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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3781-3790
Lymphoid-Restricted Development From Multipotent Candidate Murine Stem
Cells: Distinct and Complimentary Functions of the c-kit and
flt3-Ligands
By
Ole Johan Borge,
Jörgen Adolfsson, and
Annica Mårtensson,
Inga-Lill Mårtensson, and Sten E.W. Jacobsen
From the Stem Cell Laboratory, the Department of Molecular Medicine
and Gene Therapy, the Department of Internal Medicine, Institute for
Laboratory Medicine, University Hospital of Lund, Lund, Sweden; The
Scripps Research Institute, La Jolla, CA; and Developmental Immunology,
The Babraham Institute, Babraham, Cambridge, UK.
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ABSTRACT |
The two tyrosine kinase receptors, c-kit and flt3, and their
respective ligands KL and FL, have been demonstrated to play key and
nonredundant roles in regulating the earliest events in hematopoiesis.
However, their precise roles and potential interactions in promoting
early lymphoid commitment and development remain unclear. Here we show
that most if not all murine
Lin /loSca1+c-kit+ bone
marrow (BM) cells generating B220+CD19+
proB-cells in response to FL and interleukin-7 (IL-7) also have a
myeloid potential. In contrast to FL + IL-7, KL + IL-7 could not
promote proB-cell formation from
Lin/loSca1+c-kit+ cells.
However, KL potently enhanced FL + IL-7-stimulated proB-cell formation, in part through enhanced recruitment of FL + IL-7-unresponsive Lin/loSca1+c-kit+
progenitors, and in part by enhancing the growth of proB-cells. The
enhanced recruitment (4-fold) in response to KL occurred exclusively from the
Lin/loSca1+c-kit+flt3
long-term repopulating stem cell population, whereas KL had no effect
on FL + IL-7-stimulated recruitment of
Lin/loSca1+c-kit+flt3+
short-term repopulating cells. The progeny of FL + IL-7-stimulated Lin/loSca1+c-kit+ cells
lacked in vitro and in vivo myeloid potential, but efficiently reconstituted both B and T lymphopoiesis. In agreement with this FL,
but not KL, efficiently induced expression of B220 and IL-7 receptor- on
Lin/loSca1+c-kit+flt3+
cells. Thus, whereas KL appears crucial for recruitment of FL + IL-7-unresponsive candidate (c-kit+flt3)
murine stem cells, FL is essential and sufficient for development toward lymphoid restricted progenitors from a population of
(c-kit+flt3+) multipotent short-term
reconstituting progenitors.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE PROCESS BY WHICH multipotent
progenitors differentiate and commit into lymphoid restricted
progenitors is currently poorly understood. An important requisite to
be able to dissect the molecular mechanisms involved in this process is
to identify and characterize the earliest stages of lymphoid
development. Studies of mice deficient in Ikaros expression have
demonstrated a selective loss of natural killer (NK)-, T-
and B-lymphoid development implicating the potential presence of a
common lymphoid progenitor cell (CLP).1 Recent data support
the existence of such a CLP in adult murine bone marrow
(BM).2 Specifically, single
LinSca1loc-kitloIL-7R+
BM cells, lacking detectable myeloid potential, were demonstrated to
give rise to both T and B cells. In addition, others have characterized in detail the phenotype of the earliest cells committed to the B-lymphoid lineage.3-7 However, our knowledge regarding the
potential function of early acting cytokines in promoting development
from candidate stem cells with a combined myeloid and lymphoid
differentiation potential to these earliest stages of lymphoid
restricted progenitors remains limited.
Lin/loSca1+c-kit+ cells,
although representing only approximately 0.05% of adult murine BM
cells, contain virtually all long-term reconstituting stem cells (LTRC)
and represent a pure population of multipotent progenitor
cells.8-13 Whereas c-kit appears to be critically involved
in promoting sustained self-renewal and maintenance of
LTRC,14,15
Lin/loSca1+c-kit+ cells
expressing flt3 have been shown to contain less LTRC than the
flt3 subpopulation.16 Interestingly,
whereas mice deficient in flt3-receptor expression have normal numbers
of all mature cell lineages, they have a selective reduction in the
number of pro and preB-cells.17 In addition,
transplantation experiments with BM cells from flt3-deficient mice also
showed a defect in the T-cell compartment, indicating a role for flt3,
and its ligand, in early lymphoid development.17 Although
specific lymphoid defects have not been reported in c-kit-deficient mice, this might be more difficult to show because c-kit-deficient mice have severe deficiencies in their stem cell
pools.18,19 However, c-kit has been demonstrated to be
expressed in the earliest stages of B- and T-cell progenitors as well
as the CLP, and its ligand implicated in promoting early lymphoid
development.2,19-25 Specifically, both c-kit-ligand (KL)
and flt3-ligand (FL) have been shown to act as growth factors for early
B-lymphoid progenitors, and it has been clearly demonstrated that KL
can stimulate proliferation of proB, as well preB-cells, in synergy
with interleukin-7 (IL-7).19,21,22,26-29 Much less effort
has been devoted to establish to what degree KL and FL might also
promote the transition from multipotent progenitors to CLP and
subsequently to the earliest proB-cells. Whereas our previous studies
have suggested that multipotent
Lin/loSca1+c-kit+ BM cells
stimulated with FL + IL-7, (but not KL + IL-7) generate a virtually
pure population of proB-cells,30 others have suggested that
KL might promote such commitment of primitive progenitors in the BM, as
well as fetal liver.6,27,31-34 Thus, the present studies
were initiated to clarify and further dissect the roles of KL and FL in
promoting lymphoid commitment.
Here we present data on the ability of FL and KL to promote early
lymphoid development from multipotent progenitors, resolving previously
seemingly contradicting results. Importantly, despite coexpression of
flt3 and c-kit, FL is superior to KL at promoting growth, as well as
lymphoid development of
Lin/loSca1+c-kit+flt3+
cells. However, KL also has a distinct and dual effect on early lymphoid development by first allowing recruitment of FL-nonresponsive Lin/loSca1+c-kit+flt3
cells (containing most, if not all, LTRC) to generate B-lymphoid progenitors and secondly by synergistically enhancing IL-7-dependent growth of committed proB-cell progenitors. Thus, FL and KL appear to
have distinct, but complimentary roles, in promoting the earliest stages of murine lymphoid-restricted development.
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MATERIALS AND METHODS |
Hematopoietic growth factors.
Recombinant human (rh) IL-7 and recombinant murine (rm) IL-3 were from
Peprotech (Rocky Hill, NJ). Recombinant rat (rr) KL (c-kit-ligand, stem cell factor), rhMGDF (megakaryocyte growth and
development factor, thrombopoietin), rmGM-CSF (granulocyte-macrophage colony-stimulating factor) and rhG-CSF (granulocyte colony-stimulating factor) were generously provided by Amgen Corp (Thousand Oaks, CA),
whereas rhFL was a kind gift from Immunex (Seattle, WA). RhEpo
(erythropoietin) was provided by Boehringer Mannheim Corp (Mannheim,
Germany). All growth factors were used at the following predetermined
optimal concentrations: rhEpo 5 U/mL, rhFL 50 ng/mL, rhG-CSF 50 ng/mL,
rmGM-CSF 20 ng/mL, rmIL-3 10 ng/mL, rhIL-7 100 ng/mL, rrKL 50 ng/mL,
and rhMGDF 50 ng/mL.
Enrichment and purification of subpopulations of
Lin/loSca1+ BM cells based on expression
of c-kit and flt3.
Lineage-depleted (Lin/lo) BM cells were isolated
from 6- to 10-week-old C57Bl/6 mice (Ly5.2) from M&B (Ry, Denmark) or
B6SJL mice (Ly5.1) from Jackson Laboratories (Bar Harbor, ME). In some experiments cells were also isolated from transgenic mice expressing hCD25 under control of the  5-promoter.35 Briefly, femurs
and tibias were gently crushed in a mortar. Iscove's Modified
Dulbecco's Medium (IMDM; BioWhittaker, Walkersville, MD) supplemented
with 5% fetal calf serum (FCS; BioWhittaker) was used as medium
throughout the isolation. The cell suspension was filtered through a
70-µm mesh filter (Falcon, Becton Dickinson, Lincon Park, NJ), white blood cells counted in a hemacytometer, and concentrated to 400 × 106 cells/mL. The cells were incubated at 4°C for 30 minutes in a cocktail of predetermined optimal concentrations of
unconjugated antibodies: B220 (RA3-6B2), Gr-1 (RB6-8C5), Mac-1 (M1/70),
CD8 (53-6.7), CD5 (53-7.3), CD4 (H129.19), and TER-119 (all from
PharMingen, San Diego, CA).
Cells were washed once, resuspended to 250 × 106
cells/mL, and sheep antirat IgG (Fc)-conjugated immunomagnetic beads
(Dynal, Oslo, Norway) were added at a cell:bead ratio of 1:0.3 and
incubated at 4°C for 45 minutes on a mixing wheel. Magnetic beads
were removed with a magnetic particle concentrator (MPC-6, Dynal), and
unattached cells transferred to a second tube containing the same
absolute amount of magnetic beads, incubated for 30 minutes, and
processed as in the first bead separation.
Lin/lo cells recovered from the supernatant were
further purified based on the expression of stem cell antigen-1 (Sca1)
and c-kit as previously described.9,12,13,36 Briefly,
Lin/lo cells were resuspended at 100 to 400 × 106 cells/mL and incubated for 30 minutes on ice with a
phycoerythrin (PE)-conjugated goat antirat antibody (Southern
Biotechnology, Birmingham, AL). Subsequently, cells were washed and
stained with a fluorescein isothiocyanate (FITC)-conjugated rat
antimouse Ly-6A/E antibody (Sca1), and allophycocyanin (APC)-conjugated
anti-CD117 (c-kit) antibody or isotype-matched control antibodies (all
from PharMingen). The cells were washed and
Lin/loSca1+c-kit+ cells were
sorted on a FACSVantage Cell Sorter (Becton Dickinson, San Jose, CA),
equipped with an 488 nm argon and a 633 nm He-Ne laser, at a rate of
1,000 to 4,000 cells/second. Only cells expressing high, not low or
intermediate, levels of c-kit were sorted. Practically all cells
recovered after the magnetic bead isolation coexpressing Sca1 and c-kit
fell within the Lin/lo region (O.J.B. and S.E.W.J.,
unpublished observations). Reanalysis of sorted
Lin/loSca1+c-kit+ cells on a
FACSCalibur (Becton Dickinson) showed reproducibly a purity of 95% to
99%. In some experiments
Lin/loSca1c-kit,
Lin/loSca1+c-kit and
Lin/loSca1c-kit+
cells were sorted as well.
Lin/loSca1+c-kit+ cells were
subdivided into flt3+ (20% to 25% highest expressing
cells) and flt3 (those with anti-flt3
PE-fluorescence less than 20% of the maximum PE-fluorescence of the
isotype control) using Sca1-FITC, c-kit-APC, and flt3-PE (PharMingen) antibodies.
Single-cell proliferation assay.
Lin/loSca1+c-kit+ cells were
seeded in Terasaki plates (Nunc, Kamstrup, Denmark) at a concentration
of 1 cell per well in 20 µL of serum-depleted medium, as previously
described.37 The serum-depleted medium (X-vivo 15;
BioWhittaker) was supplemented with 1% detoxified bovine serum albumin
(BSA; StemCell Technologies, Vancouver, Canada), 100 U/mL penicillin
(BioWhittaker), 100 U/mL streptomycin (BioWhittaker), 3 mg/mL
L-glutamine (BioWhittaker), and freshly made 1 × 104 mol/L 2-mercaptoethanol (Sigma, St Louis, MO).
In some experiments the presence of single cells was verified by
microscopy 2 to 12 hours after plating and only wells containing 1 cell
were included. Wells were scored for cell growth ( 3 cells) after 10 to 12 days of incubation at 37°C and 5% CO2 in
humidified air. Individual colonies (covering more than 10% of the
well) were sampled and either transferred to slides using a cytospin
centrifuge (Shandon, Cheshire, UK) to be examined morphologically after
Giemsa-staining (Sigma) or stained with antibodies and analyzed by flow
cytometry (FACSCalibur, Becton Dickinson).
Semisolid clonogenic assays.
Colony-forming unit-granulocyte macrophage (CFU-GM) potential was
evaluated using semisolid culture conditions. The cells were plated in
duplicate in 1 mL IMDM with 20% FCS, 1.2% methylcellulose (MC;
Methocel, Fluka Chemie, Switzerland), L-glutamine,
penicillin/streptomycin, and 1 × 104 mol/L
2-mercaptoethanol, and supplemented with cytokines in 35-mm petri
dishes. Cultures were incubated at 37°C and 5% CO2 in
humidified air for 7 to 9 days, at which time colonies (>50 cells)
were visualized and scored using an inverted microscope.
3H-thymidine incorporation assay was used to evaluate the
proliferation potential of the progenitors derived from
Lin/loSca1+c-kit+ cells
cultured for 7, 12, and 17 days in the presence of FL + IL-7 or KL + FL + IL-7. Cells were washed 3 times to remove cytokines present during the primary culture, counted, and plated in triplicate in round bottomed 96-well plates at 5 to 40 × 103
cells per well in 100 µL serum-depleted medium, and cultured for 3 additional days before being pulsed with 1 µCi
3H-thymidine (Nycomed Amersham, Buckinghamshire, UK). After
an additional 12 hours incubation, the cells were harvested using a
Scatron cell harvester (Drammen, Norway), and the amount
incorporated 3H-thymidine determined using a scintillation
counter (LKB Wallac, Turku, Finland).
Immunophenotyping by flow cytometry was performed as previously
described30 with specified antibodies (all from PharMingen) conjugated directly to biotin, FITC, PE, or APC used at predetermined optimal concentrations. Antibodies conjugated to biotin were
subsequently stained with streptavidin-PerCP (Becton Dickinson).
In vivo reconstitution experiments.
A total of 1,000 Lin/loSca1+c-kit+ freshly
isolated or in vitro-cultured cells originating from 1,000 Lin/loSca1+c-kit+ cells was
intravenously injected in the tail vein (0.5 mL per mice) of lethally
irradiated (950 rad) C57Bl/6 (Ly5.2) mice. All mice were kept in
individually ventilated cages throughout the experiment and given
sterile food and autoclaved acidified water. Irradiated C57Bl/6 mice
were cotransplanted with 150,000 unfractionated syngeneic
(Ly5.2) BM cells to provide a competitor and survival population.
Peripheral blood cells were collected after 6 and 10 weeks, red blood
cells lyzed with ammonium chloride, and the white blood cells stained
with antibodies against Ly5.1, Ly5.2, and lineage-specific antigens
(all from PharMingen), and subsequently analyzed on a FACSCalibur.
Statistical analysis.
Student's t-test was used for statistical analysis.
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RESULTS |
High-efficiency lymphoid-restricted development in response to FL and
IL-7 is exclusively observed from the multipotent
Lin/loSca1+c-kit+ stem cell
population.
Whereas multiple studies have demonstrated that FL when acting in
combination with IL-7 can promote growth of early B- and T-cell
progenitors,23,27-29,34,38,39 much less is known about the
potential ability of FL + IL-7 to promote lymphoid commitment of
progenitor/stem cells with a combined myeloid and lymphoid development
potential. In previous studies we had demonstrated that FL + IL-7
could efficiently recruit approximately 10% of Lin/loScal+ BM cells into
proliferation.30 Furthermore, a high fraction of these FL + IL-7-recruited progenitor cells had myeloid potential, and although
some of the resulting colonies were too small to analyze, most of them
appeared to have B-lymphoid potential as well, suggesting that they at
least in part were derived from cells with combined myeloid and
lymphoid potential.30 Recent and further optimization of
the conditions for optimal myeloid as well as B-lymphoid development
from single
Lin/loSca1+c-kit+ BM cells
now allowed us to demonstrate more unequivocally that FL + IL-7-responsive
Lin/loScal+c-kit+ BM cells
have a combined lymphoid and myeloid lineage potential, and also
establish to what extent they have such a mixed lineage potential.
Toward this aim, a number of single-cell cloning experiments were
initiated. In the first set of experiments, stimulation with a
combination of myeloid growth factors (KL + FL + IL-3 + G-CSF + GM-CSF + MGDF + EPO) showed that as much as 95% (±2%) of
Lin/loSca1+c-kit+ cells
(individually deposited and identified) had a high proliferative and
myeloid potential (Table 1). In the same
experiments, 42% (±1%) of the
Lin/loScal+c-kit+ progenitor
cells proliferated in response to FL + IL-7, and resulting clones large
enough to be analyzed by flow cytometry (63% ± 4%), all showed a
proB-cell phenotype (Table 1). Accordingly, these studies clearly
established that most if not all
Lin/loScal+c-kit+ progenitor
cells that develop into proB-cells, in response to FL + IL-7, have a
combined in vitro B-lymphoid and myeloid potential.
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Table 1.
Single
Lin/loSca1+c-kit+ Cells
Undergoing Lymphoid-Restricted Development in Response to FL + IL-7
Have a Combined Myeloid and Lymphoid Differentiation Potential
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Previously, we had shown that FL + IL-7 are very efficient at promoting
formation of high numbers of B-lymphoid restricted progenitors from
Lin/loSca1+c-kit+ progenitor
cells.30 Because cells with this phenotype represent only
approximately 0.05% of the total BM cells and have been demonstrated to contain most if not all pluripotent stem cells,9,11-13
we now investigated whether FL + IL-7 also could promote such
high-efficiency proB-cell formation from other early
(Lin/lo) progenitor cell populations. If so, it
could not be excluded that the high output B-lymphoid production from
the isolated
Lin/loScal+c-kit+ progenitor
cells could be derived from contaminating cells of more abundant, but
less primitive, progenitor cell populations. Interestingly, although
representing only 2% of the total Lin/lo
population, the
Lin/loSca1+c-kit+ population
was the only Lin/lo population, which reproducibly
could produce B220+CD19+ proB-cells in response
to FL + IL-7. Thus, multipotent
Lin/loSca1+c-kit+ BM
progenitor cells are the primary targets for FL + IL-7-stimulated in
vitro proB-cell formation (Table 2).
B220+CD19 cells derived from
multipotent
Lin/loSca1+c-kit+ progenitor
cells develop almost exclusively into B-lymphoid committed progenitor
cells.
Stimulation of
Lin/loScal+c-kit+ cells by
FL + IL-7 results in generation of 2 populations of B220+
cells, 1 expressing CD19 and the other lacking CD19 expression (Fig 1).36 Whereas expression
of CD19 is believed to be B-lymphoid-specific, B220 is
not.5,40 Thus, although previous reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis of
Lin/loSca1+c-kit+-derived
B220+CD19 cells had indicated that these
cells contained B-lymphoid-committed progenitor cells,36
it remained possible that most of the
B220+CD19 cells produced in response to
FL + IL-7 were not yet committed to the B-lymphoid lineage.
Thus, in the present studies, we used transgenic mice expressing hCD25
under control of the 5-promoter to investigate the timed expression
of hCD25 as an indicator of B-lymphoid-specific 5-expression in
B220+CD19+ and
B220+CD19 cells derived from
Lin/loScal+c-kit+ (all
hCD25/5) progenitor cells in response to FL+IL-7.
B220CD195
cells dominated early in culture, but these were gradually replaced by
B220+CD19 cells (almost 40% of cells at
day 3), which initially were 5, but eventually
(after 12 days) became almost exclusively 5+, suggesting
commitment to the B-lymphoid lineage. Finally, and as expected,
B220+CD19+ cells appeared later (8 days) in
culture and were all 5+. Thus, FL+IL-7 promote formation
of B220+CD19 as well as
B220+CD19+-committed proB-cell progenitors from
multipotent
Lin/loScal+c-kit+ progenitor
cells.
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| Fig 1.
Timed expression of 5, B220, and CD19 on
Lin/loSca1+c-kit+ cells
cultured in FL + IL-7. Cultures were initiated with
Lin/loSca1+c-kit+ (all
hCD25) cells isolated from transgenic mice expressing
hCD25 under control of the 5-promoter.35 Cells were
supplemented with FL + IL-7, and at each indicated time point, cells
were analyzed for the coexpression of B220, hCD25(5), and CD19 by
flow cytometry. One representative experiment of 3 individual
experiments is shown. In initial control experiments in which
hCD25 and hCD25+ cells were sorted from FL + IL-7-stimulated cultures, endogenous 5 mRNA was detected by
RT-PCR in the hCD25+ fraction, but not in the
hCD25 fraction, demonstrating the specificity and
sensitivity of hCD25 expression as a marker for 5 expression (data
not shown).
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c-kit activation in combination with FL + IL-7 is essential for
efficient B-lymphoid development from candidate murine stem cells.
Our previous studies had suggested that FL was far superior to KL at
promoting in vitro lymphoid development from
Lin/loSca1+c-kit+ BM
progenitor cells.30 In seemingly contrast, a number of
other studies suggested that KL had an ability to enhance growth of early B-lymphoid progenitors, and thus potentially also lymphoid development from candidate murine stem
cells.6,19,21,22,27,31-34 However, the level of commitment
of the KL-responsive progenitors remained unclear in these studies. In
an effort to better uncover and resolve the effects of KL on the
earliest stages of lymphoid commitment and development, in particular
in relationship to the effects mediated by FL, we investigated to what
degree KL might enhance the ability of FL + IL-7 to promote B-lymphoid
development from
Lin/loSca1+c-kit+ progenitor cells.
In agreement with our previous studies,30 a 292-fold
cellular expansion was observed when
Lin/loSca1+c-kit+ BM cells
were cultured for 12 days in the presence of FL + IL-7 (Fig 2) and 55% of these showed a
proB-cell phenotype (B220+CD19+). In contrast,
in cultures stimulated with KL + IL-7, none of the cells produced by
day 12 had a B220+CD19+ phenotype and only 6%
of the cells were B220+CD19. Rather,
most of the cellular expansion (293-fold) observed in response to KL + IL-7 resulted from production of myeloid cells (41% ± 11%
Gr1/Mac1+). In agreement with this, single-cell cloning
experiments with Lin/loSca1+c-kit+ cells
never showed any clonal formation (0% ± 0%) of
B220+CD19+ cells in response to KL + IL-7, whereas as many as 26% of the Lin/loSca1+c-kit+ cells
generated large proB-cell clones in response to FL + IL-7 (Table 1).
Combined stimulation with KL and FL resulted in a 148-fold cell
expansion by 12 days, but as for KL + IL-7-stimulated cultures, none
of these cells expressed the B-lymphoid-specific marker, CD19 (Fig 2),
and were rather predominantly Gr1/Mac1+ (42% ± 8%).
However, when combined with FL + IL-7, KL enhanced cell expansion
5.3-fold (Fig 2), and although 4% ± 3% of these cells expressed
Gr1/Mac1, most were B220+CD19+, suggesting that
the growth-promoting effect of KL was predominantly mediated through an
increase in proB-cell formation. A more detailed cell surface
phenotyping after 17 days of culture showed a similar proB-cell
phenotype of cells generated in KL + FL + IL-7 as in FL + IL-7
(Table 3), with a fraction of the
B220+CD19+ cells becoming BP-1+ by
day 17, but with no cells expressing cytoplasmic (or cell surface) IgM.
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| Fig 2.
KL enhances FL + IL-7-stimulated proB-cell formation
from Lin/loSca1+c-kit+
progenitors. A total of 500 Lin/loSca1+c-kit+ cells/mL
was cultured in the presence of the indicated cytokines. After 12 days
of culture, total cells were counted and analyzed for expression of
B220 and CD19. The B220+ and CD19+ bars
indicate the fraction of total cells represented by B220+
and CD19+ cells, respectively. Data presented are the
mean (+standard error of mean [SEM]) of 4 individual experiments.
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Table 3.
Phenotypic Characterization of Day 17 Progeny of FL + IL-7 and KL + FL + IL-7-Stimulated
Lin/loSca1+c-kit+ Cells
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The potent ability of KL to enhance FL + IL-7-stimulated proB-cell
formation (Fig 2) could either be the result of enhanced proliferation
of cells already committed to the B-lymphoid lineage in response to FL + IL-7, and/or it could reflect recruitment of an increased number of
uncommitted
Lin/loSca1+c-kit+ cells
toward B-lymphoid development. Single-cell cloning experiments with
Lin/loSca1+c-kit+ cells
showed that KL enhanced FL + IL-7-stimulated formation of proB-cell
clones by 33% (O.J.B. and S.E.W.J., unpublished
observations). We next addressed whether this rather
limited increase in proB-cell development was a result of enhanced
recruitment from the
Lin/loSca1+c-kit+flt3+
and/or
Lin/loSca1+c-kit+flt3
subpopulations. Such a distinction is of considerable importance, as only the flt3 subpopulation of
Lin/loSca1+ cells has been
demonstrated to be highly enriched in LTRC.16 In agreement
with this,
Lin/loSca1+c-kit+flt3+
cells isolated in the present studies provided efficient short-term (4 weeks), but little long-term reconstitution (6 months) in lethally irradiated recipients, whereas
Lin/loSca1+c-kit+flt3
cells were highly enriched in LTRC (O.J.B. and S.E.W.J., unpublished observation). In support of the studies on the whole
Lin/lo Sca1+c-kit+
population, neither flt3+ nor flt3 cells
showed any proB-cell formation in response to KL + IL-7 (Fig 3). As expected, virtually all of the
FL + IL-7 responders were located in the flt3+ population,
resulting in as much as 70% of the flt3+ progenitors
forming proB-cell clones (Fig 3). This response could not be further
enhanced by KL. The successful isolation of a flt3
population was supported by as little as 3% of these progenitors forming proB-cell clones in response to FL + IL-7 (Fig 3).
Interestingly, KL enhanced FL + IL-7-stimulated proB-cell colony
formation almost 4-fold in the flt3 subpopulation
(Fig 3; P < .01). Thus, importantly, whereas FL + IL-7
efficiently promote B-lymphoid development from a
flt3+-population of short-term reconstituting stem cells,
KL appears important for early B-lymphoid development from the
Lin/loSca1+c-kit+flt3
population containing most if not all LTRC.
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| Fig 3.
KL, as well as FL, are required for optimal proB-cell
development from flt3 candidate murine stem cells.
Lin/loSca1+c-kit+ cells were
sorted into flt3+ and flt3 populations and
cultured in the presence of the indicated cytokines at a density of 1 cell per well. Fifteen to 25 days after initiation of culture,
individual colonies were picked and analyzed by flow cytometry to
verify the presence of proB-cells, as defined by combined B220 and CD19
expression. No proB-colonies were found in response to KL + IL-7.
Results represent the mean (+SEM) of 4 individual experiments. Cells
stimulated with an optimal combination of cytokines (KL + FL + MGDF + IL-3 + G-CSF + GM-CSF + Epo) served as a control for total
number of in vitro clonogenic progenitors. Three independent
single-cell experiments with flt3+ and
flt3 cells showed a high cloning frequency, 98 (1)% and
97 (1)%, respectively. Thus, the data are presented as percent
B-lymphoid colonies of the total number of clonogenic progenitors.
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Delayed addition studies were next performed to determine to what
degree FL, KL, and IL-7 were required to promote B-lymphoid development
from Lin/loSca1+c-kit+ cells
at an early stage or whether they might act in a sequential manner.
Delaying addition of IL-7 to FL-stimulated cultures for as little as 64 hours resulted in reduced formation of proB-cells by as much as 38%,
whereas a 24- or 48-hour delay had no effect (Fig 4). Thus, FL-stimulated
Lin/loSca1+c-kit+
progenitors become IL-7 responsive at an early stage of development. Interestingly, other studies demonstrated early (by 40 hours) requirements for KL as well as FL activation for optimal proB-cell formation from the
Lin/loSca1+c-kit+flt3
population (Table 4), suggesting that KL
might rapidly induce FL responsiveness in the
Lin/loSca1+c-kit+flt3
stem cell population.
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| Fig 4.
Requirement for IL-7 during early lymphoid development
from Lin/loSca1+c-kit+
progenitors. A total of 500 Lin/loSca1+c-kit+ cells/mL
was cultured in the presence of FL from initiation of culture. IL-7 was
added to the cultures at the indicated time points, whereas KL was
added to all groups 64 hours after initiation of culture. Total cell
expansion was evaluated 12 days after initiation of culture. Each data
point represents the mean (+SEM) of 3 individual experiments with
duplicate wells in each experiment.
|
|
View this table:
[in this window]
[in a new window]
|
Table 4.
Requirement for Early KL and FL Activation for Optimal
ProB-Cell Formation From
Lin/loSca1+c-kit+flt3
Cells
|
|
KL enhances FL + IL-7-stimulated proliferation of lymphoid-committed
progenitor cells derived from
Lin/loSca1+c-kit+ progenitor
cells.
Whereas 63% of fresh
Lin/loSca1+c-kit+ cells
formed myeloid colonies in methylcellulose, only 0.1% of the cells
generated after 12 days in FL + IL-7 maintained such an ability
(Fig 5A). In comparison, the frequency of
FL + IL-7-derived cells capable of forming proB-cell colonies was
260-fold higher than those with a myeloid potential and almost as high
as in the starting
Lin/loSca1+c-kit+ population
(Fig 5B).
View larger version (18K):
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| Fig 5.
In vitro myeloid and lymphoid potential of progeny
derived from FL + IL-7-stimulated
Lin/loSca1+c-kit+ cells. A
total of 500 Lin/loSca1+c-kit+ cells/mL
was cultured in the presence of FL + IL-7 for 12 days, at which time
cells were washed and either transferred to myeloid MC cultures
(duplicates) supplemented with G-CSF + GM-CSF + IL-3 + KL (A) or
plated at a density of 1 cell per well in serum-depleted medium
supplemented with FL + IL-7 (B). The CFU-GM content was analyzed 7 to
10 days after transfer and the proB-cell potential was evaluated (as
described in Fig 3) after 12 to 20 days of culture. The results
represent the mean (+SEM) of 3 individual experiments.
|
|
Because these studies clearly showed an almost exclusive development of
lymphoid-restricted progenitors in response to FL + IL-7, we next
investigated the cytokine responsiveness of these progenitors. Cells
derived from
Lin/loSca1+c-kit+ cells in
response to FL + IL-7 were washed and replated in medium with or
without fresh cytokines after 7, 12, and 17 days incubation. Whereas
fresh Lin/loSca1+c-kit+
cells showed absolutely no responsiveness to IL-7 alone (O.J.B. and
S.E.W.J., unpublished observations), day 7 cells showed a significant IL-7 responsivess (Fig 6).
However, KL (2.3-fold) and in particular FL (6.9-fold) synergistically
enhanced the IL-7 responsiveness, and KL further enhanced FL + IL-7-stimulated growth. This suggested that KL enhances FL + IL-7-stimulated proB-cell formation at 2 levels; by promoting
recruitment of multipotent Lin/loSca1+c-kit+flt3
progenitors and by enhancing growth of lymphoid-restricted progenitors. Regardless of whether day 7 cells were stimulated with IL-7, FL + IL-7,
or KL + IL-7, production of cells with a proB-cell phenotype dominated
the cultures, and no IgM+ (surface or cytoplasmic) cells
were observed (O.J.B. and S.E.W.J., unpublished
observation).
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[in a new window]
| Fig 6.
Cytokine responsiveness of progeny derived from FL + IL-7-stimulated
Lin/loSca1+c-kit+ cells.
Lin/loSca1+c-kit+ cells were
cultured in the presence of FL + IL-7. After 7 or 12 days, cells were
harvested, washed, and stimulated with the indicated cytokines for an
additional 3 days before 3H-thymidine incorporation was
determined (Materials and Methods). The data represent the mean
(+SEM) of 3 experiments, in which KL + FL + IL-7-induced
3H-thymidine uptake was used as a control (100%) in each
experiment.
|
|
Cells obtained after 12 days (Fig 6) or 17 days (O.J.B. and S.E.W.J.,
unpublished observation, March 1998) of culture in FL + IL-7 showed a dramatically different pattern of cytokine responsiveness than the starting and day 7 populations. Most importantly, cells now
responded strongly and almost optimally to IL-7 alone (Fig 6).
Flt3-expression is suggested to decrease with increased B-lymphoid differentiation,19,41-44 and in agreement with this, the
day 17 cells had no detectable flt3-expression (Table 3) and FL had no
ability to enhance IL-7-induced proliferation, whereas KL synergized slightly with IL-7 (Fig 6).
FL induces B220 and IL-7R expression on
Lin/loSca1+c-kit+flt3+
progenitor cells.
As previously demonstrated by others,2,45 freshly isolated
Lin/loSca1+c-kit+ cells did
not express detectable levels of IL-7R (O.J.B. and S.E.W.J.,
unpublished observation). However, as our delayed
addition studies suggested that FL induced IL-7 responsiveness within a few days of incubation, the ability of FL to induce expression of B220
and IL-7R on
Lin/loSca1+c-kit+flt3+
cells was investigated (Fig 7). After 7 to
8 days of stimulation with FL, 26% of the cells expressed detectable
levels of IL-7R, and most of these coexpressed B220. In addition,
38% of FL-stimulated cells expressed B220, but lacked detectable
IL-7R expression. Noteworthy, KL, inefficient at supporting
B-lymphoid development from
Lin/loSca1+c-kit+flt3+
progenitors, induced expression of IL-7R on only 6% of the cells, and most of these did not coexpress B220. Furthermore, because FL-stimulated cultures contained 8-fold more cells, the formation of
IL-7R+ cells was more than 30-fold higher in response to
FL than KL. In contrast to what was observed on flt3+
cells, KL was incapable of inducing IL-7R expression on
Lin/loSca1+c-kit+flt3
cells, suggesting that the ability of KL to enhance FL + IL-7-stimulated proB-cell formation from
Lin/loSca1+c-kit+flt3
cells is not mediated through induction of IL-7R, but more likely results from acquisition of FL responsiveness as indicated by the
delayed addition studies (Table 4).
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| Fig 7.
FL, but not KL, efficiently promotes development of
B220+IL-7R+ cells from
Lin/loSca1+c-kit+flt3+
progenitors. A total of 2 to 10,000 Lin/loSca1+c-kit+flt3+
(flt3+) and
Lin/loSca1+c-kit+flt3
(flt3) cells was cultured in serum-depleted medium
supplemented with either KL or FL, as depicted in the figure. After 7 to 8 days in culture, cells were counted and evaluated for B220 and
IL-7R expression. The figure shows 1 representative experiment of 3 individual experiments, whereas the mean (±SEM) is shown for each
quadrant. Flt3 cells cultured in FL did not survive and
thus could not be investigated.
|
|
FL + IL-7-derived progeny of multipotent
Lin/loSca1+c-kit+
progenitors have ability to short-term reconstitute lymphoid, but not
myeloid, cell lineages in vivo.
In vivo experiments were next initiated to further characterize the
progeny generated from
Lin/loSca1+c-kit+ cells in
response to FL + IL-7. The objectives of these studies were multiple.
First, we wanted to determine to what degree these cells maintained an
ability to reconstitute in vivo. Next, it was important to show that in
vitro FL + IL-7-derived cells, which were blocked at the proB-cell
stage under appropriate conditions, could develop into normal mature
circulating B cells. Furthermore, although our in vitro studies had
suggested that these cells would lack myeloid reconstituting ability,
we wanted to confirm this through in vivo reconstitution experiments.
Finally, and most importantly, we examined whether FL + IL-7-derived
cells also had an ability to regenerate T cells. This possibility was
supported by the persistence of a minor (1% to 2%) portion of
B220CD19 as well as
B220+CD19 cells lacking myeloid
potential as well as expression of B-lymphoid-specific markers after
as much as 12 days of incubation (Fig 1). Thus, the day 12 FL + IL-7-derived progeny from 1,000 Lin/loSca1+c-kit+ cells
(Ly5.1) were transplanted into lethally irradiated C57Bl/6 mice
(Ly5.2). The level of reconstitution in the peripheral blood was
assayed after 10 weeks. A high level of donor-derived Ly5.1 (mean of
12%) reconstitution was observed in the peripheral blood of all
transplanted mice. In addition to donor-derived B cells, CD3+ T cells were observed in all transplanted mice
(Fig 8). These CD3+ cells
included single positive CD4 and CD8 cells (O.J.B. and S.E.W.J.,
unpublished observations, February 1999). Importantly, myeloid cells (Gr1/Mac1+CD3
B220) could not be detected in any of the mice
analyzed, whereas myeloid cells could easily be detected in mice
transplanted with freshly isolated
Lin/loSca1+c-kit+ cells (Fig
8). Thus, FL + IL-7 specifically promote development of
lymphoid-restricted progenitors from multipotent
Lin/loSca1+c-kit+ BM cells.
View larger version (21K):
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| Fig 8.
FL + IL-7-stimulated
Lin/loSca1+c-kit+ cells
support lymphoid-restricted reconstitution in vivo. A total of 1,000 Lin/loSca1+c-kit+ cells from
B6SJL mice (Ly5.1, donor) was either transplanted directly or cultured
in the presence of FL + IL-7 for 14 days before transplantation into
lethally irradiated C57Bl/6 mice (Ly5.2, recipient), together with
150,000 (Ly5.2) fresh BM cells. Peripheral blood was analyzed for the
presence of donor and recipient-derived cells and lineage distribution
6 and 10 weeks after transplantation. Similar results were obtained at
both time points. The CD3 and B220 reconstitution (only from Ly5.1
donor cells) from 1 typical mouse is shown for fresh, as well as FL + IL-7-cultured cells. In addition, for FL + IL-7-cultured cells, the
coexpression of B220 and IgM on circulating B cells is shown. The
percentage in each quadrant shows the mean from 2 or 3 mice (in 1 of 2 representative experiments) in the fresh
Lin/loSca1+c-kit+ and FL + IL-7-culture group, respectively.
B220CD3 cells detected when transplanting
fresh Lin/loSca1+c-kit+
cells were practically all Gr1/Mac1+ myeloid cells (J.A.
and S.E.W.J., unpublished observations, February 1999).
|
|
| |
DISCUSSION |
The hematologic actions of KL and FL are predominantly restricted to
the early progenitor/stem cell compartment, and both ligands and their
corresponding tyrosine kinase receptors have been demonstrated to play
key roles in early hematopoiesis.19 Thus, physiologically
KL appears to be involved in the maintenance and expansion of long-term
repopulating stem cells and regulation of
myeloerythropoiesis,14,15,19,46 whereas FL appears to primarily play a role in early lymphoid, and in particular, early B-lymphoid development.17,19 Despite these rather clear
distinctions in nonredundant hematopoietic functions, there are several
unresolved and key questions relating to the effects mediated through
c-kit and flt3, in particular to their effects on progenitor/stem cells with a combined myeloid and lymphoid differentiation potential. Thus,
in the present studies, we specifically addressed: (1) to what degree
such multipotent progenitor/stem cells express c-kit and flt3 and (2)
whether KL and FL can promote the transition from multipotent adult BM
progenitor/stem cells to lymphoid-restricted progenitor cells.
A number of studies have clearly demonstrated that both KL and FL can
promote the growth of lymphoid-restricted progenitor cells.19-23,25-29 What remains unclear and somewhat
disputed is at which level of development these two cytokines can
promote growth and development of lymphoid progenitor cells. Thus, the
present studies focused primarily on the potential ability of these two cytokines to promote the transition from multipotent progenitor cells
(with a combined myeloid and lymphoid differentiation potential) to
lymphoid-restricted progenitors. The challenge (and, thus, often
weakness) of such studies is that firm conclusions regarding the
transition from a multipotent progenitor to a lineage-restricted progenitor require ultimate proof that the initial targets/responders had such a mixed lineage potential and that the resulting progeny are
more restricted in their lineage potential. Thus, the main limitations
with such studies are usually the heterogeneity of the multipotent
progenitor cell population, the inefficiency of the specific
progenitor/lineage assays, and the burden of proof for demonstrating
absence of a lineage potential. These are also likely explanations as
for why limited and partially conflicting data have been available with
regard to the abilities of KL and FL (as well as other cytokines) to
promote the transition from multipotent to lymphoid-restricted
progenitor cells. Although our previous studies had already suggested
that FL + IL-7 could promote such a transition,30,36 the
conclusions were somewhat limited by a low fraction of FL + IL-7
responders, an even lower fraction of responders that could be
analyzed, and by potential limitations of the assays used to
demonstrate presence and absence of lineage potentials. In the present
studies, we have overcome these challenges allowing us not only to
reach more definite conclusions, but also provide new and important
information regarding the distinctions between FL and KL in promoting
lymphoid commitment and development.
By using a purified population of
Lin/loSca1+c-kit+flt3+
BM cells, we demonstrate at the single-cell level that virtually all
(97% to 98%) starting cells have a myeloid differentiation potential and that as much as 70% of these undergo lymphoid-restricted
development in response to FL + IL-7. This unequivocally demonstrates
that the
Lin/loSca1+c-kit+flt3+
cells undergoing lymphoid-restricted development in response to FL + IL-7 originally had a mixed myeloid-lymphoid differentiation potential.
In contrast, no
Lin/loSca1+c-kit+flt3+
progenitors underwent lymphoid-restricted development in response to
KL + IL-7. Importantly, these observations were confirmed in single-cell serum-depleted cultures, excluding indirect effects of
accessory cells. In addition to pointing to a unique difference between
c-kit and flt3 in promoting lymphoid commitment from multipotent BM
progenitors, these observations are particularly intriguing in light of
the fact that the starting cells all expressed high levels of c-kit.
Thus, the inability of KL to promote lymphoid development from
Lin/loSca1+c-kit+flt3+
cells could not be explained by lack of c-kit receptor expression. Although the mechanism for lack of KL responsiveness was not addressed in these studies, it was not due to a suboptimal KL preparation, as
this efficiently promoted myeloid growth from
Lin/loSca1+c-kit+ cells
(O.J.B. and S.E.W.J., unpublished observations). Neither was it due to a blocking effect of the anti-c-kit antibody used for
isolation of
Lin/loSca1+c-kit+flt3+
cells, because the
Lin/loSca1+c-kit+flt3
cells (isolated simultaneously) responded optimally to KL and because
LinSca1+ cells that (were not further
purified based on c-kit expression) showed the same lack of lymphoid
development in response to KL+IL-7.30
The conclusion that multipotent
Lin/loSca1+c-kit+flt3+
progenitors undergo lymphoid-restricted development in response to FL + IL-7 was based on demonstration both in vitro (morphology and
phenotyping) and in vivo (phenotyping) that their progeny lacked
myeloid differentiation potential. In addition, it was demonstrated
that FL + IL-7-stimulated Lin/loSca1+c-kit+flt3+
cells efficiently produced B220+CD19+
proB-cells in vitro and that these promoted in vivo reconstitution of
surface IgM+ B-cells for as much as 10 weeks. Because a
fraction of the B220+ cells generated in vitro lacked CD19
expression, it remained possible that there might be cells generated in
response to FL + IL-7, which were not B-lymphoid committed. However, as
virtually all B220+CD19 cells generated
in vitro in response to FL + IL-7 appeared to coexpress 5, thought
to be expressed in a B-lymphoid-specific manner,4,7,35 it
appeared that most if not all cells generated from
Lin/loSca1+c-kit+flt3+
cells in response to FL + IL-7 were committed to the B-lymphoid lineage. Thus, it was surprising that all mice transplanted with FL + IL-7-stimulated
Lin/loSca1+c-kit+flt3+
cells also reconstituted with high levels of circulating mature T
cells. Although not the scope of these studies, we are currently addressing the identity of the lymphoid progenitors reconstituting T
cells. The very few B220CD19
and/or B220+CD195
cells remaining in the cultures could be the source of the observed T-cell reconstitution, however, it is tempting to also speculate that
5 might not necessarily be B-lymphoid-specific and could potentially also be expressed in a bipotent B-/T-cell progenitor. In
this regard, it is noteworthy that our kinetic studies clearly demonstrated that 5 expression on B220+ cells always
preceded CD19 expression. Also other lineage-"specific" genes
have recently been shown to be expressed before lineage commitment.47,48
LinSca1+c-kit+ BM cells have
been shown to be highly enriched in long-term reconstituting activity
when compared with
LinSca1+c-kit
cells.11-13 Previous studies have proposed that although
most LinSca1+ BM stem cells lack flt3
expression, there were also
LinSca1+flt3+
LTRC.16 In contrast, recent and more detailed studies from our laboratory suggest that long-term multilineage reconstitution is
exclusively derived from
Lin/loSca1+c-kit+flt3
and not
Lin/loSca1+c-kit+flt3+
BM cells (O.J.B. and S.E.W.J., unpublished observations).
However, we cannot exclude that there are flt3low LTRC with
flt3 expression below our level of detection, although it was evident
that the sorted flt3 cells unlike the
flt3+ cells failed to respond to FL. In that regard, it was
noteworthy that KL was found to be crucial for optimal B-lymphoid
development from the
Lin/loSca1+c-kit+flt3
candidate stem cell population. Interestingly, although showing little
or no response to FL + IL-7 alone, FL was absolutely required (together
with KL and IL-7) for lymphoid-restricted development from the
Lin/loSca1+c-kit+flt3
cells. Although not specifically investigated, it seems likely that KL
is acting by inducing flt3 expression and/or responsiveness of
Lin/loSca1+c-kit+flt3
cells, as FL had no effect on the level of recruitment of
Lin/loSca1+c-kit+flt3
progenitors seen in response to KL and as KL showed no ability to
induce IL-7R expression on
Lin/loSca1+c-kit+flt3
cells. In addition to its ability to enhance lymphoid development from
the
Lin/loSca1+c-kit+flt3
population, KL also enhanced the growth of proB-cell progenitors generated in response to FL + IL-7. These in vitro-derived proB-cells showed a normal growth and development pattern in that they remained IL-7-dependent, but gradually lost their FL and subsequently
KL-dependence.19
The finding that FL and not KL can promote lymphoid commitment from a
multipotent progenitor cell population is intriguing and deserves
further mechanistic studies. We show that coexpression of B220 and
IL-7R, both associated with early lymphoid development, are
efficiently induced on
Lin/loSca1+c-kit+flt3+
cells in response to FL, but not KL, and that KL has no ability to
induce B220 or IL-7R expression on
Lin/loSca1+c-kit+flt3
cells. These findings further point to an important role of FL and not
KL in promoting the earliest stages of lymphoid development, although
expression of IL-7R and probably also B220 follow rather than
precede lymphoid commitment. Future studies will seek to establish
whether flt3/FL might play a permissive and/or instructive role in the
lymphoid commitment process.
| |
ACKNOWLEDGMENT |
We thank Per Anders Bertilsson and Sverker Segren for expert assistance
in cell sorting and Ingbritt Åstrand-Grundström, Eva Gynnstam,
Irene Persson, and Lilian Wittman for technical assistance. We are also
grateful to Dr Koichi Akashi for important technical suggestions
regarding IL-7 staining and Drs Ian K. McNiece, Graham Molineux, and
Stewart D. Lyman for generously supplying cytokines. We thank David
Bryder, Dr Veslemøy Ramsfjell, Dr Stewart D. Lyman, and Dr Stefan
Karlsson for critically reviewing the manuscript.
| |
FOOTNOTES |
Submitted April 16, 1999; accepted July 28, 1999.
Supported by grants from the A-G. Crafoord Foundation, the Crafoord
Foundation, the Medical Faculty, University of Lund, the Swedish
Medical Research Council (MFR) and the Swedish Foundation for Strategic Research.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Sten E.W. Jacobsen, MD, Stem
Cell Laboratory, Institute for Laboratory Medicine, Department of
Internal Medicine, University of Lund, S-221 85 Lund, Sweden; e-mail:
sten.jacobsen{at}molmed.lu.se.
| |
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ABSTRACT
INTRODUCTION