1. Healthcare

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Stem Cell Factor and Interleukin-7 Synergize
Early Myelopoiesis In Vitro
to Enhance
By Cecilia Fahlman, Heidi K. Blomhoff, Ole P. Veiby, Ian K. McNiece, and Sten E.W. Jacobsen
Interleukin-7 (IL-71 has been shown to be a critical factor in
murine lymphoid development. It stimulates pre-B cells to
divide in the absence of stroma cells and it is an important
growth regulator of immature and mature T cells. IL-7 has
been shown to synergize with stem cell factor (SCF) to provide a potent growth stimulus for pre-B cells. However, the
combined effects of IL-7 and SCF on murine primitive hematopoietic cells in vitro have not been established. In thepresent study, the effects of recombinant rat (rr)SCF and recombinant human (rh) IL-7on primitive murine bone marrow
progenitors (Lin-Scal+) wereinvestigated in single-cell cloning experiments. rhlL-7 alone had no proliferative effect on
Lin-Scal+ cells, but in a dose-dependent manner directly
enhanced rrSCF-induced colony formation, with an average
increase in colony numbers of 2.7-fold. Interestingly, the
cells formed in response to SCF and IL-7 were predominately
mature granulocytes. Thus, SCF and IL-7 synergizeto stimulate early myelopoiesis in vitro.
0 1994 by The American Society of Hematology.
T
eration and differentiation of murine Lin-Scal' bone marrow progenitor cells. Lin-Scal' cells have
been shown to
be highly enriched in cells capableof long-term repopulation
of all cell lineages in the blood, and less than 100 of these
irradiated
Weshow
cellscan rescue 50% of lethally
here for the first time that SCF and IL-7 directly synergize
on these cells to promote their proliferation and primarily
myeloid development.
HE PROLIFERATION and differentiation of hemato-
poietic stem cells leads to the production ofmature
blood cells.'-3 Thisprocess is regulated by a complex system
of growth factors and mediators of cell to
cell
In
mice, interleukin-7 (IL-7) has been shown to be an essential
growth factor forB-cell de~elopment."'~
It acts as a proliferative stimulus on a subset of pre B cells that express pH
chain in thecytoplasm(Cp').
Pro-B cells (cellswithIg
genes in germline configuration) have been proposed to be
dependent on contactwith stromal cells forproliferation."."
Stem cell factor (SCF), also known as
kit-ligand and mast
cell growth factor,""' is a stromal cell-derived
factor that
can synergize with IL-7 to stimulate theproliferation of C p +
pre-B cells."~" Some studies havesuggested that pro-Bcells,
although expressing receptors for both IL-7 and SCF, donot
grow in response to thesesolublefactors
alone, requiring
direct contactwith stromal cells.13~'J.2"~21
However, it has
previously been shown that SCF plus IL-7 can
act in synergy
to stimulatethe proliferation and differentiation of pro-B
cells to pre-B cells." In contrast, a recent study suggested
that the combination of SCF and IL-7 does not stimulate the
expansion ordifferentiation of early B-cell precursors before
the expression of the B220 antigen," thought to identify all
B-lineage-committed cells in the murine bone marrow.24
We have recently observed a novel role for IL-7in myelopoiesis, in that IL-7 in synergy with colony-stimulating factors (CSFs) can directly stimulate the formation of mature
myeloid progeny from primitive murine progenitors." Because no previous studies have examined the ability of SCF
plus IL-7 to affect the growth of immature hematopoietic
progenitors, the present studies weredesigned to evaluate
the direct effects of this growth factorcombination on prolifFrom the Department of Immunology, Institute for Cancer Research, The Norwegian Radiumhospital, Oslo, N0rwa.y; Nycomed
Bioreg, Oslo, Norway; and Amgen Inc, Thousand Oaks, CA.
Submitted October 5, 1993; accepted April 25, 1994.
Supported by the Norwegian Cancer Society.
Address reprint requests to Sten E.W. Jacobsen, MD, PhD, Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
The publication cost., ofthis article were defrayed in part by page
charge payment.This article must therejbre be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/X405-00I 7$3.00/0
1450
MATERIALS AND METHODS
Enrichment and purijication of murine bone marrow cells. Linbonemarrow cells were isolated from normal C57BL mice, according to a previously described protocol.' Briefly, low-density
bone marrow cells were isolated by density centrifugation on a lymphocyte separation medium (Nycomed, Oslo, Norway). Cells were
washed twice in Iscove's modified Dulbecco's medium (IMDM;
GIBCO, Paisley, UK) and resuspended in IMDM supplemented with
20% fetal calf serum (FCS; Sera lab, Sussex, UK), 100 U/mL penicillin, and 3 mg/mL L-glutamine (complete IMDM). The cells were
incubated at 4°Cfor 30 minutes in a coctail of predetermined optimal
concentrations of antibodies: RA3-6B2 (B220 antigen; Pharmingen,
San Diego, CA), RB6-8C5 (GR-1 antigen; Pharmingen), MAC-I
(Serotec, Oxfordshire, UK), Lyt-2 (CD8; Becton Dickinson & CO,
Sunnyvale, CA), and L3T4 (CD4; Pharmingen). Cells were washed
twice and resuspended in complete IMDM. Sheep antirat IgG (Fc)conjugated immunomagnetic beads (Dynal, Oslo, Norway) were
added at a cel1:bead ratio of 1:20 and incubated at 4°C for 30 minutes.
Labeled (Lint) cells were removed by a magnetic particle concentrator (Dynal) and Lin- cells were recovered from the supernatant,
The Lin- cells were further purified on the basis of Sca- 1 expression, aspreviouslydescribed.'.''
Briefly, 4 to 6 X IO' Lin- cells
were resuspended per milliliter of complete IMDM and incubated
for 30 minutes on ice with either fluorescein isothiocyanate (FITC)conjugated rat antimouse Ly-6A/E antibody (Pharmingen) or an isotype-matched control antibody. The cells were washed twice, and
Lin-Scal' cells were collected on a Coulter Epics Elite Cell Sorter
(Coulter Electronics, Hialeah, FL) equipped with a 488 nm tuned
argon laser set to give a power of 15 mW, with a rate of 1,500 to
2,000 cellslsecond. Lin- cells falling into median right angle scatter
and median to high forward scatter were analyzed for Sca-l expression, and cells falling into both regions were selected. Light scatter
was collected through a 488 nm band pass filter and HTC fluorescence was collected through a 525-nm band pass filter. The final
recovery of Lin-Scal cells was 0.05% to 0.1% of the unfractionated
bone marrow. Reanalysis of sorted Lin-Scal+ cells showed reproducibly a purity greater than 95%.
Growthfactors.
Purified recombinant human (rh) IL-7 was
kindly supplied by Dr Steven Gillis (Immunex Corp. Seattle, WA).
Recombinant rat (TT)
SCF was expressed in Escherichia coli and
+
Blood, Vol 84, No 5 (September I),1994: pp 1450-1456
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MYELOPOIETIC EFFECTSOFSCF
1451
AND IL-7
Table 1. Direct Proliferative Effects of SCF and IL-7
on Lin-Scal+ Bone Marrow Cells
Table 2. Direct ProliferativeEffects of SCF, IL-7, and GM-CSF
on Lin-Scal- Bone Marrow Cells
No. of Positive WellsI300
No. of Positive Welld300
Growth Factor
7
1
1
1
1
2
2
2
2
3
3
3
3
Medium
1
SCF 2
IL-7
5 SCF +6IL-7
Medium
SCF
IL-7
SCF IL-7
Medium
5 SCF 7
IL-7
SCF + IL-7
+
9
Cells/
Well
Exp.
1
Exp. 2
Exp. 3
0
0
0
0
0
2
0
0
4
0
10
0
0
3
0
7
0
0
5
0
12
0
0
16
0
11
0
26
Exp. 4
Exp. 5
0
3
0
7
0
6
0
15
0
10
0
19
0
3
0
7
0
5
0
14
0
14
0
34
P Value
Growth
Factor
CellshVell
Exp. 1
<.05
SCF
IL-7
SCF + IL-7
GM-CSF
1
1
1
1
0
0
0
6
Exp. Exp.
2
3 Exp.
0
0
0
9
1
0
1
8
4
0
0
0
10
Lin-Scal- cells were separatedas described in Materials and Methods and plated in Terasaki plates at a concentrationof 1 cell per well
in 20 pL complete IMDM. BothSCF and IL-7 were used at 100 ng/mL
and GM-CSF was used at20 ng/mL. Wells were scored for cell growth
(>IO cells) after 12 days of incubation at 37°C in 5% CO? in air. The
results from four separate experiments are shown.
<.05
<.05
Lin-Scal' cells were separated as described in Materials and Methods and plated in Terasaki plates at a concentrationof 1,2, or 3 cells
per well in 20 pL complete IMDM. IL-7 and SCF were both used at
predeterminedoptimalconcentrationsof
100 ng/mL.Wellswere
scored for cell growth
( > l 0 cells) after12 days of incubation at
37°C in
5% CO2 in air. The results from five separate experiments are shown.
Statistical significance ( P value)wasdeterminedusingtheMannWhitney test.
purified to homogeneity, as described previou~ly.~'
The specific activity of rrSCF was 3 X 105 U/mg, where 1 U is defined as the
amount of rrSCF stimulating half-maximal thymidine uptake by MC9 cells.2R Recombinant murine granulocyte-macrophage-CSF
(rmGM-CSF) was from Amgen Inc (Thousand Oaks, CA).
Single-cell prolifermion assays. Lin-Scal+ cells were seeded in
Terasaki plates (Nunc, Kamstrup, Denmark) at a concentration of 1
cell per well in 20 pL complete IMDM. In addition, 2 and 3 cells
per well were seeded in 20 pL of complete IMDM. Wells were
scored for cell growth ( > I O cells) after 12 days of incubation at
37°C in 5 8 CO,.
Phenotyping of cells. Lin-Scal' cells were cultured at 5 X lo3
cells/mL in complete IMDM with SCF (100 ng/mL) or a combination of SCF and IL-7 (100 ng/mL) for 12 days at 37°C in 5% CO,
in air. ( I ) Approximately 5 X IO4 cells were cytocentrifuged onto
glass slides and stained with Giemsa. The content of granulocytes,
macrophages, and undifferentiated blast cells was determined by
microscopic examination. (2) Cells were stained for cell-surface antigen expression by standard immunofluorescence techniques using
monoclonal antibodies (MoAbs) at predetermined optimal concentrations. Granulocyte-l (GR- I ; Pharmingen) expression was shown
by FITC-conjugated antirat IgG (Cappel, Durham, NC). Ly-5 (B220;
Pharmingen) was directly conjugated to phycoerythrin (PE). Isotypematched MoAbs with irrelevant specificity served as negative controls. Fluorescence was analyzed on a FACScan flow cytometer
(Becton Dickinson).
Lin-Scal' cells were seeded in Terasaki plates at a concentration
of 1 cell per well in 20 FL complete IMDM. After 12 to 14 days
of growth, individual colonies were harvested and cytocentrifuged
onto glass slides and stained with Giemsa.
RESULTS
T
I
SCF and IL-7 directly synergize to enhance the proliferation of Lin-Scal+, but not Lin-Scal", bone marrow progeni-
Table 3. Effects of SCF and IL-7 on Differentiation
of Lin-Scal+ Bone Marrow Cells
CellslmL
Growth Factor
1.5
0.75
0
0,001
0.01
0,l
1
10
100
IL-7 (ng/nl)
Fig 1. DoseresponseofIL-7 on SCF-stimulated proliferation of
Lin-Seal+ progenitor cells. Lin-Scal' cells were separated asdescribed in Materials and Methods, plated at a density of 2 cells per
well in Terasaki plates in complete IMDM supplemented with SCF
(100 nglmLI, and exposed to increasing concentrationsof IL-7. Wells
were scored for growth ( > l 0 cells) after 12 days of incubation at
37°C in 5% CO2in air. Resultsare the mean (+SEMI of three separate
experiments with duplicate determinations.
Experiment 1
SCF
SCF + IL-7
Experiment 2
SCF
SCF IL-7
Experiment 3
SCF
SCF IL-7
(x105t
% Granulocytes
% Macrophages
% Blasts
93
87
5
5
2
8
95
88
6
4
6
4.8
1
+
3.4
+
1.1
3.9
3
94
85
3
7
8
Lin-Scal' cells were isolated as described in Materials and Methods and seededin liquid culture at 5,000 cells/mL in complete IMDM.
Cultures were stimulated with SCF (100 ng/mL) or SCF plus IL-7 (100
ng/mL) for 12 days at 37°C in 5% CO, in air. Cell morphology was
determined after Giemsa staining of cytospin preparations. "Blasts"
denotes cells with undifferentiated morphology.
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1452
FAHLMAN ET AL
Fig 2. Morphology of cells from day-12 liquid cultures of Lin-Scal' bone marrow
cells. Lin-Scal' cells were isolated as described in
Materials and Methods andseeded in liquid cultures at5,000 cells/mL in complete IMDM. Cultures were stimulated with SCF 1100 nglmLl (A)
or SCF plus IL-7 (100 ng/mL) (B) for l 2 days at 37°C in 5% CO2 in air, cytocentrifuged, and stained with Giemsa. Cytospin preparations were
photographed at x 1,000 magnification.
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1453
MYELOPOIETIC EFFECTS OF SCF AND IL-7
tor cells. To determine whether SCF plus IL-7 could synergize to stimulate immature progenitors, Lin-Scal+ cells were
isolated and cultured in the presence of SCF and/or IL-7.
Because IL-7 has been shown to potently induce cytokine
secretion from m0nocytes,2~Lin-ScaI+ cells were grown as
single isolated cells to determine the direct effects of IL-7
on the progenitor cells. IL-7 alone didnot stimulate any
colony formation of Lin-Scal+ cells (Table 1). In agreement
with other^,^.^ SCF alone induced a small number of colonies
(Table l). In five separate experiments, IL-7 at 100 ng/mL
enhanced SCF-induced colony formation from 2.3- to 5-fold.
A similar enhancement was observed when 2 or 3 cells
were plated per well (Table 1). The SCF-stimulated colony
formation was enhanced by IL-7 in a dose-dependent fashion, with maximum enhancement observed at 100 ng/mL of
IL-7 (Fig 1) and an EDSo
of 1 to 10 ng/mL. Higher concentrations of IL-7 did not further enhance the number of colonies
formed by Lin"Sca 1 cells when stimulated by SCF, because
in three separate experiments a mean of 7 (51) colonies
were formed in response to SCF alone, and IL-7 at 100 ng/
mL enhanced the number of colonies to a mean of 18 (? 1 ),
whereas 17 (52) and 14 (?2) colonies were formed in the
presence of 200 ng/mL and 500 ng/mL of IL-7, respectively.
The size of the colonies generated by Lin-Scal+ cells in
response to SCF plus IL-7, when compared with colonies
stimulated by SCF alone, was, however, not affected (data
not shown). Thus, IL-7 and SCF directly synergize to recruit
proliferating Lin-Scal' haematopoietic progenitor cells.
Most, if not all, SCF-responsive progenitors have been
shown to be contained in Lin-Scal' progenitor cell populat i o n ~Next,
. ~ we examined whether IL-7 could synergize with
SCF on the more committed Lin-Scal- progenitor cells. In
agreement with others,' we found few or no SCF responders
in the Lin-Scal- population (Table 2). In addition, no synergy was observed between SCF and IL-7 on these progenitors (Table 2). In contrast, and as previously shown,5 GMCSF stimulated colony formation of Lin-Scal- progenitor
cells (Table 2). Thus, IL-7 in combination with SCF stimulates the growth of Lin-Scal+, butnot Lin-Seal-, bone
marrow progenitor cells.
SCF and IL-7 synergistically stimulate myelopoiesis of
Lin-ScaI+progenitors. TO determine to what extent the
synergistic effects of SCF and IL-7 on the proliferation of
Lin-Scal+ cells were caused by production of lymphoid or
myeloid progeny, Lin-Scal+ cells were grown for 12 days
in the presence of either SCF alone or SCF plus JL-7. Morphologic examination of cytospin preparations showed that
SCF alone stimulated almost exclusively the formation of
mature granulocytes (>90%) as well as a small percentage
of macrophages (3% to 5%) andblast cells (1% to 3%;
Table 3 and Fig 2A), consistent with previous
The combination of SCF plus IL-7 also resulted in the production of predominantely granulocytes (85% to 88%). A
slight increase in the proportion of blast cells (6% to 8%)
was observed in response to SCF plus IL-7, when compared
with SCF alone (Table 3 and Fig 2B).
To exclude the possibility that the stimulatory effect of
IL-7 on myelopoiesis was indirect, colonies of Lin-Scal'
cells cultured individually were also examined morphologi+
cally. In agreement with a previous study from our laboratory,30 all colonies from cells stimulated with SCF alone
contained mature granulocytes and some macrophages (data
not shown). Consistent with results obtained from bulk liquid
cultures, when single cells were stimulated with SCF plus
IL-7 for 12 to 14 days, 100%of the colonies contained
predominantely granulocytes and/or macrophages (data not
shown). Thus, IL-7 in combination with SCF directly enhances myeloid colony formation of Lin-Scal' progenitor
cells.
To further define the lineage of the cells in SCF plus IL7-stimulated cultures, GR-l and B220 immunofluorescent
staining was performed. The GR-1 antigen (recognized by
MoAbs to RB6-8C5) has been shown to be a myeloid-specific differentiation antigen.3' Greater that 90% of cells stimulated with SCF expressed the GR-1 antigen, consistent with
their granulocyte morphology (Fig 3). The number of GR1' cells in cultures stimulated with SCF plus IL-7 was consistently 80% to 85%. Although no bright B220+ cells could
be observed in response to SCF plus IL-7, 14% ( 5 5 % )
B220""" cells were observed (Fig 3). As a positive control
A
B
7
Fig 3. Flow cytometric analysis of Lin-Scal' bone marrow cells.
Lin-Scal+ cells were separated as described in Materials and Methods and seeded
in liquid cultures at 5,000 ceIlslmL in complete tMDM
(100 ngl
and stimulated with SCF (100 ng/rnL) alone or SCF plus IL-7
ml) for 12 daysat 37°C in5% CO2in air. Unfractionated bonemarrow
cells were used asa positivecontrol for8220 staining. (A) Cells were
stained with a B220 or control antibody. (B) Cells were stained with
a GR-l or control antibody as described in Materials and Methods.
The results represent one of four representative experiments.
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1454
FAHLMAN ET AL
SCF + IL-7
CONTROGFITC
CONTROGFITC
Fig 4. Double-fluorescenceanalysis of OR-l and
B220 expression on Lin-Scal’ bone marrow progenitors stimulated by SCF or SCF 11-7. Lin-Scal+ cells
were cultured for 14 days at 5,000 cells/mL in complete IMDM supplemented with SCF l100 nglmL) or
SCF IL-7 (100 ng/mL). Cells were stained for GR-l
and B220 expression as described in Materials and
Methods. Negative fluorescence controls were provided by MoAbs of unrelated specificity.
+
+
GR-1 FITC
GR-1-FITC
of B220 staining, unfractionated bonemarrow cells were
stained for B220. In agreement with others,” we found that
28% of these cells stained brightly positive for B220 (Fig
3 ) . Because it previously has been shown that B220 is not
a lymphoid restricted antigen, but is also expressed on progenitors with myeloid p~tential,”.’~
we next performed double immunofluorescence staining with GR-1 and B220
MoAbs. This demonstrated that cells stimulated with SCF
plus IL-7 expressing B220 coexpressed GR-1 (Fig 4).
DISCUSSION
The role of IL-7 in B lymphopoiesis is well established,
in vitro7-I4as well as in v ~ v o , ’ ~whereas
,~~
SCF has been
shown to be a potent stimulator of erythropoiesis and
myelopoiesis, with little effects on lymphopoiesis in
vivo.4-6.16.17.37.38IL-7 alone or in combination with SCF can
stimulate the clonal proliferation of pre-B cells; however,
several reports have suggested that stromal cell-derived cytokines are required in addition to SCF plus IL-7 to stimulate
the growth of pro-B cells.13.’4*’9-21,39
We show here that IL-7 can synergize with SCF to stimulate the proliferation and
myeloid
differentiation of
Lin-Scal+ bone marrow cells, demonstrated to be highly
enriched in primitive hematopoietic progenitor
In contrast, SCF and IL-7 didnot induce proliferation of
Lin-Scal- progenitors, in agreement with previous observations, suggesting that all synergy involving SCF is observed
on the primitive Lin-Scal+ progenitor c e k 5 The single-cell
assays suggest that the effects of IL-7 are directly mediated
on the progenitor cells and not indirectly through stimulation
of contaminating accessory cells. However, because IL-7
has been shown to induce cytokine p r o d u c t i ~ nwe
, ~ ~cannot
exclude stimulation throughan IL-7-mediated autocrine
loop.
Predominantly mature granulocytes were formed from
Lin-Scal’ cells in response to SCF plus IL-7. Interestingly,
10% to 20% of these GR-l+ cells coexpressed B220, implicating, in agreement with previous reports,”,34a population
with combined myeloidand lymphoid potential. The fact
that few or no B220+GR-l- cells were observed supports
previous studies in that additional growth factors or cell to
cell contact are required to stimulate early B-cell development. In agreement with this, we have observed that SCF
plus IL-7 stimulates the formation of B cells from Lin-Scal’
cells only in the presence of stromal cells (I.K.M., unpublished observations). Although Landreth et a14’ have shown
that insulin-like growth factor-l (IGF-1) is a stromal-produced factor, capable of stimulating early B lymphopoiesis,
the combination of SCF, IL-7, plus IGF-1 is still insufficient
to stimulate B lymphopoiesis from Lin-Scal’ progenitors
(Fahlman et al, unpublished observations).
The present data are consistent with recent studies from
our own laboratory, showing that IL-7 potently synergizes
with CSFs to enhance in vitro myelopoiesis of Lin-Sca I +
progenitor^.'^ Thus, IL-7 can synergistically stimulate myelopoiesis under defined in vitro conditions and can nolonger
be regarded as a lymphoid-restricted growth factor. Although
administration of IL-7 alone in vivo shows preferential stim-
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MYELOPOIETIC EFFECTSOFSCF
AND IL-7
ulation of lymphopoiesis, effects have been observed on myelopoiesis as
and it is possible that IL-7 in combination with SCF or CSFs might have more profound effects
on myelopoiesis.
The apparent overlapping activity of SCF plus IL-7 in
myeloid and lymphoid development underscores the complex regulation of proliferation and differentiation of hematopoietic progenitor cells. As has been shown for multiple
other cytokines, the ability of a specific growth factor to
induce proliferation and differentiation of progenitor cells
along a specific lineage depends on the specific progenitor
being targeted, the nature and number of cooperating growth
factors, as well as cell-to-cell interactions.
In conclusion, the combined stimulation of SCF plus IL7 in vitro can enhance myelopoiesis as well as lymphopoiesis. The ability of this combination to stimulate early B
lymphopoiesis seems to require additional, not yet identified
factors. Identification of such factors is necessary to elucidate
the potential of SCF plus IL-7 in controlling lymphoidmyeloid committment.
ACKNOWLEDGMENT
The authors thank Drs Frede W. Jacobsen, Leiv S. Rusten, and
Erlend B. Smeland for critical review of this manuscript.
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Stem cell factor and interleukin-7 synergize to enhance early
myelopoiesis in vitro
C Fahlman, HK Blomhoff, OP Veiby, IK McNiece and SE Jacobsen
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