Bone Marrow Transplantation (2002) 30, 867–872
2002 Nature Publishing Group
All rights reserved 0268–3369/02 $25.00
W/Wvmarrow stromal cells engraft and enhance early erythropoietic
progenitors in unconditioned Sl/Sldmurine recipients
SJ Bubnic1,3, X-H Wang1, BR Clark1,4and A Keating1,2,3
1Toronto General Research Institute, Toronto, ON, Canada;2Princess Margaret Hospital/Ontario Cancer Institute, Toronto, ON,
Canada; and3Institute of Medical Science, University of Toronto, Toronto, ON, Canada
Transplantation of marrow stromal cells may provide a
means of modulating hematopoiesis and serve as a form
of cell therapy. We employed a murine transplant model
using Sl/Sldmice, which have macrocytic anemia due to
defective expression of stem cell factor (SCF) on bone
marrow stromal cells. Donor cells were derived from the
complementary mutant strain W/Wv, which also exhibit
anemia, due to mutations in c-kit, the SCF receptor
expressed on hematopoietic stem cells. The strength of
this model is that any correction of the Sl/Sldanemia
from the infusion of W/Wvstromal cells can be attri-
buted to the effect of the stromal cells and not to con-
taminating W/Wvhematopoietic stem cells, a major con-
Cultured stromal cells were infused into unconditioned
non-splenectomized Sl/Sldmice. Engraftment of donor
stromal cells reached levels of up to 1.0% of total mar-
row cells 4 months post transplant. However, stromal
engraftment was not detectable in the spleen. Recipients
of W/Wvstroma showed a significant increase in the
committed erythroid progenitors compared with those
receiving Sl/Sldstromal cells: 109 ? 26 vs 68 ? 5 CFU-
E per 105BMC, P = 0.002; 25 ? 10 vs 15 ? 5 BFU-E
per 105BMC, P = 0.037, for W/Wvand Sl/Sldstroma
erythroid progenitors, the anemia was not corrected.
Our data suggest that in this murine model, splenic
erythropoiesis may influence stromal cell therapy, and
that higher levels of marrow engraftment may be neces-
sary to obtain a clinically significant effect.
stromal cell; cell therapy; erythropoieses
this increase in
Correspondence: Dr A Keating, Princess Margaret Hospital, 610 Univer-
sity Avenue, Suite 5-211, Toronto, ON, M5G 2M9, Canada
4Current address: City of Hope National Medical Center, Duarte, CA,
Received 14 January 2002; accepted 13 September 2002
The hematopoietic microenvironment plays an important
role in the support and regulation of hematopoiesis. Stromal
cells, one component of this environment, are required for
support of long-term hematopoiesis in vitro.1These cells
are an important source of cytokines, adhesion molecules
and extracellular matrix components.2–4
The importance of a normal hematopoietic microenviron-
ment is illustrated by the Sl/Sldmouse. These mice offer a
model of abnormal hematopoiesis due to defective pro-
duction of the cytokine stem cell factor (SCF) by marrow
stromal cells.5,6In addition to macrocytic anemia, Sl/Sld
mice experience a deficiency of tissue mast cells, as well
as fertility and pigmentation defects. These defects have
been attributed to abnormal microenvironments for these
cell lineages.7The anemia of Sl/Sldmice is not corrected
by transplantation of hematopoietic cells,8unlike the comp-
lementary mutant W/Wvmouse, which responds to marrow
transplantation.9,10The W/Wvmouse has a similar disease
phenotype, but in contrast is attributed to a defect in the
receptor for SCF, c-kit.7The anemia in Sl/Sldmice responds
to transplantation of splenic tissue, which provides an intact
normal hematopoietic microenvironment. In vitro studies
by Dexter and Moore11showed that Sl/Sldadherent cells
support W/Wvhematopoiesis poorly, yet a W/Wvadherent
layer supports co-cultured Sl/Sldmarrow cells, resulting in
sustained hematopoietic cells production. Molecular studies
reveal that Sl/Sldmice have mutations in the gene for SCF,
including an intragenic deletion removing the transmem-
brane and cytoplasmic domains.12Stromal cells from these
mice are incapable of producing the membrane-bound iso-
form of SCF.5,6It is feasible that transplantation of stromal
cells with normal SCF expression into Sl/Sldmice may pro-
vide an attractive and convincing model for treating an
abnormal hematopoietic microenvironment.
Stromal cell transplantation has been achieved using
non-hematopoietic mesenchymal cells derived from long-
term bone marrow cultures,13as well as stromal cell lines.14
Anklesaria et al15showed that infusion of a transformed
stromal cell line into irradiated and splenectomized Sl/Sld
mice could partially correct their anemia. The aim of our
study is to extend this observation by transplanting normal
stromal cells, derived from long-term bone marrow cul-
tures, into unconditioned Sl/Sldrecipients as a model for
human cell therapy.
Stromal cells engraft and enhance erythropoiesis
SJ Bubnic et al
Bone Marrow Transplantation
Materials and methods
Stromal cell transplantation
Stromal cells were harvested from the adherent layer of
long-term bone marrow cultures initiated from femoral
marrow of male WBB6F1-W/Wv(W/Wv) and WCB6F1-
Sl/Sld(Sl/Sld) (Jackson Laboratory, Bar Harbor, ME, USA).
The cells were maintained in ?-medium (Gibco BRL, Bur-
lington, ON, Canada) with 10% horse serum, 10% fetal
bovine serum (HyClone Laboratories, Logan, UT, USA),
10?6m hydrocortisone (Sigma, St Louis, MO, USA), l-glu-
tamine, and antibiotics (Gibco BRL) and incubated at 37°C
in 5% CO2. Stromal cells were used for transplantation after
five or six serial passages in vitro. A sex-mismatch trans-
plant model was employed, in which male donor stromal
cells were infused into unconditioned female Sl/Sldrecipi-
ent mice according to a local IRB-approved protocol. The
cells were injected intravenously via the lateral tail vein,
and recipients were monitored for changes in hematological
parameters over 4 months until death post transplant.
Stromal cell phenotype
Expression of Mac-1 and CD45 on stromal cells was
assayed by flow cytometry using a FACScan analyzer and
CELLQuest software (Becton Dickinson, Mississauga, ON,
Canada). Stromal cells were washed with phosphate buff-
ered saline (PBS) and incubated with either FITC-conju-
gated rat anti-mouse Mac-1 antibody (Serotec, Oxford, UK)
or FITC-conjugatedrat anti-mouse
(Pharmingen, San Diego, CA, USA) at room temperature.
Appropriate isotype antibodies were used as controls.
Analyses were done in triplicate on 10 000–20 000 cells.
To assess collagen IV expression, stromal cells were grown
on Lab-Tek chamber slides and fixed with paraformal-
dehyde prior to staining (Nalge Nunc, Naperville, IL,
USA). Cells were incubated with rabbit anti-mouse col-
lagen IV antibody (Biodesign, Kennebunk, ME, USA) fol-
lowed by FITC-conjugated goat anti-rabbit antibody
(Jackson ImmunoResearch Laboratories) at room tempera-
ture. To assess both positive and negative staining cells, a
DAPI counterstain was used (4′,6-diamidine-2′-phenylin-
dole dihydrochloride) (Oncor, Gaithersburg, MD, USA).
Collagen IV-positive stromal cells were viewed using a
fluorescence microscope. Analyses were done in duplicate
on 200–700 cells.
Engraftment of male stromal cells into female recipients
Fluorescence in situ hybridization (FISH) was used to
identify the frequency of male cells in the marrow of female
recipient mice. Bone marrow suspensions were fixed in
acetic acid:methanol and cells dropped on to microscope
slides. The slides were treated with 100 ?g/ml RNase,
washed and dehydrated before denaturation at 75°C, in
50% formamide in 2 ? SSC. A biotinylated Y chromosome
paint (Cambio, Cambridge, UK) was mixed with Hybrisol
VI (Oncor) and denatured at 75°C, prior to hybridization
with the slides overnight at 37°C. The hybridized probe
was detected by incubation with FITC-labeled avidin, fol-
lowed by an amplification step involving incubation with
anti-avidin antibody and another round of FITC-avidin
(Oncor). The slides were counterstained with DAPI and
propidium iodide (PI). To determine the percentage of
donor cells, 1000 to 1500 nuclei per slide were counted
using a fluorescence microscope.
Presence of donor cells in spleen samples was determ-
ined using a duplex PCR method. This allowed identifi-
cation of a 292 bp portion of the testis determining region
on the Y chromosome (Tdy),16along with a 147 bp portion
of SCF exon 5, a region unaffected by the Sldmutation, as
an internal positive control.12,17The oligonucleotide pri-
mers used had the following sequences: TDY1 5′-GACTG
GTGACAATTGTCTAG-3′, TDY2 5′-TAAAATGCCACT
PCR involved 30 cycles at 94°C for 1 min, 56°C for 1 min,
and 72°C for 1 min. The reaction mixture consisted of
500 ng sample DNA, 4 ng/?l of each TDY primer, 2 ng/?l
of each SCF primer, 1.25 units Taq polymerase (ID Labs
Biotechnology, London, ON, Canada), 0.2 mm nucleotides,
1.5 mm MgCl2, 16 mm (NH4)2SO4, 67 mm Tris-HCl and
0.01% Tween 20. With this methodology, at least 0.5%
male DNA can be detected in a mixture of female genomic
DNA spiked with male DNA.
Erythrocyte indices were monitored on a monthly basis
from peripheral blood samples obtained from the retro-
orbital sinus while mice were anesthetized by inhalation of
isoflurane (Schein Pharmaceutical,
Canada). Littermates of Sl/Sldmice, which did not exhibit
the mutant phenotype, were used as controls for hematolog-
ical parameters. Samples were processed by a CELL-DYN
3000 automated blood cell analyzer to determine erythro-
cyte (RBC) number, mean cell volume (MCV), hemoglobin
(HGB), and leukocyte count (WBC). The mean erythrocyte
size distribution was also assessed for each treatment group,
and statistical analysis performed using a Wilcoxon rank
sum nonparametric test.
Marrow hematopoietic progenitor levels were determ-
ined by methlycellulose colony assays.18Briefly, 1 ? 105
femoral bone marrow cells were cultured in ?-medium con-
taining 1% methylcellulose (StemCell Technologies, Van-
couver, BC), 30% fetal bovine serum, 1% BSA (Boehringer
Mannheim, Laval, QC, Canada), 10?42-mercaptoethanol
medium (Gibco BRL), 2 U/ml recombinant human erythro-
poietin (Ortho Pharmaceutical, Raritan, NJ, USA), l-gluta-
mine and antibiotics and incubated at 37°C in an atmos-
phere of 5% CO2. Colony numbers (BFU-E, CFU-E, CFU-
GM) were counted between days 8 and 11 of culture. Stat-
istical comparisons were done using a Student’s t-test.
Serial passage in vitro was performed to increase the num-
ber of stromal cells, as well as deplete contaminating hema-
topoietic cells. The phenotype of stromal cells in the adher-
Stromal cells engraft and enhance erythropoiesis
SJ Bubnic et al
long-term marrow cultures
Phenotype of stromal cells derived from adherent layers of
Stroma Collagen IVMac-1 CD45
0.1 ? 0.1%
0.2 ? 0.1%
0.9 ? 1.4%
0.9 ? 0.8%
Data are means of two analyses for collagen type IV (200–700 cells) and
mean ? s.d. for Mac-1 and CD45 (n = 3, 10 000–20 000 cells).
ent layer was assessed after five or six passages. Marrow
stromal cells were predominantly non-hematopoietic, with
a small percentage of cells expressing hematopoietic mark-
ers (0.9% CD45+, less than 0.2% Mac-1+) (Table 1). Fur-
thermore, as previously reported for stromal cells,5virtually
all of the cells produced collagen IV. This level of enrich-
ment was necessary for transplantation experiments.
A sex-mismatch transplant model was employed to allow
identification of engrafted male donor stromal cells in
unconditioned female recipients. The frequency of donor
stromal cells in recipient marrow 4 months after infusion
was determined by FISH using a Y-chromosome paint.
Recipients of a dose of under 2 million cells per mouse
showed negligible levels of marrow engraftment of either
W/Wvor Sl/Sldstroma (Table 2). However, recipients of
higher doses of cells (approximately 2.5 million per mouse)
did have appreciable engraftment in the marrow. Donor
cells were found in similar ranges of frequencies in both
stromal transplant groups, with levels up to 1% of the total
marrow nucleated cell population. FISH analysis was spe-
cific because female marrow cells gave a false positive rate
of only 0.1%. The mean engraftment level of W/Wvstroma
was 0.4 ? 0.3% and 0.6 ? 0.4% for Sl/Sldstroma, with
probabilities of 0.015 and 0.0005, respectively, based on a
Poisson distribution of FISH-positive events in control
female marrow cells. A control group receiving 6 million
W/Wvwhole marrow cells had low levels of hematopoietic
cell engraftment (0.4 ? 0.3%), suggesting that potential
hematopoietic contamination in the stromal grafts would
not have any advantage over donor stromal cells.
Engraftment of donor stromal cells in recipient spleens was
not evident by PCR for the Tdy gene sequence (Figure 1).
Attempts at infusing 4 million or more stromal cells in a
single dose resulted in a high incidence of death in Sl/Sld
recipients, despite having successfully infused up to 6
million stromal cells into Balb/c mice.19
female recipient Sl/Sldmice 4 months post transplant as assayed by FISH
using a Y-chromosome paint
Engraftment of male stromal cells into the bone marrow of
Type of stromal
n Percentage FISH-
0.1 ? 0.1% (0–0.3%)
0.1 ? 0.1% (0–0.2%)
0.4 ? 0.3% (0.1–1.0%)
0.6 ? 0.4% (0.1–0.9%)
0.4 ? 0.3% (0.1–0.7%)
aMean ? s.d. with the range in parentheses.
Bone Marrow Transplantation
??? ? ??? ??? ???
donor cells. Lanes: spleen samples from transplant mice F10, G5, G6 and
J4, respectively; H2O PCR control; mixtures of female genomic DNA
spiked with male DNA ranging from 0 to 10% male. The PCR products
are a 292 bp region of TdY gene on the Y-chromosome and a 147 bp
region of the SCF gene as an internal control. A 1-kb marker was run in
the first and last lane.
Representative PCR analysis of spleen for engraftment of male
During the course of the study, erythropoiesis was moni-
tored after stromal cell transplantation including the assess-
ment of erythroid progenitor levels in the bone marrow at
death. There was a significant difference in marrow
erythroid progenitors in mice receiving 2.5 ? 106W/Wv
stromal cells (Table 3). Both CFU-E and BFU-E were elev-
ated in recipients of W/Wvstroma compared with those
receiving 2.5 ? 106Sl/Sldstroma (CFU-E: 109 ? 26 vs
68 ? 5 per 105BMC, P = 0.002; BFU-E: 25 ? 10 vs
15 ? 5 per 105BMC, P = 0.037, for W/Wvand Sl/Sldstro-
mal recipients, respectively). Levels of CFU-GM were not
(P = 0.087). Contamination of the stromal grafts by hema-
topoietic cells is not a likely source of the increased number
of erythroid progenitors. Because hematopoiesis was
assessed in recipient mice 4 months after transplant, only
contamination by stem cells could account for this increase.
Hematopoietic contamination of the stromal graft is at a
low level (Table 1), and thus not likely to contain a signifi-
cant number of stem cells, which would have an even lower
frequency. We have shown that 3 ? 106culture-derived
stromal cells had no detectable CFU-S.20In addition, whole
marrow from W/Wvdonors did not engraft well in uncon-
ditioned Sl/Sldmice (Table 2). Furthermore, transplantation
of a much higher number of W/Wvhematopoietic cells was
incapable of correcting the Sl/Sldanemia.8Thus, any
contaminating W/Wvstem cells in the stromal cell graft
plant of high dose of stromal cells (colonies per 105BMC)
Hematopoietic colony number assayed 4 months post trans-
nCFU-E BFU-E CFU-GM
109 ? 26a
68 ? 5
25 ? 10a
15 ? 5
109 ? 40
aStatistically different by t-test (P ? 0.05), compared to the Sl/Sld
Stromal cells engraft and enhance erythropoiesis
SJ Bubnic et al
Bone Marrow Transplantation
can be discounted as a source of the increase in erythroid
The mice were monitored to determine whether there
was any effect on mature erythrocytes. No change in red
cell indices (RBC number, MCV, hemoglobin) was
observed during the course of the experiment, nor were
WBC levels affected, for all stromal cell dose groups
(Table 4). A correction in the macrocytic anemia would
require an increase in erythrocyte number and a reduction
in the mean cell volume, however both indices remained
unchanged. Transplantation of W/Wvwhole marrow also
had no effect on erythrocyte parameters, consistent with
previous studies.8Analysis of the mean RBC size distri-
bution was performed for recipients receiving the higher
dose of stromal cells to determine if more subtle changes
in erythropoiesis were occurring that were not reflected in
the MCV (Figure 2). Although a slight shift towards
smaller erythrocytes in recipients of W/Wvstroma was
noted, the overall distribution was not significantly different
from the erythrocytes of Sl/Sldstromal cell recipients
(P = 0.195).
The ability of stromal cells to engraft in unconditioned
recipients has important implications for the clinical appli-
cations that do not require myeloablation. The present study
demonstrates stromal engraftment in the marrow of uncon-
ditioned Sl/Sldmice. Although engraftment did not exceed
1% of the total nucleated cell population, it should be noted
that endogenous stromal cells constitute only a minor popu-
lation and thus, the donor cells may represent a doubling
of the original number.21,22Donor cell engraftment must be
due to non-hematopoietic stromal cells because hematopo-
ietic cell contamination was negligible. Although donor
high doses of stromal cells (approximately 2.5 million cells)
Erythrocyte parameters in peripheral blood of recipients of
Type of stromal graftn Prior to
(1) RBC (?106/?l)
(2) MCV (fl)
(3) Hemoglobin (?g/?l)
(4) WBC (?103/?l)
3.71 ? 1.09
4.40 ? 0.70
3.50 ? 1.47
4.06 ? 0.77
4.16 ? 0.85
3.55 ? 1.28
74.2 ? 10.8
71.8 ? 9.4
70.3 ? 6.8
74.2 ? 7.9
72.7 ? 8.0
79.0 ? 11.3
82.9 ? 18.7
98.5 ? 9.8
76.0 ? 29.7
91.1 ? 12.3
90.9 ? 13.4
81.9 ? 23.6
7.60 ? 2.46
12.16 ? 4.36
5.17 ? 1.17
7.66 ? 2.55
7.39 ? 2.85
5.28 ? 3.23
Indices measured during the course of the transplants were: (1) Erythro-
cyte number (RBC), (2) mean erythrocyte volume (MCV), (3) hemoglobin
and (4) total white blood cell counts (WBC). Mice were the recipients of
W/Wvstromal cells, W/WvBMC or Sl/Sldstromal cells
?? ???? ?? ????
at 3 months post transplant. Plots show RBC distribution of W/Wvstromal
recipients (solid line), Sl/Sldstromal recipients (grey line) and control Steel
littermate mice (dashed line).
Mean erythrocyte size distribution for stromal treatment groups
analysis by FISH was limited to detection of male cells in
the whole marrow, the levels found were similar to those
of previous studies from our laboratory.19We have also
shown that donor cells present in whole marrow prep-
arations are detected by FISH at higher levels among the
cultured stromal cells derived from the same transplant
recipients.19Although the present study did not analyze
CFU-F chimerism, Anklesaria et al15showed that over half
of the CFU-F were donor-derived after transplantation of
a stromal cell line.
While hematopoietic cell transplantation is unable to cor-
rect the macrocytic anemia of Sl/Sldmice,8the transplan-
tation of a normal hematopoietic microenvironment in the
form of an intact spleen can treat these mice effectively.23
Thus, it could be inferred that infusion of stromal cells with
normal SCF expression into Sl/Sldmice may alleviate the
anemia, in contrast to Sl/Sldstromal cells which support in
vitro hematopoiesis poorly.11Anklesaria et al15showed that
infusion of a transformed stromal cell line into irradiated
and splenectomized Sl/Sldmice could yield a small
improvement in erythropoiesis. The aim of our study was
to investigate the role of non-transformed marrow stromal
cells in correcting the disorder in unconditioned recipients.
The use of W/Wvstromal cells has the advantage that any
contaminating hematopoietic cells are defective and would
not correct the Sl/Sldanemia. Although transplantation of
W/Wvstromal cells resulted in a significant increase in
erythroid progenitors (CFU-E, BFU-E) over the levels
found in recipients of Sl/Sldstroma, no improvement in the
anemia was observed. This increase in erythroid progeni-
tors is noteworthy given that fetal Sl/Sldmice have reduced
liver CFU-E compared with normal littermates.24The
authors speculate that the defect in these mice may arise at
the BFU-E/CFU-E transition. Other studies have also
shown the importance of SCF in regulating early erythroid
progenitors. The receptor for SCF has been identified on
Stromal cells engraft and enhance erythropoiesis
SJ Bubnic et al
erythroid progenitors,25and marrow progenitors expressing
this receptor differentiate into BFU-E in response to SCF.26
Also, injection of recombinant SCF into baboons increases
the number of primitive hematopoietic progenitors, includ-
ing BFU-E.27,28Thus, the increase in erythroid progenitors
following transplantation of W/Wvstromal cells into Sl/Sld
mice is consistent with the effect of SCF on this cell
The limited biological effect of stromal cell transplan-
tation in our model may be due to preponderance of
erythropoiesis in the spleen and contrasts with the marrow-
based erythropoiesis in humans. Transplantation of marrow
cells to the spleen results in predominantly erythroid differ-
entiation in the mouse,29and in response to phenylhydra-
zine-induce anemia, most of the erythroid expansion occurs
in the spleen and is dependent on SCF/c-kit interaction.30
Most importantly, the spleen has been shown to be a target
for correction of the Sl/Sldanemia, as demonstrated by the
imporvement in erythropoiesis following transplantation of
a normal spleen.23In the study by Anklesaria et al,15the
recipient mice were irradiated and splenectomized prior to
stromal cell infusion. Irradiation may have purturbed
endogenous marrow hematopoiesis sufficiently to allow
transplanted stromal cells to have an effect, while splen-
ectomy effectively removed a competing site of erythropo-
iesis. Our study used unconditioned host mice with intact
engraftment of donor stromal cells, although there was mar-
row engraftment. It may be that the change in the marrow
erythroid progenitor level we observed was insufficient to
overcome ongoing abnormal erythropoiesis in the spleen.
However, this limitation in the murine model is not relevant
to the clinical application of stromal cell therapy because
the spleen is not normally a site of human hematopoiesis.
Although we observed an increase in marrow erythroid
progenitors, the inability to correct the anemia suggests that
higher levels of stromal engraftment may be required. A
major improvement in erythropoiesis may require the pres-
ence of a significant amount of normal hematopoietic
microenvironment. Kapur et al31has shown that breeding
a transgenic mouse expressing a membrane-restricted SCF
cDNA, on to a Sl/Sldbackground resulted in improved
erythrocyte production compared with unmanipulated Sl/Sld
mice. In addition, the injection of recombinant SCF at phar-
macological doses also corrected the anemia, albeit transi-
ently.32Anklesaria et al15found that Sl/Sldstromal cells
suppress hematopoiesis in vitro and extrapolated that up to
80% of the Sl/Sldmarrow would need to be replaced for a
biological effect to be observed. However because Sl/Sld
mice are particularly small it was difficult to infuse very
high numbers of stromal cells in single or even multiple
Our data, and the studies of others, indicate that stromal
cell infusion can influence host hematopoiesis14,15,33,34and
suggest that this approach has possible application in cell
and/or gene therapy.35,36The use of stromal cells has the
added dimension of extensive transdifferentiation potential
to osteoblasts, chondroblasts,37,38fibroblasts,39muscle,40
astrocytes,41and neurons.42Also, stromal cells could be
used as a vehicle for gene therapy to introduce defective
or missing genes.43Based on our murine model, our studies
failedto detect splenic
Bone Marrow Transplantation
indicate that for stromal cell transplantation to become a
feasible form of cell therapy further studies are required,
particulary novel approaches that enhance stromal marrow
engraftment in unconditioned recipients.
Armand Keating holds the Gloria and Seymour Epstein Chair in
Cell Therapy and Transplantation at the University of Toronto and
the University Health Network.
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