Different effects of granulocyte colony-stimulating factor and erythropoietin on erythropoiesis
Chen et al. Stem Cell Research & Therapy
Different effects of granulocyte colony- stimulating factor and erythropoietin on erythropoiesis
Tzu-Lin Chen 0 2
Ya-Wen Chiang 0 2
Guan-Ling Lin 1
Hsin-Hou Chang 1 2
Te-Sheng Lien 2
Min-Hua Sheh 2
Der-Shan Sun 1 2
0 Equal contributors
1 Institute of Medical Sciences, Tzu-Chi University , Hualien , Taiwan
2 Department of Molecular Biology and Human Genetics, Tzu-Chi University , No. 701, Section 3, Zhong-Yang Road, Hualien 97004, Taiwan , Republic of China
Background: Red blood cells are the most abundant cells in the blood that deliver oxygen to the whole body. Erythropoietin (EPO), a positive regulator of erythropoiesis, is currently the major treatment for chronic anemia. Granulocyte colony-stimulating factor (G-CSF) is a multifunctional cytokine and a well-known regulator of hematopoietic stem cell proliferation, differentiation, and mobilization. The use of EPO in combination with G-CSF has been reported to synergistically improve erythroid responses in a group of patients with myelodysplastic syndromes who did not respond to EPO treatment alone; however, the mechanism remains unclear. Methods: C57BL/6 J mice injected with G-CSF or EPO were used to compare the erythropoiesis status and the efficiency of erythroid mobilization by flow cytometry. Results: In this study, we found that G-CSF induced more orthochromatophilic erythroblast production than did EPO in the bone marrow and spleen. In addition, in contrast to EPO treatments, G-CSF treatments enhanced the efficiency of the mobilization of newly synthesized reticulocytes into peripheral blood. Our results demonstrated that the effects of G-CSF on erythropoiesis and erythrocytic mobilization were independent of EPO secretion and, in contrast to EPO, G-CSF promoted progression of erythropoiesis through transition of early stage R2 (basophilic erythroblasts) to late stage R4 (orthochromatophilic erythroblasts). Conclusions: We demonstrate for the first time that G-CSF treatments induce a faster erythropoiesis-enhancing response than that of EPO. These findings suggest an alternative approach to treating acute anemia, especially when patients are experiencing a clinical emergency in remote areas without proper blood bank supplies.
Granulocyte colony-stimulating factor; Erythropoietin; Erythropoiesis; Reticulocyte; Erythrocytic mobilization
Red blood cells (RBCs) are the most abundant cells in
the blood and are essential for oxygen transport around
the body. After birth, the site of erythropoiesis switches
from the fetal liver to the bone marrow (BM) and spleen.
In humans, the BM is the major site for steady-state
erythropoiesis. In contrast, in mice, in addition to the
BM, the spleen plays a minor role (10%) in steady-state
]. Under stressful conditions, such as
bleeding or acute anemia, the spleen in humans and
mice plays a major role in stress erythropoiesis [
Hematopoietic stem cells (HSCs) reside in BM niches
where cytokines or signals generated by stromal cells
consisting of endothelial cells, osteoblasts, and
macrophages regulate the differentiation of various blood
lineage, including erythroid cells [
]. HSCs first
differentiate into megakaryocyte-erythroid progenitors,
subsequently into the burst-forming unit-erythroids (BFU-Es),
and finally into the colony-forming unit-erythroids
(CFU-Es). CFU-Es are more mature than BFU-Es and
appear earlier, namely at 2–3 days in mice and 5–8 days
in humans, compared with 5–8 days in mice and 10–14
days in humans for BFU-Es. In addition, CFU-Es form
smaller colonies than do BFU-Es when cultured in
]. Erythropoietin (EPO), a 30.4-kDa
glycoprotein mainly synthesized by the kidneys, is the
main regulator of erythroid cell proliferation,
differentiation, and survival . EPO production is upregulated
under hypoxic conditions through the activity of the
hypoxia-inducible transcription factor (HIF) [
EPO receptor (EPOR) is expressed dominantly on
CFUEs and gradually downregulated during erythroid
]. Upon stimulation initiated by EPO
binding to the EPORs, CFU-Es develops into
proerythroblasts, subsequently into basophilic erythroblasts, then
polychromatic erythroblasts, and finally orthrochromatic
erythroblasts. The final stage of erythroid differentiation
involves the enucleation and maturation of reticulocytes
into circulating erythrocytes [
Anemia can develop from loss of RBCs, a reduction in
RBC production, increased destruction of RBCs, or a
shorter RBC lifespan. The World Health Organization
defines anemia as a hemoglobin level lower than 12 g/dl in
women and lower than 13 g/dl in men [
]. Except for
patients with inherited hematopoietic disease, the highest
rates of anemia are observed in patients with chronic
diseases such as those of the kidney and heart, cancer,
inflammatory bowel disease, rheumatoid arthritis, and human
immunodeficiency virus (HIV) [
]. Recombinant human
EPO (rhEPO) has been used medically for more than
20 years and has generated a multibillion dollar market
annually. However, since 1998, some severe adverse effects
such as EPO-associated pure red cell aplasia by long-term
EPO injection induced neutralizing antibodies have been
]. Other side effects such as hypertension,
increased risk of venous thromboembolism, stroke, and
death have also been reported . Currently available
erythropoiesis-stimulating agents (ESAs) are variations of
EPO generated by human cells or Chinese hamster ovary
cells. New ESAs (e.g., peptide-based erythropoietic agents),
activation of endogenous EPO production through HIF
stabilization and GATA1 inhibition, and EPO gene therapy
have been developed; however, none of these new methods
have shown efficacy superior to that of existing ESAs [
Taken together, the development of new anemia therapies
with satisfactory levels of efficacy and safety and faster
action is required.
Our previous study found that granulocyte
colonystimulating factor (G-CSF) could mobilize newly
synthesized erythrocytes to the peripheral blood (PB) and promote
erythrocytic differentiation and proliferation in vitro and ex
]. The present study further investigated the
differences between G-CSF and EPO on erythropoiesis. After
mice had been treated with G-CSF and EPO, the
erythropoiesis status in the BM and spleen were compared using
flow cytometry. The stimulation of early erythroid
progenitor subsets and temporal regulation of erythroid progenitor
mobilization were characterized. The mechanism of G-CSF
promotion of erythropoiesis was also discussed.
Toxins and mice
B. anthracis lethal toxin (LT) was provided by the Institute
of Preventive Medicine, National Defense Medical Center
(Taipei, Taiwan), and purified as previously described [
EGFP mice [C57BL/6 J-Tg (Pgk1-EGFP) 03Narl] provided
by Professor Chou CK [
] and C57BL/6 J mice were
purchased from the National Laboratory Animal Center
(Taipei, Taiwan). Animals were maintained in a specific
pathogen-free environment in the experimental animal
center of Tzu Chi University (Hualien, Taiwan).
Analysis of erythropoiesis status in the BM and spleen
C57BL/6 J mice (male, 10–12 weeks old) were
retroorbitally injected with 2 IU/g rhEPO (Neorecormon®,
Roche, Mannheim, Germany) or 55 μg/kg recombinant
human G-CSF (Filgrastim, Kirin, Tokyo) once daily on 3
consecutive days. Animals treated with an identical volume of
saline served as negative controls. BM cells were isolated as
previously described [
]. Spleens were minced using the
plunger of a 50-ml syringe and resuspended using a P1000
Pipetman (Gilson, Middleton, WI, USA) to create a
singlecell suspension. Cells were blocked with RPMI-1640
medium (Gibco Laboratories) containing 5% bovine serum
albumin at 37 °C for 1 h and subsequently incubated with
rat anti-mouse fluorescein isothiocyanate-conjugated CD71
antibody (BioLegend) and rat antimouse allophycocyanin
(APC)-conjugated TER-119 antibody (BD
Immunocytometry System) at 37 °C for 1 h. After washing with
phosphatebuffered saline (PBS), the cells were resuspended in 1 ml of
PBS and analyzed using a Beckman Coulter Gallios™ flow
cytometer (Beckman Coulter, CA, USA).
Flow cytometry analysis of mobilized erythrocytes in
Enhanced green fluorescent protein (EGFP) mice (male,
8 months old) were injected with 55 μg/kg G-CSF or
2 IU/g EPO retro-orbitally once daily on 3 consecutive
days. PB was collected retro-orbitally at 20, 40, and 60 h
after initial injection. Cells were incubated with rat
antimouse APC-conjugated TER-119 antibody at 37 °C for
1 h and subsequently with 5 μM of RNA-selective dye
] at 37 °C for 30 min. After washing with PBS,
the cells were analyzed using a Beckman Coulter
Gallios™ flow cytometer.
EPO and soluble P-selectin immunoassay
C57BL/6 J mice (male, 10–12 weeks old) were treated with
55 μg/kg G-CSF administered by retro-orbital injection
once daily on 2 consecutive days for the EPO immunoassay
or 5 consecutive days for the soluble P-selectin
immunoassay. Animals treated with an identical volume of
saline served as negative controls. C57BL/6 J mice that were
retro-orbitally injected with 1.5 mg/kg LT and untreated
served as positive controls for the EPO immunoassay and
the soluble P-selectin immunoassay, respectively. PB (serum
for the EPO immunoassay and plasma for the soluble
Pselectin immunoassay) was collected retro-orbitally at 22,
44, and 66 h after initial injection for the EPO
immunoassay, and day 0 before initial injection and days 1–5 after
initial injection for the soluble P-selectin immunoassay. The
EPO and soluble P-selectin immunoassays were performed
using an enzyme-linked immunosorbent assay (ELISA) in
accordance with the manufacturers’ instructions
(Quantikine® Mouse/Rat EPO immunoassay, R&D systems; Mouse
P-selectin ELISA kit, RayBiotech, Inc., USA).
Hematopoietic parameter detection
C57BL/6 J mice (male, 10–12 weeks old) were treated
with 55 μg/kg G-CSF or 1 mg/kg recombinant mouse
Pselectin (purified mouse P-selectin–IgG fusion protein;
BD Pharmingen) once daily on 2 consecutive days
through retro-orbital injection. The hematopoietic
parameters were measured on day 2 after initial injection
using an automated hematology analyzer (XP-300,
All quantifiable data are presented as mean ± standard
deviation (SD). Statistical analysis was conducted by
one-way analysis of variance (ANOVA) followed by the
post-hoc Bonferroni-corrected t test using the SPSS
software, version 17.0 (SPSS Inc., Chicago, IL, USA). P
values lower than 0.05 indicated significant differences.
G-CSF enhanced R4 erythroblast cell production to a greater extent than EPO did in the BM and spleen
To compare the erythropoiesis status after G-CSF and
EPO treatments, C57BL/6 J mice were retro-orbitally
injected with G-CSF or EPO once daily for 3 consecutive
days. Erythropoiesis progression was analyzed in the BM
(Fig. 1a) and spleen (Fig. 2a) through flow cytometry.
Using antibodies against the erythroid markers CD71 and
TER119, erythroblasts were divided into the following
four populations from early to late sequential
differentiation stages: proerythroblasts (R1, CD71high/TER-119med),
basophilic erythroblasts (R2, CD71high/TER-119high), late
basophilic and polychromatophilic erythroblasts (R3,
CD71med/TER-119high), and orthochromatophilic
erythroblasts (R4, CD71low or −/TER-119high) (Figs. 1b and
18, 21, 23
]. In agreement with the findings of other
studies, in mouse BM, G-CSF enhanced more
nonerythroid cell populations (NE, TER-119−) [
] than EPO did
(Fig. 1c). In contrast, EPO substantially increased the total
number of erythroid cells [
] (Fig. 1d). Although total
erythroid cell counts in the BM of mice from the
G-CSFtreated groups were lower than those of mice from the
EPO-treated groups, G-CSF elicited a markedly larger R4
population than EPO did (Fig. 1e).
The spleen is a secondary organ for erythropoiesis but
not for granulopoiesis [
]. In characterizing the
differentiation progression in the mouse spleen, we found
that G-CSF treatments did not increase the nonerythroid
cell population or total erythroid cell number (Fig. 2c, d).
Compared with the responses in the BM, EPO
upregulated the percentage of early erythroblasts (R1 and R2)
and downregulated the late stages of erythroblasts (R3 and
R4) (Fig. 2e), whereas G-CSF reduced the percentage of
early R1, R2, and R3 cells and considerably increased the
number of late-stage R4 erythroblasts (Fig. 2e). These
results collectively suggest that G-CSF enhances late-stage
R4 erythroblast cell production to a greater extent than
EPO does in the BM and spleens of mice.
G-CSF mobilized more newly synthesized reticulocytes than EPO did
Using an EGFP transgenic mouse model, we found that
G-CSF caused newly synthesized erythrocytes to
mobilize to the PB and induced erythrocytes to mobilize
into the PB faster than EPO [
]. After using the same
experimental strategy (Fig. 3a), our data revealed that
the mobilization efficiencies of newly synthesized
erythrocytes (R1, EGFPhigh/TER-119high) were higher at 20 h
under G-CSF treatment than under EPO, and were
similar between G-CSF and EPO treatments at 40 and 60 h
after initial injection (Fig. 3b, c). Because G-CSF
promoted more R4 erythroblast synthesis than EPO did in
the BM and spleen (Figs. 1 and 2), we hypothesized that
G-CSF may promote reticulocyte mobilization to a
greater extent than EPO. Because only reticulocytes
exhibit residue ribonucleic acid (RNA) expression among
newly synthesized erythrocyte populations [
], F22, a
RNA-selective dye [
], combined with TER-119 was
used to discriminate reticulocytes from matured
erythrocytes in newly synthesized erythrocytes. Over two time
courses, 20 and 40 h, G-CSF preferred to mobilize newly
synthesized reticulocytes (F22high/EGFPhigh/TER-119high)
to a greater extent than EPO did (Fig. 3b, d).
G-CSF-dependent enhancement of erythropoiesis and
RBC mobilization were not mediated by eliciting EPO and the P-selectin pathway
The transcription factor HIF-1α can be upregulated by
GCSF and then binds to an EPO promoter to increase
circulating EPO levels after five consecutive G-CSF injections
]. Accordingly, we hypothesized that G-CSF may
stimulate erythropoiesis and mobilize erythrocytes by increasing
the synthesis of EPO. Our experimental design is shown in
Fig. 4a. Treatment with Bacillus anthracis, a LT that can
induce hypoxia-elicited EPO secretion in mice [
], was used
as a positive control. In agreement with a previous study
], our results revealed that circulating EPO levels
gradually increased following LT administration in mice [
contrast, circulating EPO levels were not upregulated at all
examined time points after G-CSF treatment in mice (Fig.
4b–d). These results suggest that the G-CSF-dependent
enhancement of erythropoiesis and RBC mobilization are not
mediated by eliciting EPO.
P-selectin is widely recognized as a cell adhesion
receptor that mediates leukocyte rolling through binding
to glycosylated moieties of the main ligand P-selectin
glycoprotein ligand 1 (PSGL-1) [
]. The same property
of P-selectin enables HSCs to adhere to stromal cells,
especially vesicular endothelial cells, through P-selectin
(HSC)–PSGL-1 (stroma) interactions [
induced greater and faster mobilization of myeloid cells
from BM in PSGL-1 knockout mice than in wild-type
]. Studies have also suggested that G-CSF
administration increases levels of circulating soluble
]. This evidence collectively suggests that
P-selectin/PSGL-1-mediated binding between erythroid
precursors and stromal cells may partially control the
mobilization and release of progenitor cells from BM
into the blood stream, thereby indicating the possibility
that G-CSF may enhance erythrocytic mobilization by
eliciting circulating soluble P-selectin to compete with
the P-selectin/PSGL-1-mediated precursor–stroma
interaction. To reproduce G-CSF-mediated elicitation of
circulating soluble P-selectin, G-CSF was injected into
C57BL/6 J mice on 5 consecutive days (Fig. 5a). The
levels of circulating soluble P-selectin increased
significantly after G-CSF injection (Fig. 5b). Next, we designed
an experiment to examine whether soluble P-selectin
can increase the RBC counts in the PB (Fig. 5c).
Although two doses of G-CSF increased the white blood
cell (WBC) and RBC counts in the PB, the same effect
was not observed when two equivalent doses of soluble
P-selectin were applied (Fig. 5d, e). This finding suggests
that G-CSF mobilization of erythrocytes into the PB is
not primarily mediated through the P-selectin pathway.
This study demonstrated that stimulation of
erythropoiesis in mice with G-CSF is different to that with
EPO. Treatment with G-CSF elicited greater R4
erythroblast production than did treatment with
EPO in the BM and spleen. Additionally, G-CSF
induced higher mobilization levels of newly
synthesized reticulocytes from the BM and spleen to the
PB than EPO did.
G-CSF has been a US Food and Drug Administration
(FDA)-approved drug for treating various hematopoietic
defects for many years. In contrast to EPO, elicitation of
neutralizing antibodies after G-CSF treatments has not been
reported. Myelodysplastic syndromes (MDS) are a diverse
group of diseases characterized by ineffective hematopoiesis
and peripheral cytopenia with unknown mechanisms [
]. As one of the disease hallmarks, approximately 85% of
patients with MDS manifest anemia [
between G-CSF and EPO treatments has ameliorated anemic
conditions in groups of patients with MDS who did not
respond to EPO treatments [
]. This suggests that the
erythropoiesis-enhancing effect of the combination of
GCSF and EPO has been empirically recognized by physicians
and the medical community. Studies have shown that
GCSF has antiapoptotic effects that protect erythroblasts
from cell death [
]. However, the effects and
mechanisms of G-CSF on erythropoiesis in vivo must be
elucidated. Our results demonstrate that G-CSF could promote
erythropoiesis by stimulating R4 erythroblast production
and then mobilizing newly synthesized reticulocytes to a
greater extent than EPO treatment. The effects partially
agree with the findings of our previous study that small and
medium progenitor cell colonies appeared earlier when
tested with an erythroid colony-forming cell assay . In
contrast, treatments with G-CSF alone do not exhibit
ameliorative effects on anemia in patients with MDS [
accordance with these findings, in our erythroid
colonyforming cell experiment, G-CSF exhibited erythropoietic
properties only when used with EPO . This finding
implied that G-CSF was unable to stimulate erythropoiesis
without EPO. The EPOR is expressed dominantly on
CFUEs and gradually downregulated during erythroid
]. In addition, the G-CSF receptor is expressed
on erythroid progenitors . Because late-stage
erythroblasts (R3 or R4) were upregulated after G-CSF treatment,
synergism between G-CSF and EPO is likely accomplished
by accelerating the erythropoietic process into the late stage.
Recently, papers reported that G-CSF treatments enhanced
hematopoietic stem and progenitor cell mobilization by
enhancement of dipeptidylpeptidase 4 (CD26) activities and
vascular permeability in the BM [
]. This evidence may
provide an additional explanation as to why G-CSF induced
higher mobilization levels of newly synthesized reticulocytes
from the BM and spleen to the PB than that of EPO.
Although the responses that occur in BM with multilineage
cell interactions are difficult to analyze, the mechanism
warrants further investigation.
In conclusion, although G-CSF combined with EPO has
been administered to treat anemia in patients with MDS,
aplastic anemia, and HIV for many years [
38, 39, 46, 47
the mechanism through which G-CSF promotes
erythropoiesis remains unclear. This study demonstrates that
GCSF promotes erythropoiesis independent of the secretion
of EPO and soluble P-selectin. In addition, G-CSF induces
the production of levels of R4 erythroblasts to a greater
extent than EPO does in the BM and spleen, and
treatment with G-CSF mobilizes more newly synthesized
reticulocytes to the PB than EPO does. These findings indicate
an alternative method for ameliorating anemia, especially
in situations where a patient is in immediate need of
oxygen, such as those involving infectious diseases  or
with clinical urgency in remote areas.
APC: Allophycocyanin; BFU-E: Burst-forming unit-erythroid; BM: Bone marrow;
CFU-E: Colony-forming unit-erythroid; EGFP: Enhanced green fluorescent
protein; ELISA: Enzyme-linked immunosorbent assay; EPO: Erythropoietin;
EPOR: Erythropoietin receptor; ESA: Erythropoiesis-stimulating agent;
FDA: Food and Drug Administration; G-CSF: Granulocyte colony-stimulating
factor; HIF: Hypoxia-inducible transcription factor; HIV: Human
immunodeficiency virus; HSC: Hematopoietic stem cell; LT: lethal toxin;
MDS: Myelodysplastic syndromes; PB: Peripheral blood; PBS:
Phosphatebuffered saline; PSGL-1: P-selectin glycoprotein ligand 1; RBC: Red blood cell;
rhEPO: Recombinant human erythropoietin; RNA: Ribonucleic acid;
SD: Standard deviation
We greatly appreciate Professor Chang YT (Department of Chemistry,
National University of Singapore) for kindly providing the RNA-selective
dye F22. We are grateful to Mr. Chen CC and the Department of
Laboratory Medicine (Hualien Tzu Chi Medical Center) for their assistance with
the complete blood count blood test. We thank Professor Shiue
CN (Department of Molecular Biology and Human Genetics, Tzu Chi
University) for his valuable suggestions. We thank Professors Kau JH, Hsu HL,
and Mr. Huang HH (Institute of Preventive Medicine, National Defense
Medical Center) for generously providing anthrax LT. We also thank
Professor Wang MH and his team (Experimental Animal Center, Tzu Chi
University) for maintaining the experimental animals and pathogen-free
environments. We acknowledge Wallace Academic Editing for editing
This work was supported by the Ministry of Science and Technology, Taiwan
Availability of data and materials
All data generated and/or analyzed during this study are available from the
corresponding author upon reasonable request.
TLC, YWC, GLL, TSL, and MHS contributed by performing the experiments
and analyzing the data. HHC designed the experiments and edited the
manuscript. DSS designed the experiments, composed the main manuscript,
and directed the study. All authors read and approved the final manuscript.
The research protocols associated with the experimental mice were
approved by the Institutional Animal Care and Use Committee of Tzu Chi
University (approval no. 102087) and the National Defense Medical Center
(approval no. AN-100-04).
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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