2002 100: 312-317
Edo Vellenga and Gerald de Haan
Estelle J. K. Noach, Albertina Ausema, Jan H. Dillingh, Bert Dontje, Ellen Weersing, Imre Akkerman,
increases donor cell engraftment after bone marrow transplantation in
Growth factor treatment prior to low-dose total body irradiation
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Estelle J. K. Noach, AlbertinaAusema, Jan H. Dillingh, Bert Dontje, Ellen Weersing, ImreAkkerman, Edo Vellenga, and Gerald de Haan
Low-toxicity conditioning regimens prior
to bone marrow transplantation (BMT) are
widely explored. We developed a new
protocol using hematopoietic growth fac-
tors prior to low-dose total body irradia-
tion (TBI) in recipients of autologous
transplants to establish high levels of
long-term donor cell engraftment. We hy-
pothesized that treatment of recipient
mice with growth factors would selec-
tively deplete stem cells, resulting in suc-
cessful long-term donor cell engraftment
treated for 1 or 7 days with growth factors
(stem cell factor [SCF] plus interleukin 11
[IL-11], SCF plus Flt-3 ligand [FL], or
granulocyte colony-stimulating factor [G-
CSF]) prior to low-dose TBI (4 Gy). Donor
cell chimerism was measured after trans-
High levels of donor cell engraftment
were observed in recipients pretreated
for 7 days with SCF plus IL-11 or SCF plus
FL. Although 1-day pretreatments with
these cytokines initially resulted in re-
duced donor cell engraftment, a continu-
ous increase in time was observed, finally
resulting in highly significantly increased
levels of donor cell contribution. In con-
trast, G-CSF treatment showed no benefi-
cial effects on long-term engraftment. In
vitro stem cell assays demonstrated the
effect of cytokine treatment on stem cell
numbers. Donor cell engraftment and
number of remaining recipient stem cells
after TBI were strongly inversely corre-
lated, except for groups treated for 1 day
with SCF plus IL-11 or SCF plus FL. We
conclude that long-term donor cell en-
graftment can be strongly augmented by
treatment of recipient mice prior to low-
dose TBI with hematopoietic growth fac-
tors that act on primitive cells. (Blood.
© 2002 by TheAmerican Society of Hematology
Low-toxicity conditioning regimens are increasingly applied in
settings of allogeneic or autologous bone marrow transplantation
(BMT) for an expanding variety of clinical applications.1-6Espe-
cially for transplantation protocols in gene therapy, or treatment of
autoimmune diseases, a nonmyeloablated bone marrow environ-
ment is desired for autologous stem cells to engraft effectively.7-10
ing regimens to prevent rejection of the donor graft and to create a
microenvironment in which transplanted (stem) cells are able to
migrate efficiently to the bone marrow and “home” to appropriate
niches.11-14No unequivocal vision on the mechanism of homing
and engraftment exists and discussion continues on whether
conditioning prior to BMT leads to creating actual “space” in the
bone marrow microenvironment or whether competition is induced
between remaining recipient stem cells and infused donor cells.15-19
A recent study of Ponomaryov et al20suggests that the up-
regulation of stromal derived factor-1 (SDF-1) in response to total
body irradiation (TBI) is important in the homing process, under-
scoring the role of humoral factors produced by stromal cells. In
addition, evidence has been obtained that the cell cycle status of the
transplanted cells plays a significant role and can be manipulated
with growth factor treatment.21-24
We hypothesize that a strong depletion of the recipients’
hematopoietic system prior to BMT will lead to enhanced donor
cell engraftment. Specifically, we questioned whether long-term
donor cell engraftment can be affected by pretreatment of trans-
plant recipients with various hematopoietic growth factors prior to
low-dose TBI. This concept is supported by data showing that
chemotherapy (5-fluorouracil) after growth factor treatment with
stem cell factor (SCF) can result in severe stem cell depletion.25,26
Apparently, growth factor treatment induces stem cells to prolifer-
ate and increase their sensitivity toward myeloablative treatment.
We administered growth factors affecting primitive stem cells (SCF
plus interleukin-11 [IL-11] or SCF plus Flt-3 ligand [FL]) or
progenitor cells (granulocyte colony-stimulating factor [G-
CSF]). Our results demonstrate that long-term donor cell
engraftment after low-dose TBI can be strongly augmented by
pretreatment of recipients with hematopoietic growth factors
acting on primitive cells.
Materials and methods
In all experiments female C57BL/6 mice (Harlan Nederland, Horst, The
Netherlands) were used as recipients. Male C57BL/6.SJL.Ptprcacongenic
mice (kindly provided by Ms Helen Silvius, LUMC, Leiden, The Nether-
lands) were used as donor mice. Leukocytes from these mice express the
CD45.1 antigen, whereas cells from recipients express CD45.2.Throughout
this paper, we will name recipient mice CD45.2 and donors CD45.1.
From the Department of Stem Cell Biology, University of Groningen and
Division of Hematology, University Hospital Groningen, Groningen, The
SubmittedApril 2, 2001; accepted February 12, 2002.
Supported by the Dutch Cancer Society, grant NKB1996-1204. G.d.H. is a
fellow of the Royal NetherlandsAcademy ofArts and Sciences (KNAW).
Reprints: Gerald de Haan, Department of Stem Cell Biology, University of
Groningen,ADeusinglaan 1, NL-9713AV Groningen, The Netherlands; e-mail:
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by TheAmerican Society of Hematology
312 BLOOD, 1 JULY 2002?VOLUME 100, NUMBER 1
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The following growth factors/growth factor combinations were administered for
either 1 or 7 days: G-CSF, SCF plus IL-11, or SCF plus FL. Recombinant rat
pegylated SCF and recombinant human G-CSF were donated by Amgen
(Thousand Oaks, CA). Recombinant human IL-11 was a gift from Genetics
Institute (Cambridge, MA); FL was provided by Immunex (Seattle, WA).
Growth factors were appropriately diluted, mixed, and administered either for 1
TBI, or during 7 days by subcutaneously implanted osmotic minipumps (Alzet
?g per mouse per day, IL-11 at a dose of 2.0 ?g per mouse per day, and FLat a
Two hours after growth factor treatment, mice received a single dose of
4 Gy sublethalTBI at a dose rate of 0.89 Gy/min, using a137Cs ?-irradiation
unit (IBL637, CIS Biointernational, Gif-sur-Yvette Cedex, France).
Each group consisted of 9 to 11 mice; 3 were allocated for the
cobblestone area–forming cell (CAFC) assay and the remaining animals
were used as transplant recipients. Control mice, not treated with growth
factors, were irradiated similarly.
Growth factor administration in mice leads to migration of cells from bone
marrow to spleen and liver.27-29Because we wanted to assess the extent of
stem cell depletion in recipients conditioned with TBI, we estimated the
total number of stem cells before and after irradiation with CAFC assays on
bone marrow, spleen, and liver cells, and calculated the percentage of
surviving cells after irradiation. Bone marrow suspensions were obtained
by flushing either 1 or 2 femurs of each mouse according to standard
procedures. Spleen suspensions were acquired as described.29Liver suspen-
sions were obtained by dissecting and weighing the complete liver.Apiece
of about 10% was prepared and gently pressed through a stainless steel
sieve after which liver cells were collected. Single cells were obtained by
part of the liver was weighed again and the exact fraction of used liver
material was calculated. Marrow, spleen, and liver cellularities were
determined using a Coulter Counter (Coulter Electronics, Dunstable,
United Kingdom) to calculate the appropriate cell numbers for the stem cell
assays. Femur cellularity was assumed to represent 6% of total marrow
cellularity.30,31Each data point in the CAFC assay was obtained by pooling
bone marrow, spleen, or liver suspensions of 3 mice. The total number of
CAFCs per mouse was calculated by adding the CAFC number of bone
marrow plus spleen plus liver.
The CAFC assay, a limiting dilution in vitro assay to distinguish
between different subsets of stem cells,32,33was performed exactly as
Transplantation and determination of peripheral blood
Twenty-four hours after sublethal irradiation, female CD45.2 recipient mice
received transplants of 3 ? 106freshly harvested unfractionated bone
marrow cells from male CD45.1 donor mice by intravenous injection into
the retro-orbital plexus. Peripheral blood cell chimerism in recipients was
determined in 2 independent groups of mice (n ? 3-4/group) by flow
cytometry, at first every 2 weeks (up to week 12), and thereafter every 4
weeks until week 40. In short, 50 ?Ltail blood was drawn from each mouse
and erythrocytes were eliminated by hypotonic lysis (0.16 M NH4Cl,
1.0 ? 10?4M EDTA, 0.017 M NaCl, 5 minutes at room temperature).
Leukocytes were washed and stained with donor-specific anti-CD45.1
monoclonal antibody (mAb) conjugated with phycoerythrin (PE) and either
host-specific anti-CD45.2 labeled with fluorescein isothiocyanate (FITC) or
FITC-labeled anti–Gr-1, anti-B220, or anti-CD8a (all mAbs from Pharmin-
gen, San Diego, CA). Stained cells were washed and resuspended in 200 ?L
phosphate-buffered saline plus 2% fetal calf serum. Two-color analysis
(FACSCalibur; Becton Dickinson, Palo Alto, CA) was performed to
determine the percentage of donor- and host-derived leukocytes or donor B
and T lymphocytes and granulocytes to check for multilineage
Unpaired Student t tests were performed to determine significant differ-
ences between engraftment levels of growth factor–treated mice and
untreated controls. The 95% confidence limits (CLs) were calculated for
each CAFC subset. Nonoverlapping 95% CLs were interpreted as P ? .05.
Stem cell numbers after growth factor administration
before and after TBI
To determine the effect of growth factor treatment on stem cell
pools, we performed CAFC assays on bone marrow, spleen, and
liver cells of mice treated for 1 or 7 days with SCF plus IL-11, SCF
plus FL, or G-CSF. Table 1 provides an overview of the relative
CAFC frequencies that were measured in bone marrow, spleen, and
liver cells. Figure 1 demonstrates the corresponding total CAFC
pool size in each organ and in the entire mouse. Figure 1A-C
depicts the absolute numbers of CAFCs–day 7 (progenitor cells)
per total mouse (ie, in bone marrow plus spleen plus liver). In 1-day
growth factor–treated mice no significant expansion of progenitor
cells was observed.Although the relative frequency of CAFCs-day
7 in mice treated with SCF plus IL-11 was higher than in control
mice, the cellularity in the femur of these mice was lower
(12 ? 106/femur vs 22 ? 106/femur). In contrast, after the 7-day
growth factor treatments, progenitor cells expanded 3- to 4-fold
and migrated from the bone marrow toward the liver and, in
particular, the spleen. This effect was most notable with the
combination of SCF plus IL-11.
Table 1. Progenitor and stem cell frequency in bone marrow, spleen, and liver of growth factor–treated mice
SCF ? IL-11SCF ? FLG-CSF
1 d 7 d1 d7 d 1 d7 d
Shown are the CAFC-day 7 and CAFC-day 35 frequency/105nucleated cells.
Significant differences compared to control values (nonoverlapping 95% CLs) are depicted in bold.
ND indicates not detected.
CYTOKINES BEFORE LOW-DOSE TBI IN BMT CONDITIONING 313BLOOD, 1 JULY 2002?VOLUME 100, NUMBER 1
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Primitive stem cells (CAFCs–day 35) demonstrated a different
pattern in response to growth factor treatment (Figure 1D-F).
Treatment with SCF plus IL-11 or G-CSF for 1 day resulted in a
2.5- to 3-fold expansion of stem cells. Continuation of the growth
factor treatment for an additional 6 days did not result in further
expansion, but led to a significant shift of primitive cells toward the
spleen (Table 1). In contrast, 1-day treatment with SCF plus FLdid
not result in an increased number of stem cells, but stem cells
expanded about 4-fold after a 7-day treatment. Again, most cells
resided in the spleen.
To establish the radiosensitivity of progenitors and primitive
stem cells, we calculated the percentage of surviving cells 24 hours
after irradiation per total mouse (Figure 2). Compared toTBI alone,
1 day of SCF plus IL-11 or SCF plus FL pretreatment resulted in a
4-fold reduced radiosensitivity of progenitor cells (CAFCs–day 7),
whereas radiosensitivity of primitive stem cells (CAFCs–day 35)
was reduced 2.7-fold (Figure 2A).Ashort G-CSF treatment did not
significantly change the radiosensitivity of cells compared to
controls. In contrast, after 7 days of growth factor administration,
both CAFC subsets were more severely depleted than in mice
conditioned with TBI alone (Figure 2B). Moreover, a 7-day growth
factor treatment led to a much more pronounced difference in
sensitivity between progenitors and stem cells. Whereas about 1%
of progenitor cells survived in the 7-day growth factor-treated
groups, almost complete depletion of primitive stem cells was
achieved in SCF plus FL and to a lesser extent in mice treated
with SCF plus IL-11. These findings indicate that a 7-day
treatment with SCF in combination with either IL-11 or FL
results in a considerably increased sensitivity of hematopoietic
cells toward low-dose TBI.
Donor cell engraftment after BMT
Recipient female mice received transplants of 3 ? 106unfraction-
ated congenic bone marrow cells from male hosts after low-dose
TBI. Subsequently, long-term donor cell engraftment was deter-
mined by fluorescence-activated cell-sorter (FACS) analysis of
donor and recipient leukocyte contribution. Two independent
experiments were performed and pooled data are shown in Figure
3. The transplantation was considered successful when stable or
increasing donor cell engraftment was maintained from week 12
onward (Figure 3). In 85% of control mice (17 of 20, Figure 3A-C)
chimerism was achieved, reaching values of 40% ? 4.1% (SEM)
donor leukocytes. Seven-day treatment with SCF plus IL-11
showed rapid donor cell reconstitution, resulting in significantly
higher levels compared to control mice from week 4 onward
(n ? 6, P ? 1 ? 10?8; Figure 3A). Although engraftment re-
mained high throughout the study, donor cell contribution dropped
very gradually over time. Interestingly, donor cell engraftment in
1-day SCF plus IL-11–treated mice showed a completely opposite
pattern. The first month after transplantation, a significantly lower
donor cell engraftment was observed (P ? .02), which, however,
was followed by a slow, but steady increase. Sixteen weeks after
BMT, donor leukocyte contribution was significantly higher than
control values (P ? .05).
Figure 3B demonstrates donor cell engraftment in mice treated
with SCF plus FL. Unlike 7-day, SCF-plus–IL-11–treated mice,
chimerism in animals treated with SCF plus FL did not drop in the
long-term. Almost full donor leukocyte chimerism (?90%) was
Figure 1. Progenitor and stem cell number after
growth factor treatment. Total number of CAFCs–day 7
and CAFCs–day 35 per mouse (bone marrow plus
spleen plus liver, n ? 3) after treatment with SCF plus
IL-11, SCF plus FL, or G-CSF for either 1 or 7 days. The
numbers of CAFCs–day 7 are depicted in panels A to
C, whereas panels D to F show the numbers of CAFCs–
Figure 2. Survival of CAFCs–day 7 and CAFCs–day 35 after low-dose TBI. (A)
Fraction of surviving cells 24 hours after 4 Gy irradiation in 1-day growth factor-
treated mice compared to control values. (B) Fraction of surviving cells 24 hours after
4 Gy irradiation in 7-day growth factor–treated mice.
314NOACH et alBLOOD, 1 JULY 2002?VOLUME 100, NUMBER 1
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reached (n ? 7, P ? .00001). Although a significantly lower
engraftment was found in the 1-day SCF-plus-FL–treated group
compared to controls up to 4 weeks after transplantation (P ? .01),
from week 12 onward donor leukocyte contribution was signifi-
cantly higher than in controls (n ? 8, P ? .01). Similar to the 1-day
SCF-plus–IL-11 group, engraftment slowly but continuously kept
increasing during the course of the study, reaching similar levels as
the 7-day SCF plus FLgroup at week 32 (P ? .0001).
It is apparent that 7-day G-CSF treatment prior to TBI had a
different effect on donor cell engraftment after BMT than either
SCF plus IL-11 or SCF plus FL (Figure 3C). Although increased
donor cell levels (?70%) were initially observed, (n ? 8,
P ? .00001), subsequently, 16 weeks after BMT, engraftment
gradually decreased toward control values at 24 weeks after
transplantation. Mice treated for 1 day with G-CSF initially
engrafted equally well as control animals, but all 8 mice eventually
showed a gradual decrease in engraftment.
To ensure that a true multilineage mixed chimerism was
established in all groups, we checked for donor B and T lympho-
cytes as well as granulocytes in all recipient mice 28 to 40 weeks
after BMT, using anti-B220, anti-CD8a, and anti-Gr-1 antibodies.
The contribution of total donor leukocytes, as shown in Figure 3,
was proportionally reflected in all 3 lineages (data not shown).
Stem cell depletion and long-term donor cell engraftment
We hypothesized that for successful long-term donor cell engraft-
ment after transplantation, depletion of recipient stem cells would
be required. Thus, a relationship between the extent of depletion of
host stem cells and levels of donor cell engraftment was predicted.
In Figure 4 this relationship between residual recipient CAFCs–day
35 remaining after 4 Gy TBI and donor cell engraftment at 24
weeks after transplantation is shown. Evidently, in all 7-day growth
factor-treated groups, the control group, and the 1-day G-CSF–
treated group, a strong inverse correlation (R2? 0.957) exists
between long-term donor cell engraftment and surviving CAFCs-
day 35 after TBI. Interestingly, although relatively many stem cells
survived radiation after 1-day SCF plus IL-11 or 1-day SCF plus
FLtreatment, donor cell chimerism in these mice was very high.
In this study, we show that growth factor treatment of recipient
mice prior to low-dose TBI can modulate donor cell engraftment
levels after syngeneic BMT. This modulation is dependent on the
particular growth factor or growth factor combination used, but it is
Within 4 weeks after BMT donor cell engraftment in all mice
pretreated for 7 days with either of the growth factor combinations
was dramatically increased compared to engraftment in untreated
mice. This high donor cell contribution remained stable for at least
4 months. Most pronouncedly, almost full donor chimerism (90%)
was reached in the SCF plus FL group even after a 1-day treatment
period. Also, after a short 1-day growth factor pretreatment with
SCF plus IL-11, a highly significant increase in donor leukocyte
contribution was seen, although this became apparent only several
months after transplantation. These findings are in accordance with
data that we have previously obtained by a 1-day treatment of
recipient mice with SCF alone prior to low-dose TBI.34
Interestingly, a difference in donor cell engraftment was ob-
served between mice treated with growth factors with an effect on
primitive stem cells (SCF plus IL-11 or FL) and G-CSF, which
primarily affects progenitors. Because it has been shown that
G-CSF administered before chemotherapy enhanced stem cell
damage,35,36we expected that G-CSF would also sensitize the stem
cell compartment toward TBI, resulting in higher donor leukocyte
Figure 4. Correlation between the extent of stem cell depletion and long-term
donor cell engraftment. Relationship between the absolute numbers of CAFCs–day
35 remaining in the recipient 24 hours after 4 Gy TBI and donor cell engraftment 24
weeks after transplantation.
Figure 3. Donor cell engraftment in growth factor–pretreated recipients of
transplants. Percentage of donor-derived leukocytes in peripheral blood of suble-
thally (4 Gy) irradiated host mice after transplantation of 3.0 ? 106unfractionated
donor bone marrow cells. Recipient mice were treated with growth factors for 1 day
(closed symbols) or 7 days (open symbols) before irradiation. Mice treated with SCF
plus IL-11, SCF plus FL, and G-CSF are depicted in panelsA, B, and C, respectively.
Each line represents the mean ? SEM of 2 independent experiments (n ? 4 per
CYTOKINES BEFORE LOW-DOSE TBI IN BMT CONDITIONING315 BLOOD, 1 JULY 2002?VOLUME 100, NUMBER 1
For personal use only.on October 16, 2014. by guest
contribution after transplantation. However, although 1-day
G-CSF–treated cells were equally sensitive toward low-dose TBI
as controls, the absolute number of remaining CAFCs-day 35 after
TBI was considerably higher than in controls. In agreement with
our hypothesis on the correlation between percentage donor cell
contribution and degree of stem cell depletion, donor cell engraft-
ment was considerably lower in 1-day G-CSF–treated mice than in
control mice and decreased gradually over time. Seven-day G-CSF
treatment initially resulted in a high donor cell engraftment, but
values dropped, as expected, toward controls several weeks after
transplantation. Thus, the sensitizing effect of G-CSF was not
observed. Mardiney and Malech37showed that a 5-day G-CSF
treatment prior to very low-dose TBI (1.6 Gy) and BMT led to
significantly enhanced donor cell contribution in mice. However,
they followed engraftment only up to 16 weeks after transplanta-
tion, whereas donor cell contribution in our experiment dropped
toward control values only at 24 weeks after transplantation.
In the majority of our growth factor–treated groups we observed
that the degree of stem cell depletion indeed is strongly correlated
with the degree of donor cell engraftment, as we predicted.
However, we observed an unexpectedly high donor cell chimerism
in mice pretreated for 1 day with SCF plus IL-11 or SCF plus FL.
This high level of engraftment cannot be explained by enhanced
depletion of recipient stem cells, because many CAFCs-day 35
remained after TBI. We speculate that as yet unknown microenvi-
ronmental changes in the complex bone marrow stromal system
caused by growth factor administration prior to TBI may influence
Induction of mixed chimerism after allogeneic BMT in animals
and in patients has been shown to cause increased immunocompe-
tence, increased transplantation tolerance, reduced graft-versus-
host disease, and reduced posttransplantation toxicity.2,38-40Here,
we were able to create almost full donor cell chimerism both short-
tations. It has been shown that engraftment can be slightly impaired
after transplantation of male cells into mildly conditioned female
recipients.41,42In addition, it has been shown that the CD45.1
antigen, which is expressed by the congenic C57BL/6.SJL.Ptprca
mice we used as donors, may be mildly immunogenic.43Thus, our
syngeneic, sex-mismatched transplantation model sets the stage for
testing a similar approach in allogeneic transplantation settings.
However, in these situations a growth factor pretreatment protocol
may be combined with thymic irradiation or administration of
antibodies causing a costimulatory blockade to obtain sufficient
The authors would like to thank Geert Mesander for help with the
FACS analyses and Dr Piet Wierenga for intravenous injection of
bone marrow cells.
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