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IMMUNOBIOLOGY
From the Centro Nacional de Biología
Fundamental, Instituto de Salud Carlos III (ISCIII), Majadahonda 28220, Spain; and the Centro de Biología Molecular S.O. (CBMSO),
CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
Lymphohematopoietic progenitors derived from midgestation mouse
embryos were established in long-term cultures with stromal cell
monolayers and interleukin 7 (IL-7), giving rise to B-lineage cell
lines. The initial emergence and in vitro establishment of these early
embryo cell lines were highly sensitive to IL-7-mediated signals, in
comparison to cell lines similarly obtained using precursors from
late fetal liver (> 13 days postcoitum) and adult bone marrow.
The early embryo-derived progenitors spontaneously differentiated in
vitro to CD19+IgM+ immature B cells in the
presence of optimal concentrations of IL-7, in contrast to those
progenitors obtained from late gestation and adult mice, whose
differentiation only occurred in the absence of IL-7. The newly in
vitro-generated B cells of the early embryo cell lines repopulated
adult immunodeficient severe combined immunodeficient mice on their
adoptive transfer in vivo and generated specific humoral immune
responses after immunization.
(Blood. 2001;98:1862-1871) The scenario of mouse embryo lymphohematopoiesis
has been transformed during recent years. Novel intraembryonic sites
(para-aortic splanchnopleura/aorta-gonad-mesonephros region
[P-Sp/AGM], blood, omentum) and distinctive progenitors have been
revealed in early, preliver midgestation periods (reviewed in
Morales-Alcelay et al,1 Melchers and Rolink,2
and Keller et al3). Stem cells giving rise to definitive
lymphohematopoiesis are detected in para-aortic
mesoderm,4,5 as well as in yolk sac (YS), liver, blood,
spleen and, finally, in bone marrow (BM)
microenvironments.6,7 Not only multipotential stem cells
exist, but lineage-specific gene programs are also activated at the
early ontogenic periods (days 9-12 postcoitum [dpc]); a limited
process of B lymphopoiesis occurs, as it is revealed ex vivo by the
detection of ckit+ AA4.1+ CD19+
cells, IgH DJ rearrangements, and the transcription of pre-B-specific genes (RAGs, VpreB, Ig An efficient B lymphopoiesis relies on sequentially acting
transcription factors (eg, Ikaros, Pax5, Id) that commit multipotential progenitors to the B-cell lineage, while restricting other cellular fates.17-19 Supportive cytokines (especially IL-7) and
interactions with stromal cells and components of the extracellular
matrix, maintain viability and promote growth of early B-cell
precursors (pro-B I cells) in inductive
microenvironments.20,21 The expression of pre-B-specific
genes encoding for the recombinase enzymatic complex (RAGs,
DNA-PK, Ku, others) and for the components of the surrogate light
chain (SLC) ( The work reported here focuses on the B lymphopoiesis emerging from
midgestation mouse embryo progenitors, based on the establishment of
long-term growing, untransformed CD19+ B-lineage cell
lines. The in vitro cell lines obtained from 11-dpc embryos showed a
much lower threshold of response to limiting amounts of IL-7 than the
one of cell lines derived from late fetal liver (LFL; after 13 dpc) and
BM progenitors (LFL/BM B-cell lines). Surprisingly, and in contrast
with those, the cell lines generated from 11-dpc mouse embryo
progenitors spontaneously differentiated in vitro to functional
IgM+ B cells, even in the presence of high concentrations
of IL-7. These cell lines reconstituted the B-cell compartment of adult SCID mice, giving rise to CD19+IgM+ cells in
them. On T-cell-independent immunization, the B-cell-repopulated mice
generated humoral immune responses. Midgestation mouse embryo progenitors may consequently represent an optimal source to establish long-term, in vitro growing, polyclonal B-cell lines.
Mice, microsurgery, and cell purification from ex vivo
samples
Cell cultures and limiting dilution analyses
Flow cytometry and immunofluorescence microscopy The following monoclonal antibodies (mAbs) were purified from hybridoma supernatants by affinity chromatography on protein G columns (Pharmacia, Uppsala, Sweden), and fluoresceinated or biotinylated by standard methods: anti-IgMa (RS3.1),34 anti-CD191D3,35 anti-B220 (RA3.6B2),36 anti-I-Ek, d, r, p (14-4-4S),37 anti-CD43 (S7),38 anti-CD553-7.313,39 anti-BP-1,40 and anti-PB76 (G-5-2).41 Fluoresceinated anti-IgD and biotinylated anti-IL-7 receptor chain
(IL-7R) were purchased from Pharmingen (San Diego, CA).
Fluoresceinated sheep F(ab')2 antimouse Ig(H and L)
was obtained from Silenus Lab (Hawthorn, Australia). Biotinylated antibodies were revealed with phycoerythrin (PE)-conjugated
streptavidin (Southern Biotechnology, Birmingham, AL). Two-color
stainings were performed as described.9 Cell debris and
dead cells were excluded on the basis of forward- and side-light
scatter parameters and propidium iodide-stained cells. Specific mAb
signals were defined against the background fluorescence of
isotype-matched irrelevant mAbs and after Fc block with anti-CD16/CD32
purified mAb (Pharmingen). The flow cytometry analysis of cytoplasmic
µH was done as described.24 Flow cytometry was performed
on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA)
and analyzed with the CellQuest Immunocytometry analysis system
(Becton Dickinson).
Cytospin preparations were stained as described42 with either rhodamine-labeled goat antimouse IgM or rhodamine-labeled goat antimouse IgG (Southern Biotechnology), and countersained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI; Molecular Probes, Eugene, OR). The slides were analyzed in a Leitz DMRD microscope (Leica, Wetzlar, Germany). Immunomagnetic cell subset purification B-cell precursor purification from embryo cell lines and from spleens of reconstituted SCID mice was performed by immunomagnetic cell sorting with the VarioMACS system (Miltenyi Biotec, Bergisch, Germany). IgM+ B cells were labeled with biotinylated anti-IgMa mAb and incubated later on with streptavidin-conjugated beads (Miltenyi Biotec). The degree of contamination with IgM+ cell populations was verified by reanalysis in the FACScalibur (Becton Dickinson), and was below 1%.Cell proliferation and enzyme-linked immunosorbent assays Cell proliferation was measured after a 12-hour pulse of 1 µCi 3[H]-thymidine (Amersham-Pharmacia Biotech, Buckinghamshire, United Kingdom), and incorporated radioactivity was quantified by scintillation counting (Wallac, Turku, Finland). The enzyme-linked immunosorbent assays (ELISAs) were performed as described.43 Serum IgMa, IgG2aa, and IgG1 Abs were detected by using plates coated with 3 µg/mL purified mAb specific for mouse IgMa (RS3.1), mouse IgG2aa/2ba (clone 21-48.31, Pharmingen), and mouse IgG1 (clone A85-3, Pharmingen), respectively. The assays were revealed with biotinylated goat antimouse IgM (Southern Biotechnology), biotinylated mouse antimouse IgG2aa (clone 8.3, Pharmingen), and biotinylated rat antimouse chain (clone 187.1),
respectively. Plates were then incubated with streptavidin-conjugated peroxidase (Southern Biotechnology) and developed with 0.5 M
o-phenylenediamine (Sigma). The absorption values were read at 405 nm.
A normalized IgM titer was calculated for each serum using the GraphPad
Prism 2.0 software (Graphpad Software, San Diego, CA), with the
dilution value (D50) representing the 50% of the
absorbance obtained by a given dilution of BALB/c serum. The plates
used for the anti-DNP (2,4-dinitrophenyl) IgMa ELISA were
coated with DNP-OVA (10 µg/mL), and antimouse biotinylated anti-IgMa mAb (RS3.1) was used as developing Ab. The
DNP-specific Ab titers (D50) were calculated as above.
RT-PCR Total RNA was isolated from cell pellets and the corresponding cDNAs were prepared on heat-denatured RNA (5 µg), by using 1 µg oligo-(dT) as primer and avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI), as described.44 Equal amounts of cDNA were used to amplify 5, VpreB, RAG-2, TdT,
BSAP (Pax5), and  -actin transcripts. Enzymatic amplifications were performed as described,9 with 2 U Taq DNA polymerase
(Sigma), for 30, 35, and 40 cycles (RAG-2, TdT and BSAP) or for 20, 25, and 30 cycles (5, VpreB and -actin). The annealing temperature was 63°C for 5, VpreB, and RAG-2 amplifications; 60°C for TdT and BSAP amplifications; and 52°C for -actin amplifications. The
oligonucleotides used as primers for VpreB were: sense
5'-ATGTTCTCCAGAGCCTAAGATC-3' and reverse 5'-CTGGCCTATCTCACAGGTT-3', and
for -actin sense: 5'-TTCTTGGCTATGGAATCCTGT-3'. The primers for
reverse -actin, and for 5, RAG-2, TdT, and BSAP have been
described.9,45,46 The amplification products were
separated electrophoretically on 2% agarose gels and then transferred
to Zeta-probe membranes (Bio-Rad, Hercules, CA) after treatment with
0.4 M NaOH. Hybridization was performed with probes
32P-labeled by random priming.47 The probes used
for VpreB, RAG-2, and TdT hybridizations were obtained after cloning
the PCR-amplified fragments into a modified Bluescript vector
(Stratagene, La Jolla, CA; pBS-T).48 The -actin probe
was the PstI insert of the plasmid p41AL49 and the 5
probe was the HincII/BamHI insert from the 5 cDNA clone
pZ183-1TM.33 An internal oligonucleotide was used as a
32P-labeled BSAP probe.46 PCR signal
intensities were quantified by densitometry (Fujibas-1000 detector;
Fuji, Tokyo, Japan) with the TINA software (Raytest,
Strauhenhardt, Germany).
Cloning and sequencing of genomic DNA Genomic DNA was prepared from cell pellets.50 VHDJH-rearranged alleles were amplified by PCR, with VH7183-, VHJ558-, and JH4-specific primers as previously described.51 The reaction products were separated electrophoretically on 2% agarose gels and visualized by ethidium bromide staining. Reaction aliquots containing the correct size fragments were cloned into pBS-T. White colonies obtained from the transformation of electrocompetent DH5 bacteries were
selected with X-Gal (Sigma) and checked for the size of the inserts by
specific PCR as above. Plasmid DNA was purified using the FlexiPrep Kit
(Amersham Pharmacia Biotech), and sequenced with a DNA sequencing kit
(PE Applied Biosystems, Warrington, United Kingdom), according to the
manufacturer's instructions. The final reactions were analyzed in the
ABI PRISM 377 (PE Applied Biosystems) DNA sequencer.
In vivo reconstitution and immune response studies Established BALB/c embryo-derived cell lines (2-3 months in culture) were collected from cultures and injected into low-dose irradiated CB17.SCID mice (106 cells/mouse, 2-4 mice/cell line). Control mice were injected with LFL/BM-derived cell lines or with PBS. IgMa-specific serum concentrations were sequentially analyzed by ELISA. Mice were killed at different times after adoptive cell transfers and cell suspensions were recovered from BM, peritoneum, and spleen for flow cytometry analyses. A group of mice was immunized intraperitoneally with 100 µg 2,4-dinitrophenyl-lipopolysaccharide (DNP-LPS) in complete Freund adjuvant (CFA). Serum samples were obtained weekly and tested for specific Ab titers.
Low IL-7 concentrations rescued progenitors from midgestation mouse embryos, leading to the establishment of high numbers of B-lineage cell lines Cell suspensions from 11-dpc BALB/c mouse embryo lymphohematopoietic sites, after 13 dpc LFL and adult BM were seeded in limiting dilution conditions on 96-well plates, which were previously covered with a confluent layer of ST2 cells. Titrated dilutions of rIL-7 were added at the start point of the cell cultures. Cultures harboring well-defined cell clones (> 100 cells/clone) after 7 to 10 days of culture were considered positive. As shown in Figure 1A for 11-dpc and BM progenitors, the former gave rise to higher numbers of clonable B-lineage cell lines than the latter ones, under limiting doses of rIL-7 (0.3 ng/mL, 1 of 2900 versus 1 of 10 000, respectively). Even in the absence of any rIL-7 addition a few short-lived embryo-derived clones appeared in the cultures, whereas this was never the case in cell cultures of BM progenitors (1 of 6000 versus < 1 of 105). In contrast, the clonable BM-derived cell lines that appeared in the cultures established under optimal rIL-7 supplementation (3 ng/mL) were similar to those derived from embryonic sites (1 of 2000 versus 1 of 2500, respectively). After their initial emergence, the viability of B-lineage clones underwent a fluctuant period in culture, being stabilized at 20 to 25 days and remaining so for long periods of time (1-5 months). The generated B-lineage clones could be frequently frozen, thawed, and recultured. LFL cells were established and progressed moderately better in vitro than adult BM cells, although never reaching the high recoveries of midgestation embryo-derived cells. Because the in vitro behavior of the 11-dpc embryo cell lines was markedly different from those cell lines originated both from LFL and BM, these latter 2 were considered equivalent and their individual data were pooled. Figure 1B shows the kinetics of appearance and early evolution of cell lines obtained from 11-dpc progenitors and from 15- to 18-dpc LFL and BM progenitors under 3 ng/mL rIL-7. Most of the LFL/BM-derived clones could not be maintained in long-term cultures (80%). Eleven-dpc embryo-derived clones, however, emerged 2 to 3 days later (probably due to a higher ratio of immature stem versus intermediate cells), and they were efficiently established in long-term cultures, at a frequency that was 3 times higher than the one of LFL/BM-derived progenitors. These analyses suggest that the embryo-clonable B-lineage cell progenitors were highly sensitive to IL-7 signals leading to in vitro establishment and maintenance of long-term growing cell lines. No major differences were revealed by using precursor cells from various hematopoietic 11-dpc mouse embryo locations, despite some quantitative variations in B-cell precursor frequencies: 1 of 2500 plated cells for liver, 1 of 2800 for YS, 1 of 5000 for blood, and 1 of 9000 for P-Sp/AGM (data not shown). Embryo- and LFL/BM-derived cells did not show other significant differences in proliferation or Ig secretion when exposed to non-IL-7 stimuli, such as anti-IgM, LPS, IL-4, or anti-CD40 (data not shown).
The in vitro generated cells were characterized as B-lineage cells by
the surface expression of CD19 and IL-7R
Polyclonal VHDJH repertoires in long-term, in vitro growing B-lineage cell lines derived from mouse embryo progenitors To know whether the embryo cell lines represented an in vitro model of normal B-cell lymphopoiesis that gives rise to diverse Ig repertoires, we sequenced a pool of VHDJH rearrangements in 2 11-dpc mouse embryo cell lines (Figure 3). One half of the VHDJH-joint sequences lacked nontemplated N/P nucleotides, whereas the rest had a limited number of these nucleotides at the junctions. Seven of the 16 IgH rearrangements sequenced were productive. No significant IgH gene biases were observed, except for a high usage of JH4 (14 of 16 VHDJH rearrangements). This JH4 bias of the embryo-derived VHDJH rearrangements was also noticed when DJH rearrangements were analyzed in a larger sample of embryo- and LFL/BM-derived cell lines by specific PCR.9,56 JH4 was found in 66.6% and 36.3%, respectively, of the 48 embryo- and 55 LFL/BM-derived DJH rearrangements that were detected in a total number of 10 to 12 cell lines analyzed per group (data not shown). Novel IgH rearrangements, which were previously undetected at 2 months of evolution, appeared after 5 months in culture (Figure 3, cell line no. 2.11). Yet, the number of sequences found repeated at the later time-point cultures significantly increased (none of 13 sequences at 2 months versus 17 of 20 sequences at 5 months of continuous culture), showing a tendency to restriction of IgH repertoires and oligoclonality.
Clonable B-lineage cell lines derived from midgestation mouse embryo progenitors differentiated in vitro to surface IgM+ B cells in the presence of rIL-7 The differentiation stage reached by both midgestation mouse embryo- and LFL/BM-derived B-lineage cell lines was analyzed studying the membrane IgM expression by flow cytometry (Figure 4A). The vast majority of LFL/BM-derived B-lineage cell lines were unable to generate IgM+ B cells in cultures supplemented with 3 ng/mL rIL-7, as reported.27,29 In contrast, most of early mouse embryo-derived cell lines (> 60%) contained small but clear subsets of surface IgM+ B cells (1%-10% IgM+ cells/line) at 1 month in culture. After longer periods of time (2-5 months), one half of the embryo cell lines harbored significant IgM+ B cell populations, whereas only rare "escape" IgM+ clones were detected in the long-term cultures of adult cell lines. The relative levels of B cells increased up to a mean value of 40% IgM+ B cells/IgM+ embryo cell line at 5 months.
The IgM+ B cells in vitro generated by the embryo cell
lines were small resting cells (as defined by forward side scatter), which expressed CD19, CD43, BP-1, and PB76, but were negative for
B220/6B2, MHC class II IE, CD5, and IgD Ags (Figure 4B). These B cells
therefore can be ascribed to the stage of
BP-1+IgM+IgD IL-7 selectively regulated pre-B cell numbers, but did not block the last differentiation step to sIgM+ B cells, in midgestation mouse embryo cell lines We decided then to analyze the effect of IL-7 on the populations of pre-B and B cells of embryo cell lines. When 6 representative embryo cell lines and 2 LFL/BM cell lines (Figure 5, black and dashed bars, respectively) were plated on ST2 monolayers without rIL-7, their proportions of IgM+ B cells selectively increased at 48 to 72 hours, in comparison with the same cell lines cultured with 3 ng/mL rIL-7 (Figure 5A). However, the absolute numbers of B cells recovered did not significantly change in the 11-dpc embryo-derived cell lines cultured with or without rIL-7 (3 ng/mL), whereas minor increases in B-cell numbers were observed in the LFL/BM cell lines, on rIL-7 depletion (Figure 5B). IL-7 also stimulates the proliferation of B-cell precursors, and this activity was responsible for the apparent discrepancy between relative and absolute B-cell recoveries obtained with the embryo cell lines. Both cell proliferation and total recovered cells increased in a rIL-7 dose-dependent manner to reach a plateau at the 3 ng/mL rIL-7 dose both in 11-dpc embryo- and LFL/BM-derived cell lines (Figure 5C). The total numbers of the B-cell precursor population were selectively expanded in response to rIL-7 (Figure 5D). Subsequently, the B-cell population was diluted by this expansion of pre-B cells at increasing doses of rIL-7 and was concentrated in the absence of the cytokine (Figure 5A).
To show whether there was a component of IL-7-mediated differentiation
arrest between pre-B and B cells in the embryo cell lines,
CD19+IgM
Mouse embryo-derived cell lines reconstituted the B-cell compartment of adult SCID mice and mounted humoral immune responses To elucidate the in vivo reconstitution capacity and functionality of the embryo cell lines, we transferred 8 representative cell lines from 11-dpc BALB/c (IgHa) mouse embryos to adult, low-dose irradiated CB17.SCID mice (IgHb). Transferred cells expanded in vivo, and donor-derived IgMa+ B-cell subsets were detected in BM, spleen, and peritoneal exudate of the recipient SCID mice. Two typical examples of the cell surface features of CD19+IgMa+ B cells present in the recipient mice are displayed in Figure 7A. The BM and the spleen of the recipient mice harbored significant populations of donor-derived IgMa+ B cells, and CD19+IgMa cells whose genetic origin cannot
be firmly established. We tested for the presence of cytoplasmic IgH in
the splenic CD19+IgM cell population purified
by immunomagnetic sorting from reconstituted SCID mice, and found that
one third of them were positive by both immunofluorescence microscopy
(Figure 7B) and intracellular flow cytometry (Figure 7C). The
embryo-derived B-lineage cells partially or completely up-regulated the
B220/6B2 Ag in vivo. Donor-derived IgMa+ B cells also
up-regulated MHC class II IE molecules and lost the in vitro expression
of IL-7R (data not shown). Small subsets of
CD5+IgMa T cells, most likely of endogenous
origin, due to the "leakiness" of the SCID deficiency further
stimulated by the irradiation,57 were also present in the
peritoneal exudate of recipient SCID mice. B-cell reconstitutions were
detected up to 4 months in the recipient mice. Unfortunately, the
transfer of embryo-derived cell lines into
H-2b+RAG-2-deficient mice, performed to definitively
ascertain the donor origin of the
CD19+IgMa-cµH and the
CD5+IgMa cell populations, repeatedly failed
to produce reconstitutions.
A total of 70% to 80% of the recipient mice were restored with
different levels of donor-derived, serum IgMa at 1 month
after transfer, the titers increasing later on to reach values that
were close to those of normal BALB/c mice (Figure 8A). IgG2aa and IgG1 serum
Abs were detected in 4 and 2 recipient mice of 20, respectively, at
levels 1 log below those of BALB/c mice (data not shown). A group of
2-month reconstituted SCID mice with 2 independent embryo B-cell lines
was immunized with DNP-LPS, using BALB/c and BALB/cnu/nu
mice as immunization controls. The serum levels of anti-DNP-specific IgMa antibodies were studied by means of IgM
allotype-specific ELISAs. As shown in Figure 8B (upper histogram), the
IgMa+ B cells established in vivo mounted a primary immune
response to the DNP-LPS Ag during the 2 months after immunization. The increases of serum anti-DNP Ab levels found in the reconstituted SCID
mice were roughly similar to those of athymic BALB/cnu/nu
controls. BALB/c mice, however, displayed a more robust and faster anti-DNP response, perhaps due to a minor component of T-cell help in
this B-cell immune response (Figure 8B, bottom histograms). These
findings suggest that the B-cell lines generated in vitro from
progenitors recovered from midgestation mouse embryos were not only
able to reconstitute the B-cell compartment of adult SCID mice, but
also were able to give rise to humoral immune responses after specific
immunization.
B lymphopoiesis proceeds during the whole life span of the individual, starting on early midgestational periods of the mouse embryo. The process varies throughout the mouse ontogeny related to organ location, developmentally controlled waves, microenvironment-bound signals, and peripheral cell turnover rates. Recent evidence indicates that genetic elements of B-cell development appear very close in time to the detection of the first lymphohematopoietic progenitors in the P-Sp/AGM (8.5 dpc).8-10 Whether these processes are part of bona fide B-cell differentiation or represent low-level, random gene transcription events in multipotent cell progenitors58,59 is a question open to debate. As it happens for other somatic cell lineages, the in vitro establishment of cell progenitors in conditions promoting their full maturation and functionality is an important goal, both to approach the molecular bases of cell differentiation and to develop putative therapies of substitution in selected diseases.60 The cultures of stromal cells plus IL-7 have been a powerful tool to study mouse B-cell differentiation.27,54 These cultures, however, showed an unexpected IL-7-mediated blockade at the stage of late pre-B cells, with inhibition of the emergence of functional B cells in the cultures of LFL and BM progenitors. To differentiate into IgM+ cells, IL-7 had to be removed from the cultures,27-29 which then became short-lived and self-limited. We have now established untransformed B-lineage cell lines from 11-dpc mouse lymphohematopoietic sites (liver, P-Sp/AGM, YS) in cell cultures with stroma plus IL-7. In contrast to LFL/BM cell lines, these early embryo cell lines were able to spontaneously differentiate up to the stage of CD19+IgM+ B cells in the presence of IL-7 and to efficiently reconstitute functional B-cell compartments on their adoptive transfer into adult SCID mice. The IL-7/IL-7R pair is critical for lymphoid
development.61 Although factors alternative to IL-7 may
also be involved in IL-7R signaling,62-64 the deletion of
the IL-7R A synergistic cross-talk between IL7R and PreBCR-encoding
The embryo cell lines were able to overcome the IL-7 differentiation arrest and efficiently mature to functional IgM+ B cells in the presence of saturating IL-7 concentrations. Only when cultures of purified B-cell precursors were submitted to superoptimal doses of rIL-7 (30 ng/mL), was the number of newly emerging B cells reduced. This implies that a partial IL-7-dependent differentiation arrest also existed, therefore, in embryo cell lines at the stage of late pre-B cell. A selective driving force of midgestation mouse embryo progenitors to complete differentiation along the B-cell lineage, as has been suggested with other postgastrulation hematopoietic cells,77,78 could represent an intrinsic feature of these early cells, contributing to overcome the IL-7-mediated arrest. Both the low threshold of proliferative responses to IL-7 at the immature cell stages and the strong bias to rapid differentiation may account for the distinctive behavior observed in the embryonic cell lines. The in vitro establishment of normal, untransformed B cells represents a long-searched goal for studies of humoral immunity. We consider that the approach shown in this paper can represent a substantial contribution in that sense. This work shows that polyclonal CD19+IgM+ B lymphocytes were continuously generated in vitro when progenitors from midgestation mouse embryos were established in stromal monolayer plus IL-7 cell cultures. More importantly, these B cells efficiently reconstituted adult immunodeficient mice, showed a conventional cell surface phenotype, and became able to respond to Ag immunization. It seems likely that the in vivo expansion potential of the embryo cell lines depends on minor subsets of pro-B cells remaining in the in vitro growing cell lines. Similar repopulations of BM and spleen B-cell pools are absent in the adoptive transfers of LFL- and BM-derived cell lines (data not shown).79,80 Finally, the experimental approach shown here may be useful to analyze the progenitor's potentials from different genetic backgrounds, and in particular, those of gene-manipulated mice suffering gestational death. From a biomedical viewpoint, it would be interesting to know whether developmental characteristics similar to those observed here also apply to human progenitors from sources such as the cord blood, to consider them for putative substitution therapies in certain immunodeficiencies.
We acknowledge the suggestions and critical review of the manuscript of J. Andersson and his colleagues. We also thank A. Grandien for the ST2 cells and A. G. Rolink for the IL-7-transfected 3T3 cells. The technical support of P. Ferrero and the editorial assistance of M. Messman are also recognized.
Submitted November 30, 2000; accepted May 11, 2001.
Supported by grants from the Comisión Interministerial de Ciencia y Tecnología (PM96-0072 and PM99-0104) and the CAM (08.3/0009). The CBMSO is partially founded by Fundación Ramón Areces.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: María-Luisa Gaspar, Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Spain; e-mail: mlgaspar{at}isciii.es.
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© 2001 by The American Society of Hematology.
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  B. de Andres, I. Cortegano, N. Serrano, B. del Rio, P. Martin, P. Gonzalo, M. A. R. Marcos, and M. L. Gaspar A Population of CD19highCD45R /lowCD21low B Lymphocytes Poised for Spontaneous Secretion of IgG and IgA Antibodies J. Immunol., October 15, 2007; 179(8): 5326 - 5334. [Abstract] [Full Text] [PDF]
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 B. de Andres, P. Gonzalo, S. Minguet, J. A. Martinez-Marin, P. G. Soro, M. A. R. Marcos, and M. L. Gaspar The first 3 days of B-cell development in the mouse embryo Blood, December 1, 2002; 100(12): 4074 - 4081. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
loci.