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Prepublished online as a Blood First Edition Paper on July 18, 2002; DOI 10.1182/blood-2002-03-0809.
IMMUNOBIOLOGY
From Centro Nacional de Microbiología,
Instituto de Salud Carlos III (ISCIII), Majadahonda,
Spain; and the Centro de Biología Molecular Severo
Ochoa (CBMSO), Consejo Superior de Investigaciones
Científicas-Universidad Autónoma de Madrid (CSIC-UAM),
Madrid, Spain.
B-lineage-committed cells are believed to arise in the liver
of mouse embryos at 14 days after coitus
(dpc). However, pre-B-specific gene transcripts and DJH gene
rearrangements have been detected in earlier, midgestation embryos. We
describe here a population of
c-kit+AA4.1+CD19+Pax5+
cells present in the aorta-gonad-mesonephros (AGM) area and in the
livers of 11-dpc mouse embryos. In contrast to multipotent c-kit+AA4.1+CD19 Embryonic lymphohematopoiesis is a highly dynamic
developmental process that takes place in mesoderm-derived locations
(yolksac [YS], para-aortic splanchnopleura/aorta-gonad-mesonephros
[P-Sp/AGM]), closely after mouse gastrulation (7.5-11 days after
coitus [dpc]). A transient, self-limited
myeloerythropoiesis first takes place in the early YS from 7.5 dpc to
12 dpc,1 while definitive lymphohematopoiesis arises
independently in the intraembryonic P-Sp/AGM ( Fetal hematopoietic progenitor populations have been traced with
different cell surface receptors. The receptor tyrosine kinase c-kit,
besides being present in migrating melanoblasts and germ cells, is also
expressed in hematopoietic cell progenitors.31 The AA4.1
molecule, which is involved in cell adhesion,32 is found
in all embryonic HSCs,33 and its expression is also
up-regulated in BM B-lymphoid progenitors in correlation with the onset
of Pax5 transcription, CD19 Ag expression, and loss of pro-T-cell potential.34 IL-7R This wide-ranging information about the emerging embryonic B
lymphopoiesis has mostly come from studies with either unpurified and/or mixed cell populations and has frequently been obtained from in
vitro differentiation assays of varying efficiency. We decided to
search for the earliest-restricted B-cell precursors at the cellular
level, by means of genetic and flow cytometry analyses performed on
cell populations directly purified ex vivo. In the present paper, we
describe a novel population of
c-kit+AA4.1+CD19+ cells detected in
the 11-dpc P-Sp/AGM and liver, which selectively expresses
pre-B-specific genes. In contrast to multipotent
c-kit+AA4.1+CD19 Mice and embryo cell preparations
Monoclonal antibodies and flow cytometry
Polymerase chain reaction analyses Total RNA was extracted, and cDNA prepared and amplified, with Taq-DNA Polymerase (Sigma Chemical, St Louis, MO) for rag-2, VpreB, and 5 (2 µL cDNA/reverse transcriptase-polymerase chain reaction
[RT-PCR]), and for  -actin (1 µL cDNA) as
described.42 FastStart Taq-Polymerase (Roche Diagnostics
GmbH, Mannhein, Germany) was used for Pax5 (4 µL cDNA), CD19, EBF,
and Ikaros (2 µL cDNA) amplifications. Each cycle was repeated 25 times for -actin, 35 times for VpreB and 5, and 40 times for the
other PCRs. Primers and annealing conditions have been described for
rag-2,44 VpreB, 5,42 Pax5,45
CD19,37 EBF, Ikaros,21 and
-actin.45 Amplification products (one-fifth each) were
separated electrophoretically on 2% agarose gels, transferred, and
hybridized. Rag-2, VpreB, and 5 cloned probes, oligonucleotides
5'-ACTCACCCTATGCCATTGTGC-3' (EBF-in), 5'-ATGTGGATATTGTGGCCGGA-3'
(Ikaros-in), 5'-CCAGTACGGGAATGTGCTCT-3' (CD19-in) and those described
for Pax5 and -actin45 were used as
32P-labeled probes. PCR signal intensities were quantified
by densitometry on a FLA-3000 detector (Fuji, Tokyo, Japan) using the
AIDA software (Raytest, Stauhenhardt, Germany). Relative message levels
were calculated by referring the gene-specific signal intensity values to the value obtained for -actin in the same cDNA. HC locus
transcripts (VDJCµ) were revealed by 2 rounds of amplification (35 cycles each), by using described primers43,46
(VH7183, VHJ558, and Cµ1), and a newly
designed for Cµ2 (5'-GGGAAATGGTGCTGGGCAGGAA-3'). For transcript
expressions from individual Pax5 / embryo livers, 2 µL
of the first PCR products were subjected to 35 cycles of amplification
with internal primers for rag-2, VpreB, and CD19 (rag-2-3' in:
5'-GGTTCAGGGACATCTCCTTACTAAG-3'; VpreB in:
5'-CCCACGGCACACTAATACACA-3'; CD19:
5'-TTGAGTGGAGCTGAGGAGCT-3' and the above-described CD19-in). The
nested PCR (60°C, annealing temperature) products were visualized on
ethidium bromide-stained gels. Pax5 genotypes41 were
determined by PCR assays on 11-dpc embryo tail DNA preparations, using
 -actin44 to verify DNA content. Control experiments with
sequential numbers of cycles confirmed the linear range of the
amplification process for the cDNA inputs. As additional controls for
the differential sensitivity of VpreB and 5 RT-PCR assays,
amplifications were performed on cDNA samples from serial dilutions of
the 70Z/3 cell line.
Lymphoid, myeloid, and erythroid potentials from sorted embryonic cells B-lymphoid progenitor frequencies were calculated by seeding dilutions of 50 to 500 sorted cells per well in 96-well plates bearing confluent layers of Mitomycin C (Sigma)-treated ST2 stromal cells (5 × 103 cells/well; 7-10 days of culture). The medium used was Iscove modified Dulbecco medium (IMDM) (BioWhittaker Europe, Verviers, Belgium), supplemented with 10% IL-7-conditioned medium (IL-7 sup), as previously described.42 The stromal cell cultures were refreshed every 96 hours. The B-lymphoid, myeloid, and erythroid potentials were evaluated in parallel by using a modification of described methods.12 In brief, sorted cells (100-500 cells/well, 96-well plates) were seeded on an ST2 stromal cell monolayer, and grown in IL-7 sup (10%), IL-11 (20 ng/mL; Pepro Tech, Rocky Hill, NJ), and stem cell factor (SCF) (20 ng/mL; R&D Systems, Abingdon, United Kingdom) for 7 days. The growing cell clones were then split into 3 aliquots that were set up in (1) SCF alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) (2 ng/mL; Pepro Tech) (myeloid conditions), (2) ST2 plus 1% methylcellulose, SCF, and erythropoietin (EPO, 2 U/mL) (erythroid conditions), and (3) ST2 plus IL-7 sup (B-lymphoid conditions). After an additional culture period of 10 to 14 days, wells harboring growing cells were scored and collected. Additional cultures of sorted populations were made with the Methocult GFM3434 system (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with recombinant IL-3, IL-6, SCF, and EPO to optimize the recovery of erythroid/myeloid precursors, as described16 (0.5-5 × 103 cells/well; 24-well plates). After a period of 7 to 9 days, colonies were scored on the basis of their morphology, and individually picked for flow cytometric studies. Myeloid lineage colonies were also identified by May-Grünwald-Giemsa staining of cytospin preparations.Fetal thymic organ cultures Fetal thymic organ cultures (FTOCs) were performed as described.47 Briefly, 15-dpc thymic lobes were obtained from C57BL/6-CD45.1 mouse embryos and cultured on polycarbonate filters, floating on IMDM containing 2-deoxyguanosin (1.35 mM; Sigma) for 6 days. After washing the lobes, they were separated into individual Terasaki plate wells (Sarstedt, Australia). Unfractionated 11-dpc liver cells, and sorted c-kit+AA4.1+CD19 and
c-kit+AA4.1+CD19+ populations
recovered from 11-dpc livers of C57BL/6 (CD45.2) mouse embryos were
expanded on ST2 cells with IL-7, IL-11, and SCF (500 cells/well) for 3 days. Cells from each well were added to the thymic lobes in the
Terasaki plates (250 initial cells/lobe) and were placed in hanging
drop IMDM/IL-7 cultures during the first 48 hours of cultures
maintained for 12 additional days. Cells were then pooled from several
thymic lobes (n = 5-10 lobes for each cell population) and were
stained for flow cytometry. The anti-CD45.2 mAb identified cells of
donor origin (C57BL/6).
Lymphoid-specific gene expression in 9- to 13-dpc mouse embryos Lymphohematopoietic sites of the early BALB/c mouse embryo (YS, P-Sp/AGM, liver, blood) were microdissected, cells were mechanically dissociated, and mRNA levels of rag-2, VpreB,5, and -actin genes were
evaluated in semiquantitative RT-PCR assays. A summary of the results
obtained is shown in Figure 1A. While all
these transcripts were undetectable in 8-dpc mouse samples
(n = 8 for P-Sp and n = 10 for YS; data not shown),
recombinase-encoding rag-2 gene expression was frequently
found in 9-dpc P-Sp and YS (3 of 8 and 4 of 9 independent samples,
respectively). This result is consistent with previous findings about
the simultaneous rag-1 gene transcription at these
sites.14 VpreB transcripts were found one day later in
P-Sp/AGM and liver, and their levels increased during the subsequent
days of gestation. The other surrogate light chain (SLC) component, the
5 gene, was not up-regulated until 12 dpc in the AGM and liver, 2 days later than the VpreB gene, suggesting the existence of VpreB+5 B-cell
progenitors at the earliest ontogenic stages. The asynchronous expressions of the SLC-encoding VpreB and
5 genes were not due to different
sensitivity thresholds of the PCR assays, because these revealed
positive VpreB/5 transcripts up to the single-cell level in titrated
dilutions of the SLC+ 70Z/3 pre-B-cell line. These signals
were in fact stronger for 5 gene
transcripts (Figure 1B). All of these genes were markedly up-regulated
in later liver samples (rag-2, VpreB, and 5 transcript levels
increased 5-, 18-, and 10-fold, respectively, from 12 to 15 dpc; data
not shown), while they progressively reduced in AGM and YS, probably in
relation to the exhaustion of the local lymphohematopoietic sustaining potential.
A population of c-kit+AA4.1+CD19+ cells is present in 11- to 13-dpc mouse embryos To establish whether the earliest B-lymphoid-specific gene expressions were due to low-level gene transcriptions in multipotent HSCs or if they corresponded to bona fide B-lineage-restricted precursors, we attempted to identify directly the putative B-lineage cells with specific cell surface markers and flow cytometry. We examined the appearance of the B-lineage-specific CD19 Ag, as well as the expression of B220, IL-7R, AA4.1, CD43, and other surface
receptors in mouse embryos. As shown in Figure
2A, a small population of
c-kit+CD19+ cells was clearly observed in both
AGM and liver cell suspensions of mouse embryos at 11 dpc. Tiny
CD19+ cell subsets were also detected in isolated samples
of blood (Figure 2A) and YS (data not shown). The fact that
CD19+ cells represent a higher fraction of
c-kit+ cells in AGM than in liver (5.9% and 1.2%,
respectively) is due to the presence of other nonlymphohematopoietic
c-kit+ progenitors in the latter organ, since
CD19+ cells constitute 10% to 11% of AA4.1+
hematopoietic precursors in both locations. When the percentages of
these CD19+ cells were referred to the absolute cell
recoveries obtained in the embryo lymphohematopoietic organs, we
observed that 11-dpc AGM and liver harbored 97.8 ± 58 and
480 ± 100 CD19+ cells per organ, respectively (Figure
2B; n = 6 for AGM, and 17 for liver; mean ± SD). The number of
AA4.1+ cells increased exponentially in the liver between
11 and 13 dpc, but not in the simultaneous AGM, where they declined
after 12 dpc. CD19+ cells expanded in the liver during the
same period and, to a lesser extent, in the AGM. CD19+
cells were also detected in 11-dpc livers of C57BL/6 embryos (275 ± 45 CD19+ cells per liver; n = 4). The CD19
immunofluorescent findings were confirmed and extended by CD19-specific
RT-PCR assays (Figure 2C). At 11 dpc, high levels of CD19 gene
transcripts were always found in both AGM and liver, and weaker signals
were also present in YS and blood. In contrast, CD19 transcripts were
absent from all 8-dpc samples, and they began to appear in 9-dpc (2 of
4 P-Sp and 1 of 5 YS) and 10-dpc (3 of 4 P-Sp and 3 of 5 YS) mouse
embryos (data not shown).
A more detailed characterization of 11-dpc liver
c-kit+CD19+ cells was performed (Figure 2D).
The majority of the cell population was positive for the AA4.1 and CD34
Ags, markers representative of fetal HSCs33 and very early
progenitors.48 Levels of Sca-1 Ag, which is up-regulated
at this point in mouse ontogeny,14 were not expressed by
the c-kit+CD19+ cell population. The IL-7R Selective expression of lymphoid-specific genes in purified CD19+ cells from 11-dpc embryonic liver We next investigated whether the earliest detected lymphoid gene expressions in the embryo (see the first paragraph of "Results") were restricted to the newly revealed population of CD19+ cells and/or to other embryonic hematopoietic cells. The following cell subsets were purified by electronic sorting from 11- and 13-dpc livers after 3-color stainings (c-kit, AA4.1, CD19) (Figure 3A): (R1) c-kit+AA4.1 cells, which excluded any
CD19+ cell, and represented 54 ± 6% of total liver
cells at 11 dpc (mean ± SD; n = 5), (R2)
c-kit+AA4.1+CD19 cells
(6 ± 1%; n = 5), and (R3)
c-kit+AA4.1+CD19+ cells
(0.67 ± 0.3%; n = 10), and their mRNAs were studied in RT-PCR assays. The remaining c-kitAA4.1
cells were not analyzed because we knew from previous studies that this
population was constituted by TER-119+ erythroid cells and
hepatocyte progenitors, and that it lacked any other
lymphohematopoietic progenitor potentials (S.M., M.L.G., M.A.R.M.,
unpublished data, June 2002). Pre-B-specific rag-2 and VpreB
transcripts were selectively detected in the 11-dpc liver CD19+ (R3) cell population, while the
5 gene expression was delayed up to 13 dpc in the same population (Figure 3B, top diagram). We also tested for
the expression of transcription factors involved in B-lineage
commitment, such as Ikaros (expressed by both T- and B-cell
progenitors),49 EBF (upstream regulatory gene of Pax5), and Pax5 (essential for B-cell commitment) in 11-dpc
liver c-kit+CD19 cells (R1 + R2) and in
CD19+ cells (R3) (Figure 3B, bottom diagram). The 3 genes
(Ikaros, EBF, and Pax5) were highly expressed in
CD19+ (R3) liver cells. Gene transcripts for Ikaros and EBF
(but not Pax5) were also present among
c-kit+CD19 cells. We conclude that the
expression of pro/pre-B-specific genes observed in midgestation mouse
embryos (VpreB, 5, rag-2, Pax5) is restricted to the population of
c-kit+AA4.1+CD19+ cells. Finally,
VDJCµ transcripts were negative at 11 dpc, both in the sorted
populations and in unfractionated liver samples (Figure 3B), as well as
cytoplasmic µHC (Figure 3C).
We also analyzed CD19, rag-2, and VpreB mRNA expression in 11-dpc
livers from Pax5-deficient mice, which are devoid of B-lineage cells,
as an additional proof of the B-cell-restricted character of the
embryonic c-kit+AA4.1+CD19+ cells
(Figure 3D). Isolated livers from Pax5 11-dpc c-kit+AA4.1+CD19+ liver cells are restricted to B-lineage differentiation We analyzed the lymphohematopoietic potentials of c-kit+AA4.1+CD19+ (R3) cells in comparison to those of simultaneous c-kit+AA4.1+CD19 (R2) cells from
11-dpc livers. Both cell populations were established in low-cell
density, 2-step stromal cell cultures, and in the Methocult system, as
described in "Materials and methods." Representative findings of
these experiments are shown in Figure 4.
Sorted 11-dpc c-kit+AA4.1+CD19
(R2) liver cells differentiated to populations of (1) small, round
CD19+CD90CD11bTER-119
cells (n = 200 wells; 27% positives), and (2) big, granular, macrophagelike CD19CD11b+ cells (n = 200
wells; 43% positives) in both B-lymphoid and myeloid conditions,
respectively. The selective addition of GM-CSF to the myeloid cultures
increased their proportion of Gr-1+ granulocytes with
respect to CD11b+ macrophages (data not shown). R2 cells
also gave rise to small, anucleated,
CD19TER-119+ erythrocytes in
EPO-methylcellulose cultures. They also generated several types of
colonies when seeded in the Methocult system (500-1000 erythroid, 50 to
400 myeloid/granulocyte, and 50 to 200 erythro/myeloid colonies per
105 cells; n = 4). In contrast, although well expanded in
the first culture step (ST2 + IL-7, SCF, IL-11), the
c-kit+AA4.1+CD19+ (R3) starting
cultures rapidly died when secondarily transferred to conditions in the
absence of IL-7. Purified 11-dpc liver
c-kit+AA4.1+CD19+ (R3) cells only
expanded successfully in defined B-cell conditions (ST2 + IL-7)
that allowed the establishment of 45% of the initially obtained
cultures (n = 150 wells). The frequencies of B-cell precursors obtained in limiting dilution experiments performed as
described42 were 1/500 and 1/160 for the R2 and the R3 cell
populations, respectively. The B220 Ag (which was absent in vivo) was
partially up-regulated in B-cell cultures of multipotent R2 cells, and
became completely positive in those derived from R3 cells.
B-lineage cell lines that acquired the stage of LPS responsiveness and
matured to surface IgM+ cells were derived from both R2 and
R3 cell populations (data not shown). We conclude that, in contrast to
multipotent embryonic c-kit+AA4.1+CD19 (R2)
cells, c-kit+AA4.1+CD19+ (R3) cells
represent progenitors functionally committed to the B-lymphoid lineage
and are devoid of any other myeloerythroid potentials.
Embryonic c-kit+AA4.1+CD19+ liver cells lack T-cell potential We also examined whether the embryonic CD19+ cell progenitors showed any T-cell differentiation potential by seeding them in FTOC. Purified 11-dpc liver R2 and R3 cells obtained from C57BL/6 (CD45.2+) mouse embryos were expanded for 3 days in vitro (ST2 + IL-7, IL-11, SCF) and then were seeded on 15-dpc 2-deoxyguanosin-treated thymic epithelia from C57BL/6-CD45.1+ mouse embryos. After 2 weeks, the FTOCs were dispersed and donor-derived CD45.2+ cells were analyzed by flow cytometry. Sorted R2 cells expanded in vitro and efficiently matured throughout the stages of CD4+CD8+, CD4+, and CD8+ thymocytes, up to CD3++
cells. However, only occasional donor-derived CD45.2+ cells
were seen in FTOC established with R3 cells (Figure
5). FTOC initiated with total cells from
11-dpc liver embryos were also reconstituted (Figure 5). We conclude
that the embryonic c-kit+AA4.1+CD19+ (R3) cell
progenitors are devoid of T-cell precursor potential, and thus are
selectively restricted to differentiation along the B-cell
lineage.
Primitive myeloerythropoiesis and definitive lymphohematopoiesis emerge early in the postgastrulation mouse embryo (7-8 dpc) in mesoderm-derived sites such as the YS and the P-Sp/AGM.1-3,5,6,50 From these primary sites, multipotent progenitors home to secondary hematopoietic organs (liver, spleen, BM, thymus).10 While the first definitive HSCs are found in the 8- to 9-dpc P-Sp,3,6 and erythromyeloid programs of differentiation are rapidly established in the YS,16 it was thought that restricted B-lymphopoiesis did not appear until 13 to 14 dpc, and that it occurred exclusively in the liver.12,15 These (and additional data related to other hematopoietic differentiation pathways) led to the proposal of P-Sp/AGM as being a pure organ of hematopoiesis where multipotent progenitors self-renewed and expanded, while the B-lineage differentiation program was initiated later in fetal liver.15,30 The findings of low-level lymphoid lineage-specific gene transcripts and of DJH rearrangements in early extra-liver sites14,26,27 were considered to be the result of partially accessible gene loci in "primed," but not yet committed, multipotent progenitors.30 The above scenario leaves an extended developmental window (9 to 13 dpc) where HSCs would only proliferate but not up-regulate differentiation programs, at least for the B-lymphoid lineage. The present work has isolated and characterized a novel population of
c-kit+AA4.1+CD19+ cells emerging at
10 to 11 dpc in the AGM and liver, and to a lesser extent, in the YS.
They represent a few hundred cells per organ between 11 and 13 dpc,
whose numbers increase during this period, before the later synchronous
and exponential expansion of B-cell progenitors that takes place in the
liver after 13 dpc.23,24 It is of particular note that the
B-lineage-specific gene transcripts previously detected in
hematopoietic sites of postgastrulation mouse embryos (rag-1 and -2, VpreB, We consider that the studied population of committed B-lineage
c-kit+AA4.1+CD19+ progenitors has
probably escaped detection before now, due to (1) the unusual features
of these B-lineage cells (Figure 2D), (2) the heterogeneous
experimental approaches that employed either total or variously
purified cell populations in which the
AA4.1+c-kit+CD19+ cells might
represent a minor subset, and (3) the indirect conclusions from in
vitro culture assays of variable efficiency. Although we know that
midgestation B-cell progenitors are highly sensitive to IL-7 signals
and that they can be very efficiently established in vitro on ST2 + IL-7 cell cultures,42 it may be that only a subset of
the embryonic CD19+IL-7R The 11-dpc mouse embryo
c-kit+AA4.1+CD19+B220 Although lacking the CD45R/B220 molecule, embryonic CD19+
cells were positive for the CD45 common leukocyte antigen. Other phenotypical traits of the CD19+ cells also indicate their
immature stage (AA4.1, IL-7R In conclusion, the findings of this study support the idea that
the developmental process of embryonic B lymphopoiesis begins at 10 to
11 dpc with a population of fully B-committed
c-kit+AA4.1+CD19+B220
We are grateful to M. Busslinger and C. Martínez-Alonso for critical reading of the manuscript,
to M. Busslinger for providing us the Pax5+/
Submitted March 20, 2002; accepted July 8, 2002.
Prepublished online as Blood First Edition Paper, July 18, 2002; DOI 10.1182/blood-2002-03-0809.
Supported by grants from the Comunidad Autónoma de Madrid (CAM) (08.3/0009/1997), the Ministerio de Ciencia y Tecnología (MCyT) (PM 99-0104), and ISCIII (01/34). The CBMSO is partially funded by the Fundpción Ramón Areces. B.d.A., P.G., and S.M. are supported by MCyT-Ramón y Cajal Program, ISCIII, and MCyT, respectively.
B.d.A. and P.G. contributed equally to this work.
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: M. L. Gaspar, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo km 2, Majadahonda 28220, Spain; e-mail: mlgaspar{at}isciii.es.
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Top
Abstract
Introduction
8.5 dpc), in cell
clusters related to the ventral wall of the aorta.
R+ liver cell population at
13 dpc.
gene loci, and are essential for B-cell
development.
(145-2C11). All other reagents were obtained from
BD Pharmingen (San Diego, CA) as purified, biotinylated, FITC-, or phycoerythrin (PE)-coupled antibodies: c-kit/CD117 (2B8), CD34 (RAM34), IL-7R
symbols in the figure, which
represent 
) and FL (
). Total cells, AA4.1+, and
CD19+ cells/organ are shown, the latter 2 obtained from
their relative numbers in FACS analyses and absolute cell
numbers/organ. n = 5-7 independent analyses; means ± SD are
shown. (C) CD19 gene transcripts detected in the displayed
lymphohematopoietic sites at 8 and 11 dpc. The data are presented as in
Figure 
