent cells were then collected by forceful pipetting. The cell suspension, as determined by flow cytometry, was shown to contain >95% AA4.1+ cells.
Establishment of the pre-pro-B cell line (YS-PPB) and clonal sublines
The enriched AA4.1+ cells were dispensed onto a subconfluent layer of irradiated (2500 rad, 137Cs) S17 bone marrow stromal cells in 96-well plates (two or three cells per well) at limiting dilution and cultured in the absence of growth factors. Half the medium was replaced every 5 days. Fifteen days later, mixed colonies consisting of different cells based on cell size and morphology were present in the cocultures. Individual mixed colonies were removed, transferred onto fresh irradiated S17 monolayers in 24-well plates for further cultivation, and passaged every 5 days.
Although mixed colonies generally gave rise to more mixed colonies or differentiated to produce various myeloid lineages, a culture was identified that eventually consisted of a uniform population of small lymphocytes. This culture was further expanded and passaged repeatedly to establish a stable cell line (designated YS-PPB). For cloning of the cells, 96-well plates were prepared with near-confluent, irradiated S17 stromal cells. Single yolk sac cells from the fifth passage were then deposited into each well by cell sorting in flow (FACStarPlus, Becton Dickinson, Mountain View, CA; 488 nM; 100 millwatt; Macrosort nozzle). After 5-7 days, the plates were checked for cultures showing positive growth of clones. Clones were transferred to 24-well plates and thereafter to 15 x 60 cm dishes for further expansion under the same conditions as those described above. The YS-PPB cells and selected clones have been propagated in vitro under these conditions for >1 yr. All cultures were maintained in RPMI 1640 (Life Technologies) medium supplemented with 10% FBS and 50 µM 2-ME. Cells were frozen in liquid nitrogen once every 1 to 2 mo.
Cell proliferation assay
Subconfluent irradiated S17 cell monolayers were established 1 day before addition of YS-pre-pro-B cells. Proliferation of the YS-PPB cells was measured by the increase in the number of the cells generated in the presence or the absence of murine rIL-7 (R&D System, Minneapolis, MN). Cultures were also prepared by inserting 0.4-µm pore size filters (Millicell, Millipore, Bedford, MA) into wells containing pre-established S17 feeder layers in 24-well plates. Only those cells excluding trypan blue were counted. For thymidine uptake, the cultures were pulsed for 12 h with [3H]thymidine (Amersham, Arlington Heights, IL) before the termination of each experiment. The radioactivity of incorporated cells was determined by means of a liquid scintillation counter (Beckman, Fullerton, CA).
Flow cytometric analysis
Cells were labeled sequentially with Abs diluted to the appropriate concentration in staining medium on ice for 30 min, washed, stained with second-step reagents for 30 min where required, washed, and then resuspended in FACS buffer (PBS/1% BSA). Flow cytometric analysis was conducted using a FACScan instrument (Becton Dickinson) and LYSYS-II or PC-LYSYS analysis software.
Immunofluorescence detection of cytoplasmic Ig µ-chain
Cytosmears were prepared using a Shandon Cytospin centrifuge (Shandon, Pittsburgh, PA), air-dried, and fixed (13). After washing, the slides were blotted to remove excess buffer and placed in a humidified chamber. About 10 µl of FITC-conjugated goat anti-mouse IgM (µ-chain) Abs (Caltag, San Francisco, CA) was placed on the cell smears. Slides were removed from the chamber after 30 to 40 min and washed in PBS/0.1% sodium azide. A coverslip was mounted on the stained, washed cytosmears with Fluormount G (Southern Biotechnologies, Birmingham, AL). Slides were observed under an immunofluorescence microscope and scored for FITC+ staining.
RT-PCR assay
Total RNA was extracted from 0.5 to 1x107 cells
including uncloned cultured cells, clonal YS pre-pro-B cells, S17
cells, and spleen cells from adult BALB/c mice using TRIzol reagent
(Life Technologies) according to the procedure recommended by the
manufacturer. First-strand cDNA was synthesized from 1 µg of total
RNA using Superscript (Life Technologies) RT, and random hexamer (Life
Technologies). The resulting cDNA was amplified by RT-PCR using
TdT, 5, and RGA-1 primers
(synthesized by Biotechnology Center, University of Wisconsin,
Madison), using primer sets exactly as previously reported (14, 15),
and subjected to 32 PCR cycles (2400 GeneAmp Thermal Cycler,
Perkin-Elmer, Norwalk, CT). Each cycle consisted of denaturing at
94°C for 50 s, annealing at 53°C for 60 s, and
polymerizing at 72°C for 70 s. Amplification of the ß-actin
gene using the previously described (16) standard primer set
(synthesized by Biotechnology Center, University of Wisconsin, Madison)
was used throughout as a control for template integrity and for
normalization of data to a constitutively expressed transcript.
Genomic DNA isolation and PCR assay
For PCR assay for Ig gene configuration, the genomic DNA was extracted from the same cell populations as those above using the QIAamp Blood Kit (Qiagen, Chatsworth, CA) according to the manufacturer’s directions. DNA quantity and quality were determined by OD scanning. PCR oligonucleotides described previously (12, 17), including specific primers for D-JH rearrangement (DSF, 5'-GGG(A/C)TTTTTGT(C/G)AAGG(G/T)ATCTACTACTGTG-3'; and JH4, 5'AAAGACCTGCAGAGGCCATTCTTACC-3') and for VH-D-JH rearrangement (J558-JH4, 5'-CAGGTCCAACTGCAGCAG-3' and D-JH4; see above) were coamplified with a normalizing ß-actin primer set (same as above). Two micrograms of DNA from each sample was used for PCR amplification. Conditions for PCR were 1 min of denaturation at 94.5°C, 30 s of annealing at 62°C, and 1.3 min of polymerization at 72°C. This cycle was repeated 32 times.
Hybridization analysis
Twenty microliters of each PCR reaction product were separated
by electrophoresis in a 1.5% agarose gel and transferred to Hybond
membranes (GeneScreen Plus, Boston, MA) by vacuum transfer using
VacuumGene equipment (Bio-Rad, Hercules, CA). The membranes were rinsed
in 2 x SSC, UV cross-linked, prehybridized for 1 h at 55°C
in 0.25 M Na2H4PO4 (pH 7.2)
containing 1 mM EDTA and 7% SDS, then hybridized in the same solution
as that used for the prehybridization but containing 3 pmol/ml
biotinylated oligonucleotide probes including JH4
(5'-ACCCCAGTAGTCCATAGCCATAGTAAT-3') (18), TdT
(5'-CTTCCTCTCGTGTGTGGCATAGCG-3') (19), 5
(5'-ACCAAAACTGGGGCTTAGATGG-3') (15), RAG-1
(5'-TTCTTCGGGTGCCTTTTCAA-3') (19), and ß-actin
(5'-TTCTGCATCCTGTCAGCAAT-3') (16) (synthesized by Biotechnology Center,
University of Wisconsin, Madison) at 55°C for 2 h. None of the
probes shared overlapping sequences with their corresponding
oligonucleotides used as PCR primers. After washing, the hybridized
biotinylated probes were detected by using a Southern-Light test kit
(Biotex, Life Technologies). Kodak XARl5 film exposures (Eastman Kodak,
Rochester, NY) were used to detect the chemiluminescent signals.
Cloning and sequencing
The bands of interest (putative DJH4 rearrangements or possible primer dimers) were excised and purified from 1.5% agarose gels, and then cloned directly with a TOPO TA Cloning kit (Invitrogen, San Diego, CA). Plasmid DNA was prepared from white, kanamycin-resistant colonies using the S.N.A.P. Miniprep kit (Invitrogen) and were sequenced with M13 primers (Invitrogen).
ELISAs
Limiting dilution cultures were established by plating clonal
and uncloned YS-pre-pro-B cells at a specified concentration (24 or 36
replicates/cell concentration) on irradiated S17 cell feeder layers in
96-well plates with IL-7 at 0.1 ng/ml and LPS (25 µg/ml).
Supernatants were collected after 12 to 14 days of culture. A
double-sandwich ELISA was conducted as described previously for
determination of the total Ig in culture supernatants (20). Briefly,
immuno-plates were coated with 200 µl of the appropriate dilution of
purified anti-mouse polyclonal Ab overnight at 4°C. After
washing, 200 µl of serial dilutions of mixed IgM, IgG, and IgA
standards (Sigma) in PBS/0.05% Tween 20 were added to the plates to
generate a standard curve. Dilutions of each culture supernatant were
assayed in parallel. After 2 h at room temperature, plates were
blocked with 2% BSA in PBS at 37°C for 2 h followed by washing.
Anti-mouse IgM/IgG/IgA (1/1000) coupled to alkaline phosphatase (Sigma)
was applied and incubated for an additional 1 h at 37°C. After
an additional wash, Sigma 104 phosphatase substrate (1 mg/ml) was added
in each well and held for approximately 2 h at room temperature.
The concentration of Igs in individual samples was calculated by
comparing the mean O.D. obtained for replicate wells to a semilog
standard curve of titrated standard Abs using linear regression
analysis. Supernatants containing <100 ng/ml were considered negative
for Ig secretion. Calculations of Ig-producing precursor frequency were
made as described in Figure 7
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The induction of differentiation of YS-PPB cells into sIgM+ B lymphocytes was conducted in 35-mm dishes containing irradiated S17 stromal cells and both IL-7 (0.1 ng/ml) and LPS (25 µg/ml, Sigma). Following 5 to 6 days of culture, 1 ml of fresh medium was added to each dish. After 12 to 14 days of culture, sIgM+/B220+ cells were identified by flow cytometric analysis.
Reconstitution of irradiated mice
For repopulation of lymphoid-lineage compartments of irradiated C57BL/6 Ly5.2+ mice (1200 rad, split dose, 3-4 h interval, 166 rad/min), YS-PPB clone 2 cells (BALB/c Ly5.1+, 5 x 106 cells/mouse) in 0.5 ml of PBS were injected i.v. into recipient animals 4 to 6 h after irradiation. Radioprotection was provided by injection of 2 x 105 host-syngeneic C57BL/6 Ly5.2+ bone marrow cells. Recipient animals injected with BALB/c bone marrow cells (Ly5.1+, 5 x 106 cells/mouse) or PBS only were used as positive and negative controls. Twelve to sixteen weeks post-transplantation, lymphoid repopulation of spleens was assessed by flow cytometric analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AA4.1+ yolk sac cells (AA4.1+ cells,
>95% determined by flow cytometry) were enriched by plastic
adherence, differential centrifugation, and immunocytoadherence as
described in Materials and Methods and then cultured on
irradiated S17 stromal cells without the addition of exogenous growth
factors. Half of the medium was replaced at 5-day intervals. After day
15 of culture, the formation of mixed colonies (4.2%) was observed
under low power magnification. The round nonadherent cells were gently
recovered from individual mixed colonies and then transferred onto new
irradiated S17 feeder layers in 24-well plates for further expansion.
About five passages later, the cells in one culture appeared to be
small and lymphoid-like, while macrophage-like cells were absent. For
cloning of these cells, single cells were deposited onto S17 cells in
96-well plates by FACS sorting. We found that with this protocol,
24.7% cloning efficiency could be obtained in 768 cultures conducted
(cloning frequency, about one of four cells). From 189 clones, 13
clones with high proliferation potential (>5 x 105
cells/well) were selected for further expansion on stromal cells and
were established as long term proliferating clonal sublines. Uncloned
cells and four of these clones (clones 1, 2, 7, and 11) were selected
at random for further expansion and characterization. Figure 1A shows the morphology of
YS-PPB B cells grown on S17 stromal cells. Wright-Giemsa staining of
these cells is shown in Figure 1B.
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Uncloned cells and clones 1, 2, 7, and 11 were examined for
expression of surface markers by flow cytometry as shown in Table I. These cells were positive for CD19,
CD43, AA4.1, Sca-1, HSA, IL-7R, MHC class I, and B220, but sIgM, MHC
II, CD5, and CD44 were not detected on the uncloned cells or any of the
clonal sublines. None of the cells expressed cytoplasmic Ig heavy chain
(µ-chain) proteins or surface markers associated with pre-T
(Joro37.5), mature T lymphocytes (Thy1.2, CD3, CD4, and CD8), or
myeloid lineage (Mac-1) cells. As shown in Figure 2, however, expression of c-Kit varied
with the different clones. Clones 7 and 11 were uniformly
c-Kit+ and were present on a subpopulation of the parent
line as were clones 1 and 2 (37-69%). The observed phenotypes suggest
that the yolk sac-derived, long term cultured cells have been committed
to an early stage of B cell development.
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We examined the rearrangement-associated genes TdT,
5, and RAG-1 using RT-PCR. As shown in Figure 3, all clonal sublines as well as the
uncloned parental cells expressed TdT, 5, and
RAG-1. These genes have been shown to be expressed in early
B cells such as pro-B cells (10, 21) and pre-B cells (22). In contrast,
mRNA for these same genes was not detected in mature splenic cells or
S17 cells used as controls. To further confirm the developmental
staging of the cells, IgH gene rearrangements were evaluated using PCR
amplification analysis. As shown in Figure 4, DSF primer together with a primer
complementary to a segment of DNA 3' of JH4 could amplify a
ladder of rearranged DJH products. In positive control
spleen cells, four bands of the sizes expected for rearrangements to
the four JH elements were detected with the JH4
probe. Although no DJH1-3 rearrangements was detectable, a
band seen in the pre-pro-B cell line and its clones was close in size
to the DJH4 rearrangement seen in splenic cells.
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Characterization of clonal cell growth
To determine whether contact between YS-PPB and stromal cells was
mandatory, YS-PPB cells were removed from coculture with S17 stromal
cells and replated on S17 cell monolayers (positive control), in a
diffusion chamber where the cells were prevented from direct contact
with the stromal cells, or in medium only. Cell recovery under these
different culture conditions was measured after day 3 of culture. No
cells were recovered in either transfilter cultures or in cultures
grown in the absence of stromal cells (Figs. 5 and 6).
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shows that no
proliferation was observed under these conditions. However, in the
presence of stroma the proliferation of uncloned cells and clone 7 was
augmented by IL-7, while clones 1, 2, and 11 were unresponsive to IL-7
under these conditions (Fig. 5) despite the fact that they also
expressed IL-7R. The results obtained by cell counting were in
agreement with those obtained by measuring [3H]TdR
incorporation (data not shown). The growth of uncloned or clone 7
YS-pre-pro-B cells could not, however, be achieved by combining IL-7
with S17-conditioned medium (data not shown), indicating that the
growth of these cells was directly dependent on signals mediated by
direct contact with stromal cells and/or membrane-bound growth factors
produced by these stromal cells. Frequency of Ig-producing B cell precursors
The uncloned YS-PPB cells as well as the four cloned sublines were
tested for their potential to produce Igs following stimulation in
vitro. The frequency of Ig-producing B cell precursors was determined
by limiting dilution analysis as described in Materials and
Methods. When cultured on S17 cells, all clonal sublines as well
as the uncloned cells were able to respond to dual stimulation by IL-7
and LPS to produce Igs, but none of them was able to produce Ig either
on the stroma alone or with stimulation by IL-7 or LPS alone. Among
these cells, the frequency of Ig-producing cells ranged from a high of
1 in 425 in clone 1 to a low of 1 in 2680 in clone 11 (Fig. 7).
In vitro and in vivo differentiation of YS-pre-pro-B cells
The fact that YS-PPB cells and the clonal sublines were capable of
producing Igs in response to IL-7 and LPS suggested that these cells
have the potential to differentiate into Ig-producing mature B cells.
We therefore cultured cells for a longer period of time (12-14 days
stimulation with LPS plus IL-7) and analyzed the resultant cells by
flow cytometry. The results shown in Figure 8 indicate that both the uncloned and
clonal cells could be induced to differentiate into sIgM+ B
cells, although there were differences in the percentage (11-33.7%) of
sIgM+ cells between them. Once again, however, neither IL-7
nor LPS alone had this effect on the differentiation of the pre-pro-B
cells (data not shown). In addition, expression of MHC II Ag could be
detected on the tested cells (data not shown).
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nearly all (>95%) of the donor-derived Ly5.1+ lymphocytes
expressed B220 and sIgM (Fig. 9D). Furthermore donor-derived
B220 and sIgM-positive cells (Ly5.1+) represented 71 and
54%, respectively, of the total population of B220 and sIgM-positive
cells (Fig. 9C); the remaining B220 and sIgM-bearing cells
were of host origin (Fig. 9E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Even after 1 yr, during which the cell line maintained the germline Ig gene configuration, the cells retained the capacity for B cell maturation. This was shown for both the YS-pre-pro-B cell line and several of the YS-pre-pro-B cell clones and could be observed in 2-wk cultures grown on S17 stromal cells and stimulated by IL-7 and LPS as well as following cell transfer into irradiated, histoincompatible, allogeneic mice. In contrast, whereas both freshly isolated and in vitro propagated AA4.1+yolk sac stem cells readily differentiate into T cells when grown in combination with stem cell-depleted embryonic thymus rudiments (2, 4, 23), neither the parental YS-PPB cell line nor clonal sublines were able to generate T cells in this assay system (data not shown).
Our decision to designate the YS-PPB cell line as pre-pro-B cells is
based on two major considerations: 1) the cell line and clonal sublines
derived from it are stable in the germline configuration of Ig genes,
yet the cells retain the ability to differentiate into mature B cells
in vitro and in vivo; and 2) the cell line can be maintained for
prolonged periods of time (>1 yr) when grown on stromal cells in the
absence of cytokines such as IL-7. As shown in Table II (10, 12, 24), the most critical
distinction between pre-pro-B and pro-B cells, as defined in adult bone
marrow, is the state of Ig gene rearrangement. From this standpoint our
cell line would be considered to be pre-pro-B. On the other hand,
pivotal markers such as HSA and CD19, which are first found in bone
marrow at the pro-B cell stage are present on our cell line. Other
markers such as CD43, expressed on our yolk sac cell line, are found on
both pre-pro-B cells and pro-B cells. Significantly, our yolk
sac-derived cell line manifests detectable mRNA levels of
recombination-associated genes, including RAG-1,
5, and TdT, which in bone marrow are
restricted to late (A2) pre-pro-B and pro-B cells (25, 26). Based on
these comparisons, we propose that the stable cell line that we have
described is best defined as the very latest stage of pre-pro-B cells.
To our knowledge this is the first cell line arrested at this critical
transitional stage, and for this reason it should prove to be valuable
in the study of B cell differentiation.
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We examined the possibility that our S17 cells actually produced IL-7 required for growth of YS-pre-pro-B cells. However, we (unpublished observations) and others have been unable to detect IL-7, as assessed by bioassays or PCR (31, 32), although S17 cells do produce the ligand for the c-Kit receptor (c-Kit ligand) (31) with its potentiating effect on proliferation of early B cells. We also considered the possibility that the isolated AA4.1+ yolk sac cells were contaminated with yolk sac stromal cells that produce IL-7 to support the growth of pre-pro-B cells, but such cells could not be detected by flow cytometric analysis of the starting cell population. Our results lead to the suggestion that the signals mediated by interactions between S17 stromal cells and the most primitive yolk sac hemopoietic stem cells are required for the development and the maintenance of yolk sac-derived IL-7-independent pre-pro-B cells. The critical signals could be triggered via membrane-bound factors produced by S17 stromal cells. Alternatively, this direct contact between YS-pre-pro-B cells and S17 cells may induce the production of essential, but not yet identified, factors by the stromal cells, and it is these factors that, in turn, support the growth of yolk sac-derived pre-pro-B cells. Some evidence in support of this latter possibility has been reported (33, 34). Our finding that clones 1, 2, and 11 subsequently failed to respond to IL-7, whereas the other clones became responsive suggests that clones 1, 2, and 11 represented a more primitive pre-pro-B cell population. However, it should be pointed out that all of our YS-pre-pro-B cells expressed IL-7R, so the fact that clones 1, 2, and 11 were unable to grow in response to IL-7 could reflect a block downstream of the IL-7R that renders the cells nonresponsive to IL-7 (35).
A recent report by Cumano et al. (36) that B cell precursors can be identified in the day 7.5 to 8 mouse embryo within the region destined to become the aorta/gonad/mesonephros has led to the suggestion that all hemopoietic stem cell precursors originate intraembryonically and only later migrate to the yolk sac. Our present study, initiated with day 10 yolk sac hemopoietic stem cells, cannot argue against this suggestion. However, it should be recalled that we have previously shown that as early as day 8, i.e., a stage comparable to the stage used in the recent study by Cumano et al. (36), the yolk sac contains cells capable of differentiating into T-lineage lymphocytes in vitro (23). The B cell precursors we have now identified in the yolk sac have been derived from a common precursor pool (i.e., the AA4.1+, wheat-germ agglutinin+ low density nonadherent cell). It is quite conceivable that the yolk sac pre-pro-B cell line we have established represents a lineage distinct from that described by Cumano and her colleagues.
Aside from the inherent interest in early hemopoietic stem cell development, we believe that the early, MHC-negative hemopoietic stem cells from the yolk sac may serve as a source of cells for both immune reconstitution and the introduction of genes into host recipients. The fact that these yolk sac stem cells can provide a unique population of stable pre-pro-B cells further strengthens that belief, because these cells can be expanded in long term culture, yet are subsequently able to proliferate and differentiate in vivo. Our report emphasizes the fact that not all early pro-B cells are alike, and that there may well be not only definitive points in the growth factor response of B cells but also distinct B cell lineages. Yolk sac-derived pre-pro-B cells may prove also to be useful tools for investigating the regulatory events governing Ig gene recombination and for identifying novel growth factors involved in the expansion and differentiation of hemopoietic stem cells during the early development of B cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert Auerbach, Laboratory of Developmental Biology, University of Wisconsin, 1117 W. Johnson St., Madison, WI 53706. E-mail address:
3 Abbreviations used in this paper: HSA, heat stable antigen; sIgM, surface immunoglobulin M; YS, yolk sac; YS-pre-pro-B, yolk sac-derived early progenitor B cells.
Received for publication April 21, 1997. Accepted for publication April 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5 genes rearranged in fetal liver-derived thymocytes in radiation chimera mice. Eur. J. Immunol. 23:3345.[Medline]
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