J. Biomedical Science and Engineering, 2009, 2, 200-207
Published Online June 2009 in SciRes. http://www.scirp.org/journal/jbise
JBiSE
Effects of granulocyte colony-stimulating factor
and stem cell factor, alone and in combination, on
the biological behaviours of bone marrow
mesenchymal stem cells
Feng-Ping Tang1, Xing-Huo Wu2, Xi-Lin Yu1, Shu-Hua Yang 2, Wei-Hua Xu 2, Jin Li 2
1Wuhan Medical & Health Center for Women and Children, Wuhan, China; 2Department of Orthopaedics, Union Hospital, Tongji
Medical College, Science and Technology of Huazhong University, Wuhan, China; Corresponding author: Xing-Huo Wu, Fax:
086-027-85351627.
Received 6 January 2009; revised 20 March 2009; accepted 9 April 2009.
ABSTRACT
Aim: The effects of granulocyte colony- stimu-
lating factor (G-CSF) and stem cell factor (SCF)
on the proliferation and osteogenic differentia-
tion capacity of bone marrow mesenchymal
stem cells (MSCs) were studied in the experi-
ment. Methods: Bone marrow MSCs were col-
lected from rabbits successfully, and treated
with various concentrations of G-CSF, SCF or a
combination of the two. Flow cytometric ana-
lyse, MTT test, CFU-F assay, and alkaline
phosphatase (ALP) activity measurement were
employed. Results: The results of flow cytome-
try showed that immunophenotype of the cells
were CD29+/CD45-, CD105+/ CD34–, CD90+/
HLADR–. MSCs were shown to constitutively
express low levels of c-kit which could be en-
hanced by SCF. G-CSF and SCF had an obvious
facilitative effect on the proliferation of MSCs in
a dose-dependent fashion. In addition, G-CSF
and SCF would be effective in reversibly pre-
venting their differentiation, as showed by the
decrease of ALP activity, leading to self-renewal
rather than differentiative cell divisions. The
effects of G-CSF were superior to SCF. And
cells in the group treated with combination of
G-CSF and SCF showed more powerful effects
than the groups treated with G-CS, SCF, or non e
of the two. Conclusion: On the whole, these
studies demonstrated that MSCs responsed to
G-CSF, SCF, and to G-CSF plus SCF in a manner
that suppressed differentiation, and promotes
proliferation and self-renewal, and support the
view that the se fac tors cou ld act sy nergisti cally .
Keywords: Granulocyte Colony-Stimulating Factor;
Stem Cell Factor; Synergistic Effect; Bone Marrow
Mesenchymal Stem Cells
1. INTRODUCTION
Bone marrow is composed of various types of cells of
specific phenotypes and function. Bone marrow cells can
be transplanted either as total, unfractionated bone mar-
row or as a well-defined subpopulation of bone marrow
mesenchymal stem cells (MSCs) [1,2]. MSC is a group
of multipotent cells that can expand, self-replicate, and
differentiate into many cell types under appropriate con-
ditions [3,4]; their progeny includes chondrocytes, ten-
don cells, haematopoiesis-support stromal cells, adipo-
cytes and osteoblasts [5,6,7]. MSCs, similar to other
stem cells, have an essential role in the regeneration/
maintenance of the adult tissues submitted to physio-
logical modelling/turnover or following injury. At pre-
sent, MSCs show great promise for use in a variety of
cell-based therapies, include repair of defects in cardio-
vascular muscle, spinal cord , bone, and cartilage.
Recently, enhancement of bone repair in the necrotic
zone by bone marrow MSCs has been highlighted for the
treatment of osteonecrosis before collapse of the head
[8,9]. MSCs can be delivered into the injured tissue ei-
ther by invasive or by noninvasive means. Of primary
importance to the success of such a strategy is the pro-
duction of viable, reproducible protocols for stem cell
population expansion. Invasive method is done on a sur-
gically exposed necrotic head. Isolated primary mesen-
chymal stem cells are low in numbers, in vitro expansion
is necessary. Although it is known that adult bone mar-
row MSCs can be rapidly expanded in vitro, migrate,
and differentiate into multiple tissues in vivo. However,
the expansion potential is limited and in vitro aging
leads to loss of multipotency and replicative senescence.
In addition, many transplanted cells die shortly after
implantation as a result of physical stress from the im-
plantation procedure itself, inflammation, or hypoxia.
Under consideration of noninvasive methods of targeting

F. P. Tang et al. / J. Biomedical Science and Engineering 2 (2009) 202-207 201
SciRes Copyright © 2009 JBiSE
the injured tissue with stem cells that take advantage of
endogenous mechanisms. Recent studies in a rat model
showed that endogenous signaling via cytokines can
enhance mobilization, homing, and transdifferentiation
of stem cells [10]. MSCs can be mobilized from the bone
marrow (central pool of stem cells) and directed to the
injured tissue or organ. Currently, little is known about
the signals involved in the mobilization and homing of
stem cells to the injured tissue. It is believed that the
cytokines SCF, G-CSF, and stromal-cell-derived factor-1
(SDF-1) and their receptors play a major role (1).
MSCs constitutively expressed mRNA for interleukin
(IL), colony-stimulating factor (CSF), and stem cell fac-
tor (SCF) [11]. G-CSF is a polypeptide hematopoietic
factor that stimulates survival, proliferation, and matura-
tion of neutrophilic granulocyte progenitors and en-
hances their functions. Stem cell factor (SCF) is a potent
costimulatory molecule for many cytokines. Its synergy
with granulocyte colony-stimulating factor (G-CSF) re-
sults in important biologic and clinical effects, although
the mechanism by which this occurs remains poorly un-
derstood [12]. Notwithstanding, cytokine-induced mobi-
lization of bone marrow stem / progenitor cells in the
necrotic foci may represent a promising strategy for re-
placing necrotic bone. A better understanding of the ki-
netics of MSC and MSC derived progenitor cell prolif-
eration and differentiation is of great current interest
from both a clinical and tissue engineering perspective
[13,14,15]. Consequently, in the present study, we inves-
tigated the biological effects of G-CSF and SCF, alone
and in combination, on proliferation and osteogenic dif-
ferentiation capacity of bone marrow MSCs.
2. MATERIALS AND METHODS
2.1. Generation of Rabbit BM-MSCs
The BM-MSCs were prepared as described previously
with slight modification [16]. Bone marrow cells were
harvested from iliac crest aspirates from healthy
3-month old New Zealand white rabbits, and the proce-
dures were used in accordance with the procedures ap-
proved by the animal experimentation and ethics com-
mittees of Tongji Medical College. Approximately 10
ml of iliac bone marrow was aspirated and suspended in
20 ml of DMEM-LG medium (Gibco) containing 2000
U of heparin sodium. Mononuclear cells (MNCs) were
separated on Ficoll-Paque density gradient (1.077g/mL)
and washed in PBS. Then MNCs were seeded at a den-
sity of 1x106 cells/cm2 in growth medium containing
DMEM- LG and 10% FBS (HyClone) and incubated at
37°C in 5% CO2/95% air. Medium was changed first
after 24 h and then every 3 days. MSCs were used at
passage 3 to 4.
2.2. Flow Cytometric Analyses
To evaluate the lineage and surface marker phenotype of
passage 3 cultures of MSCs, cells were detached and
incubated in phosphate-buffered saline containing 1%
bovine serum albumin with the following fluorescent
antibodies: anti-human CD29 (integrin b1 chain)–PE,
anti-huaman CD90 (Thy-1)-FITC, anti-human CD105
(endoglin)-PE, anti-human CD34-FITC, anti-human
CD45-FITC, anti-human HLA-DR-PE(Santa Cruze,
USA), and were analyzed by FACS caliber flow cy-
tometry (BD, USA). To examine the expression of G-
CSFR(G-CSF receptor) and c-kit (SCF receptor) on the
cultured MSCs, PE- conjugated mouse anti-human
G-CSFR (CD114) monoclonal antibody (Becton- Dick-
inson, America), mouse anti-human c-kit(CD117)
monoclonal antibody and secondary antibodies conju-
gated with FITC (Zymed，America）were used. Isotype-
identical antibodies served as controls. Meanwhile, im-
munofluorescence staining was used to test cultured
MSCs as well. Primary antibody was anti-vimentin, and
secondary antibody was anti-gout polyvalent-Cy3 con-
jugate (Sigma).
2.3. Cell Proliferation of MSCs
MSCs of passage 3 were harvested by treatment of the
cells with Trysin/EDTA and washed twice with
DMEM. The cells were then resuspended (1×104 cells
per ml) in DMEM containing 10% FBS and plated in
96-well culture plates (100ul /well). After 48 h culture,
culture medium and nonadherent cells were removed.
Every experimental group was given 100ul growth
medium containing DMEM-LG, 2% FBS, and various
concentration of SCF and G-CSF, and cultured in the
CO2 incubator. Assessment of cell proliferation was
measured in terms of optical absorbance (OD) per well
by a semi-automated tetrazolium- based colorimetric
assay using MTT. The growth rate was calculated ac-
cording to the formula: (OD treated/OD control -1)×100%.
And the cell growth curves were drawn with the cul-
ture time (d) as the abscissa and the mean OD valu e as
the ordinate.
2.4. Colony-Forming Unit-Fibroblast (CFU-F)
Assay
The frequency of CFU-F was measured using the
method of Castro-Malaspina with slight modification
[17]. BMSCs (at 5×105 cells/ml) were suspended in
growth medium containing DMEM-LG, 10% FBS, anti-
biotics, and various concentration of SCF and G-CSF
(0.1,1,10,100, and 1000 ng/mL), and cultur ed in the CO2
incubator. Each flask contained 1×106 cells. The me-
dium was completely renewed every 3 days. The fibro-
blast colonies were counted on day 12 of culture. Cell
clusters containing > 50 cells were scored as CFU-F
colonies. Based on the number of colonies generated in
the various concentrations of CSF, a dose-response
curve to each growth factor was graphed.

202 F. P. Tang et al. / J. Biomedical Science and Engineering 2 (2009) 200-207
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2.5. Alkaline Phosphatase (ALP) Detection
of MSCs 2.6. Statistical Analysis
Data were expressed as mean ± SEM of at least three
separate experiments. Comparisons were made using 2-
tailed unpaired Student t test and Mann-Whitney signed
rank test as appropriate. A P value of <0.05 was consid-
ered significant.
MSC (3×106 cells per well) were plated in 48-well
culture plates, then being induced by an osteogenic
supplement (1×10-7mol/L dexamethasone, 5.0 mmol/L
b- glycerophosphate, 50 mg/L ascorbic acid) and
treated with G-CSF and/or SCF at final concentrations
of 0.1, 1, 10, 100, and 1000 ng/ml for 5d at 37°C in an
atmosphere containing 5% CO2. Culture was washed
with phosphate-buffered saline (PBS), fixed in a
solution of cold 70% ethanol for 15 min and stained
for alkaline phosphatase (ALP) activity. For quantita-
tive analysis, the plates were washed thrice with
ice-cold PBS and lysed by two cycles of freezing and
thaw. Aliquots of supernatants were subjected to alka-
line phosphatase activity using an alkaline phosphatase
kit (Nanjing Jiancheng Bioengineering Institute,
China). The osteogenic differentiation rate was calcu-
lated according to the formula: (ALP activitytreated/ALP
activitycontrol -1)×100%.
3. RESULTS
3.1. Identification of MSCs
The expression of stem cell markers assessed with flow
cytometric ana lyses showed that after passag e 3 thes e cells
were nearly completely negative for haematopoietic cell
markers (CD34, CD45, and HLADR) and positive for
CD29, CD90 and CD105, which were markers of MSCs.
MSCs presented as a homogeneous fibroblast-like cell
population. They were positive in immuno-cytochemical
staining with anti-vimentin antibody. This population was
considered to be MSC based on its immunophenotype
profile (Figur e 1).
Figure 1. Immunophenotype of cultured MSCs. A. Immunophenotype of bone marrow MSCs by
FACS analysis. The immunophenotype was CD29+/CD45– (a),CD105+/CD34– (b), CD90+/
HLADR– (c). B. Immunofluorescence staining of vimentin were observed in all cells (d).
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F. P. Tang et al. / J. Biomedical Science and Engineering 2 (2009) 202-207 203
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3.2. The Expression of G-CSFR a nd C-Kit on
Surface of MSCs
We analysed the expression of receptors for G-CSF
(G-CSFR) and SCF (c-kit) on rabbit bone marrow MSCs.
Flow cytometric analysis showed that G- CSFR was
expressed at extremely level in MSCs (1.2 ±0.5%). Al-
though the expression of G-CSFR was higher after
G-CSF administration (4.2±1.6%), the difference was
not statistically significant (P>0.05). MSCs were shown
to constitutively express low levels of c-kit at the cell
surface, as shown by flow cytometric analysis. SCF
treatment induced a significant increase in the number of
c-kit+ cells. The number of c-kit+cells was significantly
larger in the SCF-treated group (28.4±4.8%) than in the
control group (13.6±3.6%) (P<0.05 vs. control group)
(Figure 2).
3.3. Effects of G-CSF/SCF on the Prolifera-
tion of MSCs
As well, we also found that G-CSF and SCF could pro-
mote MSCs proliferation significantly (*P<0.05,
**P<0.01). Treating MSCs with G-CSF and SCF
(0.1~100 ng/ml) resulted in a positive dose-dependent
increase in cell proliferation, and the maximal growth
rate was 42.2% and 34.2%, respectively. When the dose
reached 1000ng/ml, the growth rate stepped down, in-
stead. As time proceeded, cells in the group treated
with combination of G-CSF and SCF growed more
faster than the groups treated with G-CSF,SCF , or none
of them, and the same cell population was advanced over
2 to 3 days. Moreover, a shift to the left in the growth
curve and a advance in multiplication point was ob-
served. A comparison for the promotion of cell prolifera-
tion, the combination of G-CSF and SCF was superior to
the better of the two agents given alone (Figure 3).
3.4. CFU-F-Colony Formation of MSCs Re-
sponse to Different Doses of G-SCF
and SCF
MSCs have been recognized to derive from single-cell
suspensions of bone marrow by the selective growth of
plastic-adherent fibroblast-like cell colonies. Such a colony
of adherent marrow stromal cells, each derived from a sin-
gle precursor cell, is termed a CFU-fibroblast (CFU-F) [7].
The number of CFU-Fs formed per 1 x 106 MSCs plated
varied among groups. Treating MSCs with G-CSF and
SCF (0.1~100 ng/ml) resulted in a positive dose-dependent
increase in the formation of CFU-F. G-CSF and SCF sig-
nificantly increased the number of CFU-F compared with
control. The effect of G-CSF was powerful than SCF, and
the maximal CFU-F formation occurred with exposure to
the combination of G-CSF (100 ng/mL) and SCF (100
ng/mL). (*P<0.05, **P<0.01) (Figure 4).
Figure 2. Immunophenotyping of cultured MSCs by flow cytometric analysis. The histo-
grams show specific mAbs(CD114/CD117) in control group and treated group.(a) Control
group; (b) G-CSF treated group;(c)Control group; (D) SCF treated group.

204 F. P. Tang et al. / J. Biomedical Science and Engineering 2 (2009) 200-207
SciRes Copyright © 2009 JBiSE
Figure 3. Effects of G-CSF/SCF on the proliferation of MSCs . A. The effect of G-CSF and SCF on the proliferation rate
of MSCs. G-CSF and SCF at final concentrations of 0.1, 1, 10, 100, and 1000 ng/ml . B. To examine the effects of G-CSF,
SCF, alone or in combination on MSCs growth curve. G-CSF,100 ng/ml; SCF, 100 ng/ml .Values represent means
±SEM .The asterisk indicates statistical difference (*P < 0.05, **P < 0.01).
Figure 4. Colony-forming unit-fibroblast (CFU-F) assay.
CFU-F- colony formation by MSCs on re sponse t o differen t
doses of colony-stimulatin g factors (G-CSF and SCF, alone
or in combination). The optimal dose of SCF and G-CSF
was 100 ng/ml. The combination of G-CSF and SCF had the
best activity. C= control group. The results are presented a s
the number of CFU-F (mean±SEM). The asterisk indicates
statistical difference (*P < 0.05, **P < 0.01).
3.5. Effects of G-CSF and SCF on the Os-
teogenic Differentiation of MSC
In repeated ex vivo experiments, we observed that G-
CSF and SCF had obvious inhibitory action on the os-
teogenic differentiation of MSC. High Alkaline phos-
phatase (ALP) activity was an osteoblastic phenotype.
When cultured in osteogenic conditions, MSCs acquired
an osteoblastic morphology demonstrated by an
upregulation of ALP activity (Figure 5(a), 5(b)). ALP
activity in every group was measured quantitativly at
different time point. ALP activity of cells cultured with
G- CSF and SCF was low at one day, and increased
time-dependently. Inhibitory effect of G-CSF on ALP
activity superior to SCF, and the combination of G-CSF
and SCF had the most powerful effect, as compared to
control (P<0.05) (Figure 5(c)). The concentration-effect
relationship of G-CSF and SCF was described. It wasn’t
shown here that dose-response measurements generated
a linear plot of inhibitor concentration. The calibration
curve was validated and linear over the concentration
range from 0.1 to 100 ng/ml, and the maximal inhibitio n
rate was obtained at a dosage of 100 ng /ml (54.12％ and
34.38％, respectively)and did not increase more at the
dosage of 1000 ng/ml (*P<0.05,**P<0.01) (Figure
5(d)).
4. DISCUSSION
In addition to hematopoietic stem cells, it is now clear
that adult bone marrow contains a rare population of
mesenchymal stem/progenitor cells (MSCs) (0.01% to
0.001%) (18). MSCs are of great therapeutic potential
because of their ability to self-renew and differentiate
into multiple tissues. They can be extensively expanded
in vitro and, when cultured under specific permissive
conditions, retain their ability to differentiate into multi-
ple lineages including bone, cartilage, tendon, muscle,
nerve, and stromal cells [18,19]. There is increasing
evidence of the potential use of MSC infusion for clini-
cal purposes, such as hematopoietic support, tissue repair,
immunosuppressive cell therapy, and anticancer gene
therapy [20,21,22,23,24]. Thus, it is of great interest to
study which factors may have a role in MSC adhesion,
migration, expansion, maintenance of MSC stem cell
plasticity, and interaction with normal and pathologic
cells once the MSCs are recruited and included in prolif-
erating tissues.
The cytokine G-CSF is widely used to mobilize
stem/ progenitor cells. How G-CSF mobilizes stem
cells and progenitor cells from the bone marrow into
the circulation is not clear. In addition, G-CSF plays an
essential role in proliferation, survival, and differentia-
tion of granulocyte precursors in the marrow. Generally,
G-CSF acts by binding to its receptor (G-CSFR), a sin-
gle-chain member of the cytokine receptor superfamily,
which lacks tyrosine kinase activity. Binding of G-CSF
to its receptor induces the tyrosine phosphorylation of a

F. P. Tang et al. / J. Biomedical Science and Engineering 2 (2009) 202-207 205
SciRes Copyright © 2009 JBiSE
Figure 5. Effects of G-CSF and SCF on the osteogenic differentiation of MSC. (A,B) Alkaline phos-
phatase(ALP) staining was carried out using an Ca–Co staining method. Representative images of ALP
staining were shown. A, control group; B, G-CSF/SCF treated group. C. The effects of G-CSF/SCF on the
ALP activity in BMSCs (mU/mg protein). G-CSF,100 ng/ml; SCF, 100 ng/ml. D. Osteogenic inhibition
rate of G-CSF/SCF on MSCs. G-CSF and SCF at final concentrations of 0.1, 1, 10, 100, and 1000 ng/ml .
Values represent means ±SEM .The asterisk indicates statistical difference (*P < 0.05, **P < 0.01).
number of cellular proteins and activates signal trans-
duction pathways, including Ras/Raf/MAPK, PI3- kinase,
and JAK/STAT cascades [25,26,27]. But confusing re-
sults trickled in, flow cytometric analysis showed that
bone marrow MSCs expressed very low levels of
G-CSFR, even treated with G-CSF. This suggested that
an indirect mechanism might exist. For example, G-CSF
stimulation potentiates the homing abilities of cyto-
kine-stimulated BMSCs, an action that can be inhibited
by pretreatment with anti-CXCR4 antibodies [28]. In
contrast to the G-CSF receptor, the receptor for SCF,
possesses intrinsic tyrosine kinase activity. Binding of
SCF to c-kit induces kinase activation and transphos-
phorylation of the receptor chains. Recent exciting evi-
dence has shown the central role of SCF, c-kit, and ma-
trix metalloproteinase- 9 in the mobilization of stem and
progenitor cells from the bone marrow [29]. As shown
by flow cytometric analysis, MSCs were shown to con-
stitutively express c-kit at the cell surface and which
could be increased materially by SCF intervention. The
increasing evidence showed that the combination of
G-CSF and SCF could generate synergistic effect.
Bodine, et al. [30] found that mice bone marrow cells
collected 14 days after in vivo administration of G-CSF
and SCF have a 10 times greater ability to repopulate
than untreated bone marrow. Cell cycle analysis re-
vealed that the enhanced proliferative state induced by
SCF and G-CSF cotreatment was associated with a direct
effect of these cytokines on cell cycle distribution, spe-
cifically a marked shortening of the duration of G0/G1
[11]. Despite increased understanding of G-CSF and
SCF signaling pathways, the mechanism by which this
biologically and clinically important interaction between
SCF and G-CSF occurs remains poorly understood.
In vitro isolation and characterization of MSCs is
based on their adherence, rapid expansion in serum-
containing medium, expression of specific cell surface
antigens as well as their ability to differentiate into
various mesodermal tissues such as fat, bone, cartilage
and muscle [31,32,33,34]. Morphologically, MSCs in
their undifferentiated state are spindle shaped and resem-
ble fibroblasts. They do not express hematopoietic mark-
ers, but a specific pattern of molecules. At flow cytometry,
the isolated cells showed a homogeneous expression of

206 F. P. Tang et al. / J. Biomedical Science and Engineering 2 (2009) 200-207
SciRes Copyright © 2009 JBiSE
markers commonly used to identify hMSCs, i.e., CD29,
CD90, CD105 positivities, and CD34, CD45, HLADR
negativities, consistent with that reported for bone mar-
row-derived MSCs [20,34]. Although these markers have
been used by various groups, there is still no general
consensus on th e optimal marker combination for MSCs.
At present we did not know how long MSCs will main-
tain innate characteristics, so we used early MSCs not
exceeding 4 passages based on the assumption that early
passage cells would be more likely to have the innate
characteristics of MSCs.
Cell proliferation was determined using MTT assay,
which showed the combination of G-CSF and SCF was
superior to the better of the two agents given alone.
Usually, MSCs were typically defined by their capacity
to adhere on plastic and form a fibroblastic colony
(CFU-F). The colony forming unit fibroblast (CFU-F)
assay was a well-established method for the quantifica-
tion of marrow stromal cells (MSCs). We observed that
G-CSF/SCF enhanced ex vivo MSC proliferation, and
treating MSCs with G-CSF and SCF (0.1~100 ng/ml)
resulted in a positive dose-dependent increase in the
formation of CFU-F. G-CSF/SCF proliferative effect on
MSCs was direct, dose dependent, long lasting. In addi-
tion, ALP activity of cells cultured with G-CSF and SCF
was low at one day, and increased time-dependently.
MSC differentiation potential was not affected obviously
by the enhancement of self-renewal, as the proliferative
effect was not associated with induced differentiation.
On the whole, these studies demonstrated that MSCs
responsed to G-CSF, SCF, and to G-CSF plus SCF in a
manner that suppressed differentiation, and promoted
proliferation and self-renewal, and supported the view
that these factors could act synergistically. And the ef-
fects of G-CSF/SCF on MSCs give the cu e to understand
better the biology and the role of MSCs. However, the
biochemical mechanism underlying this activity remains
to be resolved .
5. ACKNOWLEDGEMENTS
The authors appreciate the help of the members of the Center Labora-
tory and the Osteonecrosis research team at the Department of Ortho-
pedic Surgery of Union Hospital, Tongji Medical College.
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