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Prepublished online as a Blood First Edition Paper on November 21, 2002; DOI 10.1182/blood-2002-07-2244.
 Previous Article | Table of Contents | Next Article 
Blood, 1 May 2003, Vol. 101, No. 9, pp. 3424-3430
HEMATOPOIESIS
Human cord blood CD34+Pax-5+ B-cell
progenitors: single-cell analyses of their gene expression
profiles
Eva Sanz,
Melchor Alvarez-Mon,
Carlos Martínez-A, and
Antonio de
la Hera
From the Laboratory of Immunology and Oncology, Consejo
Superior de Investigaciones Científicas (CSIC)-Alcalá
University Research Associated Unit, Madrid; the Immune System Disease
and Oncology Unit, Príncipe de Asturias University Hospital,
Department of Medicine, Alcalá University, Madrid; and the
Department of Immunology and Oncology, Centro Nacional de
Biotecnología, CSIC, Campus de Cantoblanco, Madrid,
Spain.
 |
Abstract |
Circulating CD34+ cells are used in reparative medicine
as a stem cell source, but they contain cells already committed to different lineages. Many think that B-cell progenitors (BCPs) are
confined to bone marrow (BM) niches until they differentiate into B
cells and that they do not circulate in blood. The prevailing convention is that BCP transit a
CD34+CD19 10+
early-B CD34+CD19+CD10+
B-cell progenitor
(pro-B)CD34CD19+CD10+ B-cell
precursor (pre-B) differentiation pathway within BM. However, populations of CD34+CD10+ and
CD34+CD19+ cells circulate in adult peripheral
blood and neonatal umbilical cord blood (CB) that are operationally
taken as BCPs on the basis of their phenotypes, although they have not
been submitted to a systematic characterization of their gene
expression profiles. Here, conventional
CD34+CD19+CD10+ and novel
CD34+CD19+CD10 BCP populations
are characterized in CB by single-cell sorting and multiplex analyses
of gene expression patterns. Circulating BCP are Pax-5+
cells that span the early-B, pro-B, and pre-B developmental stages, defined by the profiles of rearranged V-D-JH, CD79, VpreB,
recombination activating gene (RAG), and terminal
deoxynucleotidyl transferase (TdT) expression. Contrary to the
expectation, circulating
CD34+CD19CD10+ cells are
essentially devoid of Pax-5+ BCP. Interestingly, the novel
CD34+CD19+CD10 BCP appears to be
the normal counterpart of circulating preleukemic BCPs that undergo
chromosomal translocations in utero months or years before their
promotion into infant acute lymphoblastic B-cell leukemia after
secondary postnatal mutations. The results underscore the power of
single-cell analyses to characterize the gene expression profiles in a
minor population of rare cells, which has broad implications in biomedicine.
(Blood. 2003;101:3424-3430)
© 2003 by The American Society of Hematology.
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Introduction |
Circulating blood CD34+ cells are
proposed as a totipotent stem cell source in organ and gene
regenerative medicine.1-5 CD34+ cells are a
heterogeneous population, however, because they also contain early
progenitors already committed to distinct lineages.5-9 Early in life, a major subset of bone marrow (BM) CD34+
cells are B-cell progenitors (BCPs).8,10 In contrast,
CD34+ BCPs were undetectable in fetal and term umbilical
cord blood (CB) in many independent phenotypic studies.6-8
The latter reports support the accepted idea that BCPs are retained in
the BM and fetal liver until they acquire the surface immunoglobulin
(sIg), IgM/CD79 antigen receptor complex, and that B-lineage emigrants to peripheral blood are solely immature sIgM+ B cells
selected for tolerance to self-antigens.11-13 The paradigm that BCPs do not emigrate to the periphery has been challenged by
authors who report that rare populations of CD34+ cells
that express either CD10 or CD19 develop in blood.7,14 Although gene expression profiles of the
CD34+CD10+ and
CD34+CD19+ blood cells have not been
characterized, as was done for BM and in vitro-differentiated
BCPs,8-10,15,16 they are often operationally defined as
circulating BCPs on the basis of their surface
phenotypes.2,17 (The prevailing convention is that
B-lineage-committed cells pass through a
CD34+CD19CD10+
early-BCD34+CD19+CD10+
pro-BCD34CD19+CD10+
pre-B-cell development pathway; reviewed in LeBien9.) The use of surface phenotypes to unambiguously define the lineage affiliation and differentiation stage of early CD34+
progenitors may, however, be an oversimplification.9 The
CD34+CD19CD10+ phenotype is not
B-lineage specific,9 and blood
CD34+CD19+ cells markedly outnumber the
CD34+CD10+ subset,14 suggesting
that yet uncharacterized
CD34+CD19+CD10 BCP occurs.
Single-cell analyses of the expression pattern of genes and proteins
implicated in the developmental specification of the
B-lineage,8-10,15-17 conducted by fluorescence activated cell sorting (FACS) and multiplex reverse transcription-polymerase chain reaction (RT-PCR) study of the gene expression profiles in the
individually separated cells, has allowed us to unambiguously characterize BM
CD34+CD19+CD10+ cells as
pro-B and pre-BI cells.10 Here, we address whether CB
CD34+ cells contain BCP and define their phenotype and
differentiation stage by single-cell molecular analyses.
B-cell differentiation is characterized by a commitment event and a
series of specification steps.18-20 The commitment event requires expression of the Pax5 gene. Experiments using
Pax5/ mouse cells have shown that
considerable progress down the B-cell specification pathway is possible
in the absence of commitment.18 CD19 expression depends
strictly on Pax-5,20 making its surface expression a
natural reporter for the transcription of a gene essential in B-lineage
commitment.19 We are not aware of single-cell studies on
CD34+CD19 human early-B-cell candidates. The
expression of surface CD10 and sterile transcripts of the IgH locus or
CD79, terminal deoxynucleotidyl transferase (TdT), or VpreB mRNA have
been used to define early-B cells in bulk
CD34+CD19 BM populations.9,21-23
In Pax-5 cells, however, these are considered priming
events in the specification of hemopoietic progenitors because they do
not impose a B-cell fate, a role reserved for the Pax-5 gene
product.18,19 The BM pro-B cell is the earliest BCP stage
defined by RT-PCR in single cells.10
CD34+CD10+CD19+ pro-B cells do not
bear rearranged IgH or L gene products, but they express enzymes
belonging to the immunoglobulin gene rearrangement and diversification
machinery (RAG-1, RAG-2, TdT) and the surrogate light chain subunits
(VpreB and  5,  L).8-10,15,16 Progression to the pre-B-cell stage is marked by the synthesis of a rearranged IgH
product. V-D-JH rearrangement is an imprecise process, and the IgH products are nonfunctional in most pre-B-cell clones and are
retained within the cytoplasm (cµH+). However, pre-BI
cells that bear in-frame productive V-D-JH transport the
µH chains to the membrane associated with L and CD79, which
signals several rounds of division.15,16 The large cycling
pre-BI cells that bear functional µH down-regulate CD34, TdT, RAG-1,
and RAG-2, and maintain expression of L, CD10, and CD19
genes.10,15,16 To allow for Ig  or L gene
recombination, RAG-1 and RAG-2 expression are reinduced in small
resting CD34CD19+CD10+ pre-BII
cells.10 Functional IgL recombination leads to the surface
expression of Ig(H+L)2/CD79 complexes that mark the pre-BII to newly formed B-cell transition.8-10
Here, single-cell multiplex RT-PCR analyses show that the
CD34+ population circulating in CB includes sizable
Pax-5+ subsets with early-B, pro-B, and pre-B cell
genotypes. Surface expression of CD19 and CD10 is markedly asynchronous
in CB CD34+ cells. Notably, the circulating
CD34+CD19+sIgH cell subset,
independently of its CD10 expression status
(CD34+CD19+CD10+sIgH
or
CD34+CD19+CD10sIgH),
includes most circulating CD34+ BCPs, whereas the
CD34+CD10+CD19sIgH
cells are essentially devoid of Pax-5+ BCP.
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Materials and methods |
Mononuclear cell purification and CD34+ progenitor
cell enrichment
Neonatal umbilical CB, obtained after informed consent from 49 donors at 38- to 41-weeks' gestation, was placed in sterile heparin-containing tubes (10 U/mL) and processed within 24 hours. Mononuclear cells were isolated by Ficoll-Hypaque gradient (density, 1.077 g/mL) (Pharmacia, Uppsala, Sweden). CD34+ progenitors
from mononuclear cells were enriched using the immunomagnetic CD34
Progenitor Cell Selection System (Dynal, Oslo, Norway), according to
the manufacturer's instructions. Cells detached from the paramagnetic beads were typically 85% to 95% CD34+ by flow cytometry.
Circulating CD34 T-cell precursors were purified as
reported.24
Flow cytometry and single-cell sorting
Surface staining10,25 of CB mononuclear cells and
CD34+-enriched cells was performed using a panel of
monoclonal antibodies including fluorescein isothiocyanate
(FITC)-labeled anti-CD19, cychrome-anti-CD34 (both from Pharmingen,
San Diego, CA), phycoerythrin (PE)-anti-CD10 (Caltag, San Francisco,
CA), and biotin-anti-human µH (SA-DA4; Southern Biotech, Birmingham,
AL) combined with streptavidin-allophycocyanin (APC) (Becton
Dickinson, San Jose, CA). FITC-labeled anti-CD3, -CD14, -CD16, and
-CD56 and PE-anti-CD4 antibodies were all from Becton Dickinson, and
CD79a was from DAKO (Glostrup, Denmark). Three- and 4-color
immunofluorescence and flow cytometry analyses were performed on an
EPICS-XL (Coulter-Beckman, Hialeah, FL) and a FACSCalibur flow
cytometer (Becton Dickinson), respectively. Four-color
immunofluorescence and single-cell sorting was performed on a
dual-laser FACStar Plus cell sorter (Becton Dickinson), using its
automated cell deposition unit (ACDU). Only cells exhibiting low
forward-angle and low right-angle scatter properties (lymphoid gate)
were analyzed and sorted when needed. Routinely, 2 × 105
events within the lymphoid gate were acquired for flow cytometry analyses. Isotype-matched antibody controls (Pharmingen) were used to
set backgrounds. Data from all flow cytometers were displayed and
analyzed using FlowJo-3 software (San Carlos, CA). There is a major
difference between the single-cell analyses performed here in CB and in
our previous study in BM.10 Here the expression profiles
of 4 CD34+sIg CB subsets were studied (ie,
CD19+CD10+,
CD19+CD10,
CD19CD10+, or
CD19CD10), whereas only the
CD10+ subpopulation was sorted in the BM.10
That experimental design difference reveals the novel
CD34+CD19+CD10 BCP population and
the distinct gene expression profile of
CD34+CD19+CD10+ and
CD34+CD19CD10+ cells.
Multiplex RT-PCR on single sorted cells
A total of 216 individual cells were analyzed in each
population, obtained from 8 different blood samples (range, 18-36 cells per donor and population). Single cells from the selected populations were placed by the ACDU into 0.2 mL PCR tubes containing 2.5 µL phosphate-buffered saline solution (PBS) and immediately
frozen on dry ice. All reactions were performed on a GeneAmp PCR System 9700 (PE Applied Biosystems, Branchburg, NJ). A 2-step strategy was
used to detect mRNA from a single cell.10 This involves an
initial one-step multiplex RT-PCR, followed by a second PCR in
individual aliquots of the initial PCR reaction that use the panel of
primers specific for each gene separately. Samples were heated (2 minutes, 65°C), then chilled on ice before the addition of the
multiplex RT-PCR reaction mix. RT-PCR reactions were performed using
SuperScript One-Step RT-PCR System (Life Technologies, Paisley, United
Kingdom). Each RT-PCR reaction consisted of one cycle of reverse
transcription (50°C, 30 minutes) and a denaturation step (94°C, 2 minutes) linked to 30 cycles of PCR amplification, each at 94°C for
20 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final
extension cycle at 72°C for 7 minutes. The multiplex RT-PCR reaction
mix included up to 8 primer pairs, specific for each gene amplified:
GAPDH, Pax-5, VpreB, TdT, RAG-1, mb-1, VH, and preT .
Oligonucleotides used for the multiplex RT-PCR were GAPDH
sense, 5'-GAAGGTGAAGGTCGGAGTC-3', GAPDH antisense,
5'-GAAGATGGTGATGGGATTTC-3'; Pax5 primers, designed by Ryan
et al,22 VpreB sense, 5'-TTTGTCTACTGCACAGGTTGTGG-3', VpreB
antisense, 5'-TGCAGTGGGTTCCATTTCTTCC-3'; RAG-1 sense,
5'-CCAAATTGCAGACATCTCAAC-3', RAG-1 antisense,
5'-CAACATCTGCCTTCACATCGATCC-3'; TdT sense,
5'-GCCGTCAGTGTGCTGGTTAAAGAGG-3', TdT antisense,
5'-TCTGCTTTGAGGAATATCCTCTTGG-3'; mb-1 sense,
5'-TCCAAGCTCTGCCTGCCACCAT-3', mb-1 antisense,
5'-GACTGCTGGTATGACTCGTTGC-3'; VH sense (VH
framework III consensus), 5'-GACACGGCCGTGTATTACTG-3', VH
antisense (CHµ), 5'-GGAATTCTCACAGGAGACGAG-3'; preT
sense, 5'-GGCACACCCTTTCCTTCTCTG-3', preT antisense,
5'-GCAGGTCCTGGCTGTAGAAGC-3'. A second PCR amplification was performed
with 1 µL first RT-PCR reaction, using the primer pair specific for
each gene in individual reactions. The second PCR consisted of 30 cycles at 94°C for 20 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final extension cycle at 72°C for 7 minutes. Nested
amplifications were used for VpreB, RAG-1, TdT and mb-1, using the
following internal oligonucleotides: VpreB antisense,
5'-GTAATACATAGCCTCGTCCTCAGG-3'; RAG-1 antisense,
5'-ACCATCCACAGGACCATGGACTGG-3'; TdT antisense,
5'-AGAATCATCTTCCGCTCATGTGTGG-3'; mb-1 antisense, 5'-AGAACTCAGGGGGCCACGTGTA-3'. PCR primers were designed to allow for
discrimination between cDNA and contaminating DNA amplification. The
method for the detection of specific DNA or RNA in single cells is an
established one.10 The RT-PCR end point of mRNA detection
was checked by limiting-dilution analyses of riboprobes (cRNA) for 8 different genes, and we found that single RNA copies are indeed
detected after optimization of the primers, RNA reverse transcription,
and DNA polymerase amplification conditions in preliminary experiments.
RNA from riboprobes and cell lines were next mixed to find that the
sensitivity was maintained in the presence of whole-cell lysates.
Finally, the primer pairs were incorporated one by one into the
reaction, and we checked that sensitivity was preserved in the
multiplex RT-PCR. Here, the GAPDH housekeeping gene was the
control for cell-sorting yield rather than the B-cell-specific gene
CD79b, used for that purpose in BM.10 When
single cells were manually deposed into tubes, under visual control all
tubes gave a positive GAPDH reaction. We programmed the FACS to depose
the single droplets calculated to contain the desired cells into the
reaction tubes under conditions optimized for purity and
"sort-abort" when neighbor droplets contained cells. Rare,
occasional tubes can, however, receive cell-free droplets, leading to a
negative reaction for GAPDH and all other genes. All PCR products were
sequenced to confirm the specificity of the gene amplifications using
dye terminator technology and an automated DNA sequencer as
indicated.10
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Results |
Cord blood CD34+ progenitor subsets express CD19, CD10,
and VpreB markers
The CD34+ population of human CB cells was analyzed
using antibodies against 2 surface antigens classically associated with the CD34+ B-lineage progenitors, CD19 and CD10. After
isolation of CB mononuclear cells by Ficoll-Hypaque gradient, 3-color
staining, and gating for lymphoid cells, analyses of the
CD34+ cells (range, 0.7%-3.5%; median, 2.58%) show that
minor subpopulations bear CD10 or CD19 molecules (Figure
1A). We also examined VpreB gene transcription in CB cell subpopulations, purified by FACS using
lineage-specific surface markers and submitted to a sensitive RT-PCR
able to detect mRNA and single-copy cDNA from single
cells.10 VpreB mRNA was readily detectable in
CD34+ cells (approximately 2 × 102 cells)
and was rare in the CD19+ B-cell lineage pool
(5 × 103 to 2 × 104 cells). It was
undetectable, however, in replicates of 104 T
(CD3+) cells, natural killer (CD16+ or
CD56+) cells, or monocytes (CD14+). VpreB was
originally considered a BCP-specific gene, but a population of mature B
cells (ie, sµH/ or positive) was recently shown to coexpress
VpreB and other genes previously used to characterize BCP.9,26 Therefore, 4-color staining with CD34, CD19,
CD10, and IgM-specific antibodies was performed to determine the
coexpression pattern of CD10 and CD19 molecules on the surfaces of
CD34+ cells and to exclude the latter sIg+ B
cells that are editing their immunoglobulin
genes26 from our BCP analyses. The contour-plot analyses
of CD19 versus CD10 expression (Figure 1B) were obtained after gating
of the lymphoid cells, selection of the surface µH-negative cell
region that excluded the sµHdull or bright populations
present in CB, and selection of the CD34+ cells.
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| Figure 1.
B-cell progenitors circulate in cord blood: flow
cytometry identification and purification of candidate populations.
(A) Three-color surface immunofluorescence studies of lymphoid cells
(forward scatter × side scatter gate) show minor CB subsets that
coexpress CD34 and either CD10 (0.5%) or CD19 (0.9%), above the
background indicated by the quadrant bars, set using isotype-matched
antibodies (less than 0.01%; see "Materials and methods"). (B)
After gating on the IgM CD34+ cells (2.57%,
top panel), the contour plot correlated distribution of CD19 versus
CD10 is shown (bottom panel). Four hemopoietic progenitor populations
(CD34+) are found in CB:
CD34+CD19CD10+ (3.3%),
CD34+CD19+CD10+ (6.7%),
CD34+CD19CD10 (74%), and
CD34+CD19+CD10 (16%). Please
note that Coulter flow cytometers display contour plots in a different
format than FACS analyzers. The data are not overcompensated. (C)
Single cells in the 4 populations are purified using the R1-R4 sorting
regions indicated in the bottom panel. CD34+ cells are
enriched before the sorting step (90%; top panel). (D) On reanalyses
to define the sort purity, most cells (more than 99.5%) in every
population fall within the predefined sort regions.
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The results demonstrate that a
CD34+CD19+CD10+
"triple-positive" population circulates in CB (range, 0.3%-18% of
the CD34+ cells; median, 12%). Two populations of
cells that bear CD34 but express either CD10 or CD19 in a mutually
exclusive manner (Figure 1B) are also evident in CB
(CD34+CD19+CD10; range,
7.7%-21%; median, 15% of CD34+ cells; and
CD34+CD19CD10+; range,
1.2%-16%; median, 3.4% of CD34+ cells).
CD79, VpreB, RAG-1, and TdT mRNA expression patterns in
CD34+CD19+CD10+/µH
cells allow for the characterization of circulating early-B- and
pro-B-cell subsets
Here we addressed the correlated study of recombined
V-D-JH, VpreB, TdT, RAG-1, CD79a, and preT mRNA
expression in individual cells belonging to 4 CD34+SµH populations that were defined by
their CD19 and CD10 expression patterns (Figure
2; Table
1). In these experiments, CB
mononuclear cells were enriched in CD34+ cells before
sorting (Figure 1C) with the use of magnetic beads coated with CD34
antibodies. The sort regions, boxes labeled R1 to R4 in the dot-plot
graphs of CD19 versus CD10 expression (Figure 1C), were drawn after
gating on the lymphoid cells, selection of the surface
µH cell region, and selection of the
CD34+ cell region, similar to the analyses shown in Figure
1B. The 4 sorted subsets,
CD34+CD19CD10+ (R1),
CD34+CD19+CD10+ (R2),
CD34+CD19CD10(R3), and
CD34+CD19+CD10 (R4) cells, were
99.5% to 99.9% pure upon reanalysis (Figure 1D). No surface
µH+, surface L+, or surface
L+ cells were present in the 4 CD34+
subpopulations thus analyzed (data not shown).
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| Figure 2.
Gene expression profiles in single cells from distinct
B-lineage stages.
Surface CD34+CD19+IgM (A) and
CD34CD19+IgM+ (B) sorted cells
are analyzed for mRNA expression of the indicated genes after multiplex
RT-PCR. In the left panel, the bands in each of the twelve tracks
(numbers 1-12), aligned in the 6 electrophoresis gels, show correlated
amplification of gene products from individual
CD34+CD19+IgM cells (ie, cell 1 is VpreB+, TdT+, RAG-1+,
mb-1/CD79+, recombined V-D-J ). The right
panel shows electrophoresis of the gene products amplified from tubes
containing titrated numbers of
CD34CD19+IgM+ B cells: tracks 1 to 6, one cell; tracks 7 to 9, 10 cells; tracks 10 to 12, 100 cells.
Similarly processed positive controls (+) correspond to individual
Nalm-6 pre-B leukemia single cells, whereas no cells are added in the
negative controls ( ). The identity of the amplified gene products is
ascertained by direct sequencing of the cDNA in the excised bands and
by molecular weight estimation (ladders). Note that the lower bands in
the V-D-J gel in panel A correspond to primer artifacts and do not
contain V-D-JH products, as determined by sequencing. L
indicates ladder.
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Table 1.
Gene expression profiles in individual cells from defined
CD34+ subpopulations circulating in neonatal cord blood
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The results indicate that VpreB is expressed in a large percentage of
CD34+CD19+ circulating hemopoietic progenitors,
whether they are CD10+ or CD10. Although
there is a clear trend toward coexpression of VpreB, RAG-1, TdT, and
mb-1 in these populations, their mRNA expression is not synchronous in
every single CD34+CD19+ cell. It is noteworthy
that one third of CD34+CD19+CD10
cells express TdT but lack VpreB, RAG-1, and mb-1 (Table 1), which is
consistent with their phenotype as early-B cells.9 The
TdT+VpreBRAG-1mb-1
early-B-cell subset is, however, rare and is not readily evident among
the CD34+CD19+CD10+ cells. Despite
the heterogeneity, the largest portion of circulating CD34+CD19+ cells, both in the CD10+
and the CD10 subset, are BCPs that coexpress the
recombination and diversification machinery enzymes (RAG-1+
and TdT+), VpreB, and the immunoglobulin receptor
transduction subunit CD79a (mb-1), although most (82%) of them do not
yet express mRNA for recombined V-D-J µH genes; all are features of
pro-B cells.9,10,15,16 In control experiments, functional
µH mRNA of different lengths is readily amplified in 100% of mature
IgM+ B cells (Figure 2B), which are homogeneously
CD79a+ but typically do not express VpreB, RAG-1 or TdT.
Intracellular staining of the
CD34+CD19+CD10+/ cells revealed
that 47% to 53% of the cells in the 2 populations bound a CD79a
antibody but that none (less than 0.5%) revealed an isotype-matched
CD3 antibody (not shown). Altogether, the results indicate that subsets
of circulating CD34+CD19+CD10+/
cells that do not express µH mRNA have features of early-B and pro-B cells.
VH-D-JH µH mRNA analyses in blood
CD34+CD19+CD10+/µH
cells show a circulating pre-B-cell subpopulation
We also characterized the minor subset of circulating
CD34+CD19+IgM cells that express
recombined VH-D-JH µH mRNA, but not surface IgM H or L chains (Figure 2A; range, 15%-28%; median, 18%
CD34+CD19+IgM cells from
individual CB samples). The result of the µH sequence analyses in
individual CD34+CD19+IgM cells
shows that two thirds of VH-D-JH µH
rearrangements are out-of-frame, rendering the µH product
nonfunctional because of stop codons. This is a feature of nonselected
pre-B cells.9,16 Five percent to 10% of
CD34+CD19+IgM cells express
cytoplasmic µH protein, but none bear Ig L protein (
and ) in immunofluorescence analyses of permeabilized
cells. These results are consistent with the translation of µH in the
5% to 10% of CD34+CD19+IgM
cells containing VH-D-JH mRNA from in-frame
rearrangements, and they define this subset as early cµH+
CD34+ pre-BI cells.9,10 The analyses of D and
JH usage indicates that the cells belong to independent
clones, and the D-JH and VH-D coding joint N
diversity shows TdT contribution to IgH hypervariable region 3 diversity.27 There is preferential JH4 usage
(data not shown), as found after birth, but not the JH or D
usage bias found in B cells with edited receptors26 or in
fetal B-cell development.28 We conclude that the CB
CD34+CD19+CD10+/ cells bearing
V-D-JH transcripts represent pre-B cells and that they
include a cµH+ cell subset as well as a subpopulation
carrying out-of-frame rearrangements that do not bear cµH protein.
CD34+CD19CD10+µH
circulating cells show a distinct gene expression profile: most cells
in this population do not bear features typical of early-B-committed
progenitors
One fifth of the
CD34+CD19CD10+ cells express the
mb-1 gene (CD79a), but, in contrast to the
CD34+CD19+CD10+/ subsets, they do
not express VpreB, RAG-1, or TdT mRNA (Table 1).
CD34+CD19CD10 cells do not
express VpreB, RAG-1, or TdT mRNA either, and only 3.3% are
CD79a+ (Table 1). To address whether the
CD34+CD19CD10+ cells that express
CD79a are committed to the B lineage, we further incorporated the
study of Pax-5 mRNA expression to the multiplex RT-PCR analyses of
the circulating CD34+sIg cells.
Interestingly, most circulating
CD34+CD19CD10+ cells do not
express Pax-5 (Figure 3; 95% or more
CD34+CD19CD10+ CB single cells
are Pax-5). In addition, unlike the BCP subsets, they are
largely VpreB, RAG-1, and
TdT, as shown in Table 1. The expression of Pax-5
is also rare in single
CD34+CD19CD10 cells. The latter
results are in stark contrast to the expression of Pax-5 in most cells
from either the CD34+CD19+CD10+ or
the CD34+CD19+CD10 BCP
populations (Figure 3). Our results show that
CD34+CD19CD10+ cells show a
distinct gene expression profile, regarding the expression of B-cell
specification and commitment genes, from CD34+CD19+CD10+/
Pax-5+ BCP subsets. Only a minor subset (less than 5%)
appears to be committed to the B lineage, as ascertained by
Pax5 gene expression.
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| Figure 3.
Pax5 expression in circulating CD34+sIg
subsets correlates with CD19 but not with CD10 surface expression.
Surface
IgMCD34+CD19CD10,
IgMCD34+CD19CD10+,
IgMCD34+CD19+CD10,
and
IgMCD34+CD19+ CD10+
sorted single cells were analyzed for mRNA expression of
Pax5 after multiplex RT-PCR. For simplicity, the results for
GAPDH, VpreB, TdT, RAG-1, and CD79a genes, covered in Figure 2 and
Table 1, are not included. In each of the 4 IgM
CD34+ subsets, 19 of 20 (95%) tracks showed a neat
GAPDH+ amplification. RT-PCR tubes receiving one FACS
droplet were labeled numbers 1 to 20. Similarly processed positive
controls (+) corresponded to individual Nalm-6 pre-B leukemia single
cells, whereas all reagents without cells were added in the negative
controls (). Results are representative of 5 similar experiments that
analyzed the 4 CB populations from independent donors. Approximately
80% of the cells showed neat Pax5 mRNA amplifications in the
IgMCD34+CD19+CD10+/
subset, but less than 5% (less than 1 of 19) cells in the
CD34+CD19CD10+/ subsets bore
Pax-5 mRNA. RT-PCR fidelity was ascertained by sequencing of the cDNA
in the excised bands and by molecular weight estimation. L
indicates ladder.
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Our multiplex RT-PCR studies of CD34+ hemopoietic cells
included the study of surrogate TCR chain (preT, Table 1) and
VpreB mRNA expression to monitor early-T versus early-B lymphoid
lineage specification, and they rule out illegitimate gene
transcription. PreT expression occurs in CD10+ early
T/DC multilineage progenitors devoid of B-lineage
potential.24,29,30 Notably, our single-cell analyses show
that the frequency of preT+ cells is low in the
CD34+CD19CD10+ cell subset (4.2%
of individual cells), very low (1.4%) in the CD34+CD19CD10cells, and
undetectable in the
CD34+CD19+CD10+/ cells. Although
this study is intended to characterize CD34+ BCP, the
results are consistent with those of previous studies in bulk
circulating preT+ "pre-T cells," which found scarce
or no expression of preT mRNA in circulating CB and adult
CD34+ cells, respectively.24,29 Notably, the
major circulating T-cell precursor population, surface
CD34TCR CB cells that uniformly
co-express preT, TdT, RAG-1, and CD3 transduction
molecules,24 do not bear VpreB mRNA (E.S., A. de la H.,
unpublished results, June 2000). Hence, VpreB and preT expression occur in separate cell subsets in both
CD34+ and CD34 cells. The 19%
CD79a+ and 4% pre-T+
CD34+CD19CD10+ cells (Table 1)
are also in different subsets, because CD79a and preT
expression occur in separate individual cells. We conclude that
CD34+CD19CD10+/ cells are a
heterogeneous population and that most cells (more than 80%-95%) are
not B-lineage-committed progenitors by available gene expression
criteria.9
| |
Discussion |
Here we identify CD34+ BCPs circulating in CB and
characterize their gene expression profiles with unprecedented
resolution using a combination of FACS and single-cell multiplex
RT-PCR. These BCPs are
CD34+CD19+CD10+/ cells that span
early-B, pro-B, and pre-B differentiation stages. B-lineage commitment
in the CB CD34+ populations is indicated by Pax5
gene expression, which correlates with CD19 but not CD10 surface
expression. The specification of B-cell development stages was
ascertained by the expression patterns of the rearranged
V-D-JH, CD79a, VpreB, RAG-1, and TdT gene products, according to widely accepted conventions.8-20
Our results show that CD34+ BCPs from BM and CB differ in
the distribution of CD19 and CD10. In BM, approximately 95% of
CD34+CD10+ cells are pro-B/pre-B cells that
coexpress CD19. A minor
CD34+CD19CD10+ subset has been
characterized as early-B cells, but the
CD34+CD19+CD10 subset is not
evident in BM.9 In a phenotypic comparison of healthy BM
and circulating CD34+ subpopulations, Bender et
al14 already noted that the proportion of
CD34+CD19+ cells tripled that of
CD34+CD10+ cells in adult peripheral blood (ie,
10% vs 3% of CD34+ cells), whereas the
CD34+CD10+ cells outnumbered the
CD34+CD19+ cells in BM (17% vs 14% of
CD34+ cells). In our analyses, more than 95% of CB cells
in the novel CD34+CD19+CD10
population are Pax-5+ early-B, pro-B, or pre-B cells.
Notably, early-B cells represent approximately 30% to 40% of
CD34+CD19+CD10 cells but are not
evident in the CD34+CD19+CD10+
population. In BM, CD10 decreases as early-B cells progress down the
B-cell development pathway,9 an additional indication that CD10 surface levels are not a good surface marker for early BCP in CB.
In BM, CD34+CD19CD10+ cells
express Pax-5, CD79, RAG-1, and TdT,9,21,22 but in CB they
are heterogeneous, lack Pax-5, RAG-1, and TdT, and express CD79a and
preT in only one fifth and one twentieth of the cells, respectively.
The latter genetic profile does not appear to be a consequence of the
separation of circulating CD34+ cells from BM stroma. In
cultures of CD34+ cells with BM stroma,15 more
than 95% of the single CD19CD10+ CB cells
express neither Pax-5 nor VpreB, TdT, CD79, RAG-1, or preT (E.S., A. de la H., unpublished results, January 2001). Early-B cells are
proposed to be the progeny of CD34+ multilymphoid
progenitors, which express CD10 in BM.9,31 Galy et
al31 described a
CD34+LinCD38+CD10+
progenitor population in BM that had T/B/NK/DC potential but was
severely depleted of myelo-erythroid potential. However, Hao et
al32 recently reported that CD10 expression alone does not discriminate between progenitors with lymphoid and myeloid potential in
CD34+CD38/lowLinCD10+
cells from CB. It is noteworthy that the populations used in the latter
report differ not only in the tissue origin (CB vs BM) but in the
selection for primitive, CD38, CD34+
progenitors (cells are Lin, CD19 in both
reports).31,32 Interestingly enough, Hao et al showed that
60% to 70% of
CD34+CD38/lowLin
CD10+ cells, which lack CD7, Pax-5, and TdT, contain
progenitors that retain both myelo-erythroid and lymphoid potential in
clonal analyses of single cells. Herein we study CD34+ CB
cells that are more than 95% CD38+. Our preliminary
analyses of the distribution of CD38 show that it is heterogeneous in
distinct CD34+ CB subsets. Whereas approximately 95% of
CD34+CD19+CD10+/ cells in our
samples were CD38+, two thirds of the
CD34+CD19CD10+ cells were
CD38/low and one third were CD38+ (not
shown). Further studies are needed to more extensively readdress the
gene expression profiles and multipotency of CB
CD34+CD19CD10+ cells in
single-cell assays. The
CD34+LinCD10+ phenotype has been
operationally used before to identify BCPs, such as circulating and
BM-mobilized BCPs.2 It appears now safe to consider that
CD34 and CD10 alone do not discriminate among multilineage,
multilymphoid, or B-lineage progenitors in cord blood and that CD10
will have to be combined with several other surface markers to define
the putative hemopoietic stage and fate.32
The finding of circulating early-B, pro-B, and pre-B cells appears
striking because it is in stark contrast to 3 concepts that have
dominated the thinking about B-cell development.11-13 One
postulates that BCPs are retained in the BM until they acquire surface
immunoglobulin and that the emigrants to peripheral blood are immature
IgM+ B cells. The second idea is that BCPs may gradually
die if they are deprived of the "protective" signals delivered by
microenvironment niches such as BM. The third notion is that such
protective BM niches play an essential role during IgM receptor editing
and selection for tolerance to self in pre-B/immature B cells. Recent experimental evidence partly challenges the paradigm, however, because
of the following. First, mouse BM CD19+Vpre-B+
Pro-B/pre-BI cells placed in single-cell cultures to isolate them from
the BM environment proliferate spontaneously and differentiate efficiently into sIg+ immature B cells.33
Second, when BCPs are mobilized into blood in CXCR4/ KO
mice, they survive and continue to generate B cells.11
Similarly, analyses of sorted CD34+ CB cells cocultured
with BM stroma15 show that early-B
CD19+CD10 cells differentiate into
CD19+CD10+ pro-B/pre-B cells that generate B
cells (E.S., A. de la H., unpublished data, July 2001). Third,
the expression of VpreB, RAG, and TdT has been reported in circulating
B cells, which undergo secondary V(D)J rearrangements to edit their
sIg/CD79 antigen receptor specificity.26 The latter mature
B cells are different from the CB BCPs in that they lack CD34 and CD10
and that they homogeneously express productive µH mRNA message and
surface and cytoplasmic H and L.26,34 Taken together,
available evidence indicates that circulating BCPs may be biologically
relevant. The presence of BCPs in CB poses the question on the origin
and sIg repertoire selection mechanisms for circulating
RAG+ B-lineage cells.
The mosaic expression of genes (variegation) reported here in
CD34+ early-B and pro-B cells are consistent with current
stochastic/selective and hybrid selective/instructive models of
lymphoid development.19,35,36 As individual cells progress
down the differentiation pathway, their B-lineage specification pattern
shows a progressive fit with the genotype proposed after bulk
population analyses of BCP stages,9,15-19 as best
exemplified by mature B cells that show a homogeneous gene expression
profile consistent with the prevalent deterministic patterns. Notably,
the CD34+CD19+CD10+ CB population
reported here resembles the population referred to as pro-B/pre-BI
cells after single-cell analyses in BM.10 We characterize
a novel CD34+CD19+CD10 BCP not
considered in normal B-cell development
schemes8-10,15 21-23 but long ago
implicated in BCP cancer.37-40 Acute lymphoblastic leukemias (ALLs) are classified in subtypes considered to indicate the
stage of development at which tumor transformation
occurs.9,38 Notably, the CD10 progenitor
B-ALL subtype occurs in infants and typically bears the
CD34+CD19+CD10 genotype/phenotype
discovered here in CB.39,40 We show that healthy BCP
counterparts for the common CD10+CD19+ pro-B,
pre-B, and B-cell ALL9,25,40 also circulate in CB. Interestingly, we find that
CD34+CD19CD10+ early-B cells are
rare or do not circulate in CB, and a clonogenic leukemic counterpart
for healthy CD34+CD19CD10+
early-B cells characterized in fetal liver and BM9,41 was not found among malignant cells from B-ALL patients
either.41 In B-ALL, BCPs experience an initiating
translocation event in utero, circulate in newborn blood as preleukemic
cells, and can remain in the circulation several years before a second
mutation event promotes the conversion of the fetal preleukemic clone
into a leukemia.37,38 The observation that healthy BCPs
circulate in the CB of healthy persons may provide a physiologic
mechanism for the blood-borne dissemination of fetal preleukemic
cells.42 B-ALL represents 25% of all childhood cancers,
and chromosomal translocations are implicated in the ALL
pathogeny.37-40 Here we show that a significant fraction
of healthy CB CD34+ cells express RAG. RAG can mediate
translocation by transpositional mechanisms,41 which
raises concern regarding the potential risk for chromosomal
translocation36-38,43 in reparative medicine protocols that involve gene manipulation in CD34+
cells.4 Studies are under way to experimentally address
this possibility.
| |
Acknowledgments |
We thank M. A. R. Marcos and M.-L. Gaspar for critical review of
the manuscript, J. Monserrat for superb sortings, V. Parrillas and the
nursing staff at the San Francisco de Asís Hospital for assistance with cytoplasmic staining and for obtaining cord blood samples, and C. Mark for editorial assistance.
The Department of Immunology and Oncology was founded and is supported
by the CSIC and by Pharmacia Corporation.
| |
Footnotes |
Submitted July 24, 2002; accepted October 29, 2002.
Prepublished online
as Blood First Edition Paper, November 21, 2002; DOI
10.1182/blood- 2002-07-2244.
Supported by the Comisión Interministerial de Ciencia y
Tecnología grants 2FD97-2226, SAF-2001-2453 and
GEN2001-4856-C13. E.S. is supported by the Ministry of Science and
Technology as a Ramón y Cajal Research Scientist.
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: Antonio de la Hera, Laboratory of Immunology
and Oncology, CSIC Associated Unit, Facultad de Medicina, Universidad
de Alcalá, Alcalá de Henares, E-28871 Madrid, Spain;
e-mail: adelahera{at}cib.csic.es.
| |
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Top
Abstract
Introduction