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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1557-1566
Organ-Selective Homing Defines Engraftment Kinetics of Murine
Hematopoietic Stem Cells and Is Compromised by Ex Vivo Expansion
By
Stephen J. Szilvassy,
Michael J. Bass,
Gary Van Zant, and
Barry Grimes
From the Blood and Marrow Transplant Program, and the Division of
Hematology/Oncology, Lucille P. Markey Cancer Center, University of
Kentucky, Lexington, KY.
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ABSTRACT |
Hematopoietic reconstitution of ablated recipients requires that
intravenously (IV) transplanted stem and progenitor cells "home"
to organs that support their proliferation and differentiation. To
examine the possible relationship between homing properties and
subsequent engraftment potential, murine bone marrow (BM) cells were
labeled with fluorescent PKH26 dye and injected into lethally
irradiated hosts. PKH26+ cells homing to marrow or spleen
were then isolated by fluorescence-activated cell sorting and assayed
for in vitro colony-forming cells (CFCs). Progenitors accumulated
rapidly in the spleen, but declined to only 6% of input numbers after
24 hours. Although egress from this organ was accompanied by a
simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8%
of input after 3 hours), spleen cells remained enriched in donor CFCs
compared with marrow during this time. To determine whether this
differential homing of clonogenic cells to the marrow and spleen
influenced their contribution to short-term or long-term hematopoiesis
in vivo, PKH26+ cells were sorted from each organ 3 hours
after transplantation and injected into lethally irradiated Ly-5
congenic mice. Cells that had homed initially to the spleen regenerated
circulating leukocytes (20% of normal counts) approximately 2 weeks
faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of "spleen-homed" cells also contained approximately 50% higher numbers of CFCs per
femur than recipients of "BM-homed" cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1+c-kit+Lin
cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem
cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were
then stained with PKH26 and assayed as above. Strikingly, CFCs
generated in vitro exhibited a 10-fold reduction in homing capacity
compared with fresh progenitors. These studies demonstrate that
clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline
in the homing capacity of progenitors generated in vitro underscores
critical qualitative changes that may compromise their biologic
function and potential clinical utility, despite their efficient
numerical expansion.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE MOST PRIMITIVE HEMATOPOIETIC stem
cells (HSCS), identified by their capacity to repopulate lymphoid and
myeloid lineages upon transplantation into myeloablated or
immunocompromised hosts, reside in the bone marrow (BM) and are closely
associated with mesenchymal cells lining the bone
endosteum.1,2 Within this microenvironment, HSC
self-renewal, proliferation, and differentiation are thought to be
regulated through specific interactions with a heterogeneous population
of stromal cells, and the extracellular matrix components and cytokines
they produce. When HSCs are collected from BM or peripheral blood for
hematopoietic rescue of patients with hematologic disease, efficient
and timely reconstitution requires that at least some intravenously
transplanted stem and progenitor cells reseed niches that have been
ablated by radiation or chemotherapy to eliminate malignant cells. This
migration of stem cells through the circulation and back to a
supportive hematopoietic microenvironment is referred to as
"homing."
Classical experiments by Wolf and Trentin have long implicated the role
of the hematopoietic microenvironment in influencing the lineage
commitment of HSCs in vivo.3 Recently, the existence of
unique niches that promote either short-term or long-term hematopoiesis has been affirmed by the isolation of murine stromal cell lines that
differ markedly in their ability to maintain enriched populations of
repopulating stem cells in vitro.4,5 Some stromal cells sustain clonogenic cells only transiently, while others support high
levels of primitive cell proliferation, but only after prolonged culture.4,6 These data suggest that clonogenic cells with early hematopoietic reconstitution potential may home to different niches than HSCs which sustain long-term hematopoiesis in vivo. This
concept is supported by studies demonstrating that the precursors of
murine spleen colony-forming units (pre-CFU-S) and stem cells with
marrow repopulating ability migrate first to the BM after transplantation, where they generate progeny CFU-S that emigrate to the
spleen.7,8 Since most CFU-S are separable from more primitive stem cells with long-term repopulating ability,9 it seems reasonable to speculate that the observed differences in the
repopulating potential of these two classes of cells might be due to
differences in their localization in vivo.
Manipulations directed at improving the homing of HSCs to sites that
support their rapid proliferation may provide a means to overcome the
delayed engraftment that compromises the safe recovery of patients
following stem cell transplantation. However, a potential relationship
between stem cell homing and engraftment kinetics has not previously
been examined, due in part to the lack of a suitable assay to monitor
the fate of limited numbers of transplanted stem cells in the whole
animal. To address this problem, Hendrikx et al exploited the
availability of brightly fluorescent aliphatic dyes, such as PKH26, to
label murine hematopoietic cells enriched for CFU-S.10 The
homing of fluorescent cells to different hematopoietic organs following
transplantation was then monitored by flow cytometry. These studies
established the feasibility of this approach to track HSCs in vivo, but
no functional assays were performed to compare the biology of
hematopoietic cells that had homed to different organs. Recently, two
groups have reported such studies. Oostendorp and Eaves studied murine BM cells labeled with 5-(and 6-) carboxyfluorescein diacetate succinimydl ester (CFSE) and demonstrated that progenitors identified by their potential to generate colonies of mature blood cells in vitro
homed predominantly to the marrow by 24 hours after transplantation, where they appeared to divide more slowly than in the
spleen.11 Primitive stem cells enriched by counterflow
centrifugal elutriation have also been stained with PKH26 and tracked
to the marrow of recipient animals up to 96 hours after
transplantation. PKH26+ cells that were sorted and
retransplanted into secondary hosts could give rise to stable
hematopoietic engraftment up to 3 months later, demonstrating the
preservation of their initial potential after the labeling and homing
procedure.12
We now report the refinement of the general technique of fluorescently
labeling cells before transplantation in the development of a
quantitative assay to measure HSC and progenitor cell homing in vivo.
Temporal analyses indicate a preferential concentration of
colony-forming cells (CFCs) in the spleen compared with marrow within
the first few hours after transplantation. To determine whether this
differential accumulation of CFCs in these organs influenced their
subsequent contribution to early and sustained hematopoiesis, labeled
cells were sorted from marrow and spleen and retransplanted into
lethally irradiated Ly-5 congenic mice. Interestingly, cells that had
homed initially to the spleen regenerated circulating leukocytes almost
2 weeks faster than cells that homed to marrow. Both primary and
secondary recipients of "spleen-homed" cells also recovered
higher numbers of CFCs per femur than recipients of "BM-homed"
cells. Thus, clonogenic cells with differential homing properties
appear to contribute variably to early and late hematopoiesis in vivo.
In a practical application of this assay, we also evaluated the homing
properties of progenitor cells generated by ex vivo expansion. It has
been suggested that by culturing HSCs in a mixture of hematopoietic
growth factors in vitro, their expanded and partially differentiated
progeny may regenerate circulating neutrophils and/or platelets
more quickly than unmanipulated HSCs in a clinical setting.13-16 Support for this hypothesis was provided
initially by Muench et al, who demonstrated that lethally irradiated
mice transplanted with BM cells that had been cultured for 1 week in interleukin (IL)-1 plus stem cell factor (SCF) regenerated twofold to
fivefold higher peripheral blood cell counts 4 to 10 days after transplantation than recipients of fresh marrow.17 We have
also recently shown that the delayed engraftment of myeloablated mice injected with 103 highly enriched
Thy-1loSca-1+Lin stem cells
is accelerated by cotransplantation of their partially differentiated
progeny generated after 1 week of culture in IL-3, IL-6, granulocyte
colony-stimulating factor (G-CSF), and SCF.18 However,
despite the fact that expanded cells contained several hundred-fold
more day 12 CFU-S and CFCs than noncultured stem cells, they were not
as effective at hematopoietic reconstitution as might be predicted from
their composition. HSCs and progenitor cells that have been cultured in
vitro typically exhibit a diminished capacity for long-term
reconstitution, although the severity of this defect is clearly related
to the source of HSCs and the culture conditions used.19-23
This has led some investigators to speculate that changes in adhesion
molecule expression in response to pancytopenia or cytokine elaboration
may alter HSC homing properties and reduce engraftment efficiency by
mechanisms that may include (1) conferring a propensity to migrate to
tissues not permissive for hematopoiesis, or (2) reducing the
localization of progenitors to, or extravasation of their progeny from,
a permissive microenvironment once inside the marrow. As a first step
to address these questions, we now show using the homing assay
described herein that progenitor cells generated by the in vitro
expansion of HSCs exhibit an approximately 10-fold reduction in homing
efficiency compared with unmanipulated cells. Our data suggest that
qualitative changes in transplantation potential may pose significant
obstacles to the clinical application of cultured hematopoietic cells,
despite their successful numerical expansion.
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MATERIALS AND METHODS |
Animals.
Five- to 8-week-old C57BL/6 (B6) mice (Ptprcb
[Ly-5.2]) were used as marrow donors. Age-matched B6 or B6.SJL
(Ptprca [Ly-5.1]) mice were used as recipients as
indicated. Mice were purchased from commercial suppliers and maintained
under specific pathogen-free conditions at the animal facility of the
Chandler Medical Center until use.
Enrichment of HSCs.
Donor B6 mice were injected intravenously (IV) with a sterile solution
of 5-fluorouracil (5-FU; Roche Laboratories, Nutley, NJ) in
phosphate-buffered saline at a dose of 150 mg/kg body weight. Twenty-four hours later, 5-FU-treated BM (FUBM) cells were flushed into Hank's balanced salt solution (HBSS) containing 2% fetal calf
serum (FCS) (HF medium) using a 21-gauge needle. Erythrocytes were
eliminated by hypotonic lysis, and the remaining leukocytes incubated
with saturating amounts of biotinylated rat monoclonal antibodies
(MoAbs) specific for murine CD3 (clone KT3.1), CD5 (clone 53-7.3), CD8
(clone 53-6.72), CD11b/Mac-1 (clone M1/70), CD45R/B220 (clone RA3-6B2),
and Ly-6G/Gr-1 (clone RB6-8C5). The labeled cells were then incubated
with goat antirat IgG paramagnetic beads (Dynal Inc, Lake Success,
NY) at a bead:cell ratio of approximately 4:1, and mature
lymphoid and myeloid cells were removed by exposure to a magnetic
field. Lineage-depleted cells (~6% of initial numbers) were blocked
with anti-CD16/32 (clone 2.4G2, Fc Block) MoAb, and then stained with
phycoerythrin (PE)-conjugated anti-Ly-6A/E (Sca-1; clone E13-161.7)
and allophycocyanin (APC)-conjugated anti-CD117 (c-kit; clone
2B8) MoAbs and streptavidin~fluorescein isothiocyanate (FITC) (all
from Pharmingen, San Diego, CA). Stained cells were washed and
resuspended in HF containing 5 µg/mL propidium iodide (PI). Unstained
FUBM cells or lineage-depleted cells stained with appropriate
fluorochrome-conjugated isotypic control MoAbs (Pharmingen) or
streptavidin~FITC were used as background controls. PI-negative Sca-1+c-kit+Lin FUBM
cells, representing 0.013% ± 0.003% of whole FUBM, were sorted
using a dual-laser FACS Vantage instrument (Becton Dickinson Immunocytometry Systems, San Jose, CA). Reanalysis of the sorted cells immediately after sorting indicated a purity typically
greater than 90%.
Ex vivo expansion of unfractionated marrow or sorted stem cells.
A quantity of 106 unfractionated normal BM or
103 sorted
Sca-1+c-kit+Lin
cells were cultured per milliliter of StemPro-34 serum-free medium (Life Technologies, Gaithersburg, MD) containing 50 U/mL penicillin, 50 µg/mL streptomycin, 2 mmol/L L-glutamine, 0.05 mmol/L
2-mercaptoethanol (2-ME), supplemented with the following recombinant
cytokines: 10 ng/mL murine IL-3, IL-6 and human G-CSF, and 100 ng/mL
murine SCF for cultures initiated with whole BM cells; 10 ng/mL murine IL-6 and human G-CSF, 50 ng/mL murine IL-11, and 100 ng/mL murine SCF,
murine thrombopoietin (TPO), and human FLK for cultures initiated with
Sca-1+c-kit+Lin
cells. All cytokines were purchased from R&D Systems (Minneapolis, MN),
except G-CSF (Amgen Inc, Thousand Oaks, CA). Cultures were incubated
undisturbed at 37°C for 7 days. From this time onward, half of the
medium was replaced daily and all cells harvested and counted after 9 days.
In vivo homing assay.
Fresh or cultured BM cells were labeled with PKH26 dye according to the
manufacturer's (Sigma Chemical Co, St Louis, MO) instructions with
some modifications. Briefly, cells were resuspended in Diluent C at a
concentration of 2 to 8 × 107/mL, combined with an
equal volume of PKH26 dye freshly prepared at 10 µmol/L in Diluent C,
and incubated at room temperature for approximately 10 minutes with
periodic gentle mixing. Staining was terminated by addition of an equal
volume of FCS for 1 minute, and the labeled cells then washed
thoroughly in HF. The average cell recovery after this procedure was
approximately 65%. An aliquot of cells before and after PKH26 staining
was assayed for CFCs as described below. The remaining cells were then
injected IV into lethally irradiated syngeneic B6 recipients
(106 to 108 cells per mouse). Total body
 -irradiation (9 Gy) was administered approximately 19 hours before
transplant in a single dose at a rate of 2.2 Gy/min from a
137Cs source (J.L. Shepard & Assoc, San Fernando, CA). One
to 24 hours after transplantation, all of the cells in each spleen and both femora and tibiae were procured in HF. After washing and red blood
cell lysis, BM and spleen cells were suspended in HF containing PI for
flow cytometric analysis or sorting of the originally transplanted
PKH26+ population. Cell counts performed to determine
whole-organ cellularity were based on the assumption that two femora
and two tibiae represent 25% of total marrow.24 After
PKH26+ cells were sorted, an aliquot was always reanalyzed
and the percent purity used to correct PKH26+ cell counts
before assay.
CFC assays.
Unseparated, sorted or ex vivo-expanded BM cells were assayed for CFCs
as previously described.18
Measurement of in vivo hematopoietic reconstitution kinetics.
B6.SJL mice were exposed to 9 Gy total-body -irradiation
(administered in two doses of 4.5 Gy ~3 hours apart just before transplantation) and injected with 106 PKH26-labeled but
otherwise unmanipulated B6 BM cells, or 106
PKH26+ BM cells that had been isolated by
fluorescence-activated cell sorting (FACS) from the marrow or spleen of
lethally irradiated B6 mice following a 3-hour homing step. Mice were
bled from the retro-orbital sinus at 6, 9, 12, 15, 18, 25, and 32 days
after transplantation. Until day 25, only half of the mice in each
cohort were analyzed alternately at each time so that no individual
animal was bled more frequently than every 7 days. Circulating
leukocyte, erythrocyte, and platelet counts were measured by analysis
of 40 µL of blood using a System 9118+ Hematology Series
Cell Counter (BioChem ImmunoSystems Inc, Allentown, PA). At selected
times, blood samples were also stained with a donor-specific
anti-Ly-5.2~FITC MoAb (clone ALI4A2) and PE-conjugated MoAbs
specific for B (anti-CD45R/B220; clone RA3-6B2) or T lymphocytes (anti-Thy-1.2; clone 30H12), or granulocytes (anti-Ly6G/Gr-1; clone
RB6-8C5) and macrophages (anti-CD11b/Mac-1; clone M1/70). Multilineage
progeny of transplanted stem cells were quantitated using a FACScan
instrument (Becton Dickinson). After 120 days, all mice were euthanized
and marrow cells were assayed for CFCs, and injected into lethally
irradiated B6.SJL mice (0.5 femurs per mouse) for secondary
repopulation. Secondary mice were analyzed 10 weeks later for
donor-derived peripheral blood leukocytes and BM CFCs as described earlier.
Statistical analysis.
The statistical significance of differences between means was assessed
using the two-tailed t-test.
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RESULTS |
Development of a homing assay for transplanted hematopoietic cells.
To track the localization of IV-transplanted BM cells to various
hematopoietic organs in vivo, we used a well-established procedure for
fluorescently labeling cells with the fluorescent dye PKH26. Using the
staining procedure described in Materials and Methods, 99% of BM cells
could be brightly stained with a median fluorescence intensity
approximately 1,000-fold higher than that of unlabeled control cells
(Fig 1). This degree of separation was
found to be critical for the sorting of pure populations of labeled
cells from hematopoietic organs after transplantation (see later), and
homing assays were only performed when this bright staining was
achieved. As reported previously,10 PKH26 staining did not
have any deleterious effect on the cloning efficiency of hematopoietic
progenitors detected by their capacity to generate myeloid, erythroid,
or mixed colonies in vitro. Labeled BM cells generated comparable
numbers of total colonies as unstained control cells; 75 ± 4 (n = 16) and 60 ± 5 (n = 14) per 3 × 104 cells,
respectively. This indicates that PKH26 does not alter the functional
activity of hematopoietic progenitors and is suitable to track their
homing in vivo.
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| Fig 1.
Tracking of PKH26-labeled BM cells in vivo. Murine marrow
cells (A) were labeled with PKH26 (B) and 108 cells were
injected into a lethally irradiated syngeneic mouse. Three hours later,
PKH26+ cells that had "homed" to the bone marrow
(C; 11% labeled) or spleen (D; 44% labeled) were identified by flow
cytometry and sorted according to the gates depicted by the vertical
lines.
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Initial experiments were conducted to establish the time after
transplantation required to recover maximal numbers of
PKH26+ cells in the two primary hematopoietic organs of the
adult mouse: the BM and spleen. Irradiated animals were injected with
approximately 108 PKH26-labeled marrow cells. BM and spleen
cells were harvested from different recipients 1, 3, or 24 hours later
and the frequency and absolute number of PKH26+ cells in
each organ determined by flow cytometry. Marrow recoveries were
calculated based on the assumption that two femora and two tibiae
represent 25% of total BM.24 Figure
2A shows that transplanted cells homed
rapidly to the spleen (8.6% ± 3.9% of input recovered after 1 hour), but their numbers declined thereafter to 5.9% ± 0.4% and only 2.3% ± 0.5% of input after 3 and 24 hours,
respectively. Although egress from this organ was accompanied initially
by an approximately equivalent accumulation of PKH26+ cells
in the marrow, the recovery of transplanted cells in marrow plateaued
at approximately 5% of input numbers after 3 hours despite continued
migration out of the spleen. Overall, it was notable that a maximum of
only approximately 12% of all injected cells were recovered in these
two organs, the remainder likely having accumulated in highly perfused
nonhematopoietic organs such as the lungs and liver, where they are
presumably removed by cells of the reticuloendothelial system.
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| Fig 2.
Homing of murine BM cells. 108 PKH26-labeled
BM cells were injected into lethally irradiated hosts. At the indicated
times, recipient BM and spleen cells were counted to determine
whole-organ cellularity, and the frequency of PKH26+
cells in each determined by FACS. Labeled cells were sorted and assayed
for CFCs together with an aliquot of labeled cells from the
pretransplant suspension. Shown are the mean ± SEM percent recoveries
of total cells (A) and CFCs (B) in BM ( ) and spleen ( ) for pooled
data from 3 experiments (3 mice/time point). Values are derived from
the formulae: % Cell Recovery = (% PKH26+ by FACS × whole-organ cellularity)/108, and % CFC Recovery = (CFC
frequency in sorted PKH26+ cells × No.
PKH26+ cells per organ)/(CFC frequency in pretransplant
BM × 108). Differences in the recovery of total cells and
CFCs is significant at 24 hours (P < .05); differences in the
recovery of CFCs at 3 hours is significant to P = .1.
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To directly quantitate the homing of functional hematopoietic
progenitors to the marrow and spleen, PKH26+ cells were
sorted from each organ by FACS and assayed for their potential to
generate colonies of mature blood cells in semisolid medium. Figure 1
shows the gates used for donor cell selection from one mouse injected 3 hours previously with 108 PKH26-stained BM cells.
Transplanted donor cells were readily detectable in the marrow (11%
PKH26+) and spleen (44% PKH26+) and could be
sorted to greater than 98% homogeneity for subsequent assays. In some
experiments, PKH26 cells of lethally irradiated host
origin were also sorted as a negative control, but, as expected, failed
to generate any colonies in vitro (data not shown). Similar to total
nucleated cells described earlier, CFCs homed rapidly to the spleen
(~13% of input recovered after 1 hour), but declined thereafter to
approximately 11% and only 6% of input numbers after 3 and 24 hours,
respectively (Fig 2B). Progenitors accumulated simultaneously in the BM
plateauing at 6% to 8% of input after 3 hours. When the number of
PKH26+ CFCs per organ was divided by the total number of
PKH26+ cells, it was found that CFCs were moderately
concentrated among cells homing to the spleen compared with marrow at 3 hours after transplant: 390 ± 50 versus 230 ± 20 CFCs per
105 PKH26+ cells, respectively. No such
differences were noted at the 1-hour time point (260 ± 50 CFCs per
105 PKH26+ cells in both organs), but by 24 hours the relative difference in CFC concentration in the spleen versus
BM was still 1.4-fold. Thus, more clonogenic progenitors were recovered
in the spleen than in the BM within the first few hours after
transplantation, and appeared preferentially concentrated in the former
after 3 hours.
Linearity of the homing assay.
In order for the homing assay to be quantitative, it was necessary to
establish (1) that the frequency and absolute number of transplanted
cells measured in the BM and spleen were directly proportional to the
number of labeled cells injected, and (2) that transplanted cell dose
did not influence progenitor homing within the range of cell doses
normally infused. To define these assay parameters, irradiated mice
were injected with increasing numbers of PKH26-labeled BM cells
(106 to 108 per mouse) and marrow and spleen
cells analyzed 3 hours later to determine the frequency of
PKH26+ cells in each organ. Figure
3A shows that labeled cells homed to both
organs in linear proportion to the number transplanted (correlation
coefficient, r = .91 for BM and .92 for spleen), and
their frequency was maintained at a constant ratio of approximately 4:1
in spleen versus marrow at all cell doses tested. The overall recovery
of transplanted cells in marrow or spleen was also not significantly
different and was unaffected by transplanted cell dose; an average of
5% to 10% of the injected cells were recovered in each organ 3 hours
after transplantation irrespective of the number of cells infused (Fig
3B). As an additional means to exclude the possibility that stem cell
homing might be competitively inhibited at high cell doses, irradiated
mice were cotransplanted with 106 or 107
PKH26-labeled BM cells with or without a ninefold excess of unlabeled BM cells (107 and 108 total cells injected,
respectively). No difference was observed in the frequency of
PKH26+ cells in BM (0.08%) or spleen (0.3%) of mice
injected with 106 labeled cells with or without unlabeled
competitors. Consistent with the results in Fig 3A, mice injected with
107 PKH26+ cells contained approximately
10-fold more fluorescent cells in BM (0.74%) and spleen (3.9%), but
again no differences in homing were observed in animals transplanted
with 9 × 107 unstained BM cells. Taken together,
these data demonstrate that even after infusing up to 108
cells per mouse, putative niches available to sequester IV-transplanted hematopoietic cells are not yet saturated, a condition that may lead to
increased nonspecific localization at these or secondary sites.
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| Fig 3.
Linearity of the homing assay. Lethally irradiated mice
were injected with 106 to 1.2 × 108
PKH26-stained BM cells. Three hours later, BM () and spleen ()
cells were analyzed by flow cytometry. (A) The percent of labeled cells
in each organ is shown for 32 individual mice from 18 experiments.
Correlation coefficients >0.9 demonstrate a linear relationship
between hematopoietic cell homing and transplanted cell dose. (B) The
values in A were multiplied by organ cellularity in each case to
calculate the fraction of cells homing to BM or spleen relative to the
number transplanted. The essentially flat scatter of points over the
x-axis demonstrates that hematopoietic cell homing is largely
independent of graft size over the range of cell doses normally
transplanted.
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Organ-selective homing of early engrafting hematopoietic cells.
The data in Fig 2 indicate that more clonogenic progenitors home to the
spleen than BM within 3 hours after IV transplantation, and that CFCs
are almost twofold more concentrated within the donor compartment in
spleen at this time. To compare the early and long-term reconstitution
potential of these populations with differential homing properties,
PKH26+ cells that had homed for 3 hours to the BM
("BM-homed") or spleen ("SPL-homed") of irradiated mice
were sorted and retransplanted into lethally irradiated Ly-5 congenic
hosts (106 cells per mouse). A third group of control
B6.SJL mice were each transplanted with 106 B6 BM cells
that had been labeled with PKH26, but not subsequently selected with an
in vivo homing step ("nonhomed"). Peripheral blood was counted
once or twice per week from 6 to 32 days after transplantation to
measure early hematopoietic reconstitution kinetics. The levels of
leukocytes (6 to 9.2 × 103/µL), erythrocytes (9.6 to 11.4 × 106/µL), and platelets (10.9 to 13.8 × 105/µL) in normal B6.SJL mice analyzed in
parallel are shown by the shaded areas in the upper portion of Fig
4B and C (normal range for leukocytes in
Fig 4A is off scale). The decline in blood cell counts after
irradiation is indicated by the thick gray lines in each panel. In
contrast to 11 irradiated but untransplanted control animals, which all
died by day 12 after transplantation, all recipients of 106
BM-homed (five mice), SPL-homed (14 mice), or nonhomed (eight mice)
cells survived for the entire period of analysis. Interestingly, mice
that were transplanted with BM cells that had homed initially to the
spleen exhibited approximately 30% higher white blood cell counts at
all times during the first month after transplantation compared with
recipients of 106 BM-homed cells (Fig 4A). As a result,
SPL-homed cells regenerated circulating leukocytes to 20% of normal
counts approximately 13 days faster (ie, in 12 days) than cells that
had homed initially to the marrow (~25 days), a finding that
correlates with the approximately 1.7-fold higher CFC content of the
former noted above (3,900 ± 500 v 2,300 ± 200, respectively). Nonhomed control BM cells required 18 days to engraft to
this level. No differences in the rate of platelet or erythrocyte
regeneration were observed in mice transplanted with cells that had
homed to the BM versus the spleen (Fig 4B and C).
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| Fig 4.
Early hematopoietic reconstitution kinetics of
BM cells selected pretransplantation on the basis of marrow or splenic
homing. Lethally irradiated mice were injected with 108
PKH26-stained syngeneic BM cells. Three hours later,
PKH26+ cells that had homed to the marrow () or spleen
() were isolated by FACS and transplanted into lethally irradiated
Ly-5 congenic mice (106 cells/mouse). Control animals were
injected with 106 PKH26-stained BM cells that had not been
selected by a prior homing step ( ). Peripheral blood leukocytes (A),
erythrocytes (B), and platelets (C) were counted on the indicated days.
The normal range of blood cell counts (except leukocytes, which are off
scale) and their decline after irradiation are defined by the shaded
areas and thick gray lines. Shown are the mean ± SEM values
for pooled data from 3 experiments (5 to 14 mice/population). Errors
not shown are too small for the scale used. Differences in leukocyte
counts in recipients of BM-homed or SPL-homed cells are significant on
days 25 and 32 (P < .05).
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To confirm that leukocytes were regenerated by donor-derived
progenitors in these mice, and to determine whether there might be
differences in the distribution of the progeny of SPL-homed versus
BM-homed cells to lymphoid and myeloid lineages, blood cells were also
stained on days 15 and 18 with an Ly-5.2-specific MoAb together with
MoAbs reactive to B cells, T cells, granulocytes, or monocytes. As
expected, white blood cells were predominantly of donor type (87% ± 6%) in all mice from the three groups, and were distributed at
normal ratios of 73% ± 2% to the B lineage, 18% ± 1% to the
T lineage, and 13% ± 1% to the myeloid lineage (data not shown).
Therefore, no differences in the differentiation potential of BM-homed
versus SPL-homed cells were noted that could explain their differential
leukocyte reconstitution kinetics.
Superior long-term hematopoiesis in mice transplanted with BM cells
selected on the basis of splenic homing.
Four months after transplant, all animals recovered normal levels of
circulating blood cells that were virtually completely regenerated from
donor stem cells (blood 92% to 95% Ly-5.2+ in all three
groups, data not shown). Marrow cellularity had recovered to
approximately 80% of that of age-matched normal control mice and did
not differ among recipients of 106 BM-homed, SPL-homed, or
nonhomed marrow cells (Fig 5A). However, mice transplanted with SPL-homed cells contained slightly more (~38%; P < .05) CFCs per femur than mice
injected with BM-homed cells (Fig 5B). Marrow cells from these primary
animals were also retransplanted into lethally irradiated secondary
B6.SJL mice (0.5 femurs per mouse). Ten weeks later, SPL-homed cells
had regenerated approximately 43% more cells per secondary femur than
BM-homed cells (Fig 5A). Although all three groups of secondary
recipients declined in femoral CFC content relative to the primary
marrow's, recipients of SPL-homed cells contained 46% (P < .05) and 55% (P < .05) more CFCs per femur than secondary
recipients of nonhomed and BM-homed cells, respectively (Fig 5B). Taken
together, the data presented in Figs 4 and 5 suggest that BM cells that
home initially to the spleen are, as a population, enriched for HSCs and progenitor cells with early leukocyte and long-term multilineage repopulating potential in myeloablated primary and secondary hosts. In
contrast, BM cells that home to the marrow within 3 hours after transplantation do not behave significantly differently than
PKH26-stained cells that had not been selected by an in vivo homing
step before assay.
View larger version (42K):
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[in a new window]
| Fig 5.
Long-term regeneration of marrow cellularity and CFCs in
primary and secondary recipients of cells selected on the basis of
marrow or splenic homing. Shown are the mean ± SEM number of cells
(A) and CFCs (B) per femur in lethally irradiated primary mice ( )
injected 120 days previously with BM-homed, SPL-homed, or nonhomed
cells. Lethally irradiated secondary mice were transplanted with 0.5 femur/mouse and analyzed 10 weeks later ( ). Hematologic parameters
for age-matched normal B6.SJL mice are shown for comparison ().
Pooled data from 5 to 14 mice/population. Differences in femoral
cellularity between secondary recipients of BM-homed and SPL-homed
cells are significant to P = .1; differences in femoral CFC
content between primary recipients of BM-homed and SPL-homed cells are
significant to P < .05, and between secondary recipients of
SPL-homed cells and either BM-homed or nonhomed cells are significant
to P < .05.
|
|
Ex vivo expansion of HSCs generates progenitors with diminished in
vivo homing capacity.
Having developed a quantitative assay to measure stem/progenitor cell
homing in vivo, we next sought to employ it to measure the homing of
CFCs generated by ex vivo expansion. Several investigators have
demonstrated that highly enriched HSCs can be partially differentiated ex vivo to generate large numbers of functional hematopoietic progenitors.18,25-27 Although expanded cells can regenerate
circulating blood cells approximately 1 to 2 weeks faster (depending on
the lineage) than freshly isolated HSCs when transplanted into
myeloablated mice, the rate of hematopoietic reconstitution is not as
rapid as might be predicted from their clonogenic cell
content.17,18 This finding suggests that the homing
properties of primitive cells may be altered following their expansion
in potent combinations of hematopoietic growth factors in vitro. To
test this hypothesis, serum-free suspension cultures containing IL-3,
IL-6, G-CSF, and SCF were initiated with 20 × 106
normal BM cells (106/mL; one experiment). In two additional
experiments, cultures containing IL-6, IL-11, G-CSF, SCF, FLK, and TPO
were seeded with 16 to 18 × 103
Sca-1+c-kit+Lin
cells (103/mL) enriched by FACS from the BM of B6 mice
injected 1 day previously with 5-FU. This combination of cytokines was
determined in preliminary studies to support optimal expansion of total
nucleated cells and CFCs from enriched stem cells (S.J.S., unpublished
data, July 1997). Limiting dilution competitive
repopulation assays established that
Sca-1+c-kit+Lin
cells are highly enriched in stem cells with long-term lymphomyeloid repopulating potential and contain 1 competitive repopulating unit
(CRU) per 15 (95% confidence limits, 1 per 12 to 1 per 23) cells
(S.J.S., unpublished data, October 1997). In contrast,
more mature progenitor cells such as in vitro CFCs (59 ± 6 per
103 cells; five experiments) and CFU-S (2.6 ± 0.4 day
12 and 0 day 8 CFU-S per 103 cells; three experiments) are
relatively depleted. After 9 days of culture, cells and CFCs were
expanded 1.2-fold (P > .05) and 7.5-fold (P < .05),
respectively, in the experiment initiated with unfractionated BM cells,
and 900 ± 100-fold (P < .05) and 420 ± 230-fold (P < .05), respectively, in the two experiments initiated with purified
Sca-1+c-kit+Lin
cells. Expanded cells were stained with PKH26 as described earlier, and
injected into lethally irradiated syngeneic mice. Three hours later, an
average of 0.53% ± 0.13% and 0.91% ± 0.43% of the cultured cells had homed to the BM and spleen, respectively (Table
1; data pooled from all three experiments).
Similar fractions of the input number of CFCs were recovered in each
organ: 0.56% ± 0.27% in marrow and 1.67%± 0.98% in spleen
(Table 1). When these data are compared with the results of experiments
performed above with freshly isolated BM cells, it is clear that the
homing capacity of clonogenic progenitors generated in vitro is reduced
approximately 10-fold (P < .05) compared with normal (Fig
6). The data are consistent with historic
observations of the diminished engraftment potential of cultured
hematopoietic cells in both mouse and humans.
View larger version (17K):
[in this window]
[in a new window]
| Fig 6.
Reduced homing capacity of hematopoietic progenitors
generated in vitro. Lethally irradiated mice were transplanted with
~108 PKH26-labeled normal BM cells or ~107
PKH26-labeled progeny of unfractionated or
Sca-1+c-kit+Lin- BM
cells generated after 9 days of expansion in hematopoietic growth
factors (see Table 1 for details). Three hours later,
PKH26+ cells that had homed to the BM () or spleen
( ) were quantitated by flow cytometry and isolated by FACS for CFC
assays. Shown are the mean ± SEM percent recovery of donor-derived
cells (left) and CFCs (right) per organ.
|
|
| |
DISCUSSION |
In this report, we describe a quantitative assay to monitor the homing
of transplanted hematopoietic cells in vivo, and more importantly,
which facilitates their isolation from various hematopoietic organs for
subsequent functional analysis. Quantitation of progenitor homing is
achieved by multiplying the percentage of cells in a particular organ
that bear the fluorescent marker of the transplanted population
(PKH26+) by whole-organ cellularity at the time of assay.
This calculation provides the absolute number of donor cells in each
organ of interest; the absolute number of a particular class of stem or
progenitor cells within the donor population is then determined by
functionally assaying their frequency among PKH26+ cells
sorted by FACS. As is important for any quantitative assay, we have
established that the frequency of donor cells in BM and spleen is
linearly related to the number of cells transplanted between
106 and 1.2 × 108 cells per mouse (Fig
3). The fraction of input cells recovered in these organs is also
essentially constant at these cell doses (~5% to 10% at 3 hours),
and is not diminished by the cotransplantation of a ninefold excess of
unlabeled competitor cells. Taken together, the data suggest that the
number of niches available for seeding by transplanted hematopoietic
cells is not limiting in our model.
We have used this assay to demonstrate that in vitro CFCs derived from
normal adult mouse BM accumulate in the marrow and spleen of irradiated
recipients within 1 hour after IV injection. Thereafter, the number of
CFCs in the spleen declines for at least 24 hours and progenitors
accumulate in the BM until their numbers are largely stabilized at
approximately 3 hours after transplant. Interestingly, CFCs are
moderately concentrated among donor cells that home to the spleen
compared with BM, so that after 3 hours PKH26+ cells sorted
from each organ differ by almost twofold in CFC content. When
106 SPL-homed and BM-homed cells, containing a mean of
3,900 ± 500 or 2,300 ± 200 CFCs, respectively, were assayed for
their potential to rapidly reconstitute hematopoiesis in lethally
irradiated Ly-5 congenic hosts, we observed that SPL-homed cells
regenerated 20% normal numbers of circulating leukocytes almost 2 weeks faster than BM-homed cells (Fig 4). Primary recipients of
SPL-homed cells also recovered 38% more CFCs per femur by 17 weeks
after transplant than recipients of BM-homed cells, which resulted in a
55% higher recovery of clonogenic progenitors in secondary mice
injected 10 weeks previously with a 0.5 femur equivalent of primary
marrow. These differences in transplantation potential may be due
simply to differences in the number of in vitro CFCs present in each fraction. However, this conclusion is not supported by recent studies
that suggest such cells are probably not responsible for early
hematopoietic reconstitution in vivo.28,29 Instead, our data suggest either (1) that BM-homed and SPL-homed cells differ in
their content of some other class of clonogenic cells able to
regenerate and maintain hematopoiesis upon transplantation, or (2) that
3-hour homing to spleen segregates cells with functional properties
that differ from those homing to the marrow. In the latter case, it
would be important to determine whether these characteristics preexist
in the marrow population before transplant, or are imposed upon the
cells during their short residence in the organ to which they homed.
These alternative possibilities could be resolved by normalizing
PKH26+ cells isolated from each organ after homing for
equivalent numbers of some functionally defined class of clonogenic
cells (ie, CFCs or more primitive CRU), instead of total nucleated
cells pretransplant. If the differences we have observed between
BM-homed and SPL-homed cells are due to differences in their content of
otherwise functionally equivalent stem/progenitor cells, then the two
homed populations should perform similarly. If, however, differential
homing selects for cells that contribute variably to hematopoiesis,
then the present differences observed at the level of the whole
populations should be maintained. Studies to test these possibilities
are currently in progress. Furthermore, it remains to be demonstrated whether the differences we have documented in the activity of hematopoietic cells homing to the spleen or BM 3 hours after
transplantation are reproduced at later times. We and
others11 have shown that in contrast to the 3-hour time
point, after 24 hours a greater proportion of injected cells (including
CFCs) are recovered in the BM than the spleen. However, because the
relative frequency of CFCs among PKH26+ cells in the
spleen was still 1.4-fold higher in spleen than BM at this time, we
would not predict that extending the assay end point at least to 24 hours should alter our results. Nonetheless, the present assay appears
well suited to further exploring the dynamics of HSC localization in
vivo during the first few days after transplantation.
In a useful experimental application of the homing assay, we evaluated
the homing properties of hematopoietic progenitors generated in vitro
by the serum-free expansion of either unfractionated normal BM cells in
IL-3, IL-6, G-CSF, and SCF, or highly enriched Sca-1+c-kit+Lin
cells in IL-6, IL-11, G-CSF, SCF, FLK, and TPO. After 9 days, CFCs were
expanded 7.5-fold and up to 644-fold, respectively, thereby providing
very large numbers of clonogenic cells for transplantation studies.
However, when expanded CFCs were labeled with PKH26 and injected into
lethally irradiated mice, we found that only approximately 0.6% and
1.7% of injected progenitors localized to the marrow and spleen,
respectively, after 3 hours. This represents an approximate 10-fold
reduction in the homing capacity of CFCs compared with freshly isolated
marrow. Sca-1+Lin BM cells that were
expanded in vitro for 7 days in serum-containing cultures supplemented
with IL-1 , IL-3, IL-6, and SCF have previously been shown to
generate classes of progenitor cells with different repopulating
potentials depending on their replicative history.21 Cells
that had divided fewer than one or two times retained the highest
frequency of repopulating cells, and repopulating potential was
significantly diminished after four cell divisions in vitro. These data
suggest that proliferation of transplantable cells may affect their
ability to engraft myeloablated hosts. A similar decline in the seeding
efficiency of murine CFU-S isolated from regenerating marrow following
hydroxyurea treatment was noted more than 10 years ago.30
More recently, it was reported that preincubation of murine BM cells
with IL-3, or IL-3 plus IL-12 and SCF, for as short as 2 hours led to a
substantial decrease in the seeding of all subsets of cobblestone
area-forming cells (CAFC) to both marrow and spleen.31
Taken together with our present results, these data suggest that
qualitative changes in the homing properties of stem and progenitor
cells that have been stimulated to proliferate in response to cytokine
exposure may nullify the potential clinical benefits of their
quantitative expansion in vitro. Although we have not yet examined
whether the loss of CFC homing that we have observed correlates with
changes in their expression of particular adhesion molecules, a
functional association between the expression of CD43, CD44, and
L-selectin and both rapid hematopoietic reconstitution potential and
long-term marrow repopulating ability has previously been
noted.32,33 The challenge for the future will clearly be to
overcome the decline in homing capacity of stem and progenitor cells
manipulated ex vivo through continued exploration of alternative
culture conditions that promote their growth. In this regard, more
serious consideration should probably be paid to the importance of
various integrins and extracellular matrix molecules in stem cell
cultures. These may facilitate the selective expansion of
transplantable cells expressing adhesion molecules critical for in vivo
homing. Alternatively, efforts to improve stem cell homing through
treatment of transplant recipients with small peptides that promote the
attachment of clonogenic cells to stroma may also provide a route
to improving the engraftment properties of ex vivo expanded
hematopoietic cells.34 Regardless of the strategies to be
adopted, the present model system should prove useful for future
studies of the homing and engraftment properties of hematopoietic cells
from a variety of sources.
| |
ACKNOWLEDGMENT |
The authors thank Dr Craig Jordan (University of Kentucky) for
stimulating discussions and for critically reviewing the manuscript.
| |
FOOTNOTES |
Submitted July 30, 1998; accepted October 22, 1998.
Supported by the University of Kentucky Hospital and the Department of
Internal Medicine, and by Grant No. 85-001-12-IRG to S.J.S. from the
American Cancer Society. S.J.S. is the recipient of a Junior Faculty
Scholar Award from The American Society of Hematology.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Stephen J. Szilvassy, PhD,
Hematology/Oncology-BMT, Lucille P. Markey Cancer Center, Room CC414,
800 Rose St, Lexington, KY 40536-0093.
| |
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101(1):
221 - 226.
[Abstract]
[Full Text]
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L. M. Scott, G. V. Priestley, and T. Papayannopoulou
Deletion of {alpha}4 Integrins from Adult Hematopoietic Cells Reveals Roles in Homeostasis, Regeneration, and Homing
Mol. Cell. Biol.,
December 15, 2003;
23(24):
9349 - 9360.
[Abstract]
[Full Text]
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P. A. Plett, S. M. Frankovitz, and C. M. Orschell
Distribution of marrow repopulating cells between bone marrow and spleen early after transplantation
Blood,
September 15, 2003;
102(6):
2285 - 2291.
[Abstract]
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A. Bel, E. Messas, O. Agbulut, P. Richard, J. L. Samuel, P. Bruneval, A. A. Hagege, and P. Menasche
Transplantation of Autologous Fresh Bone Marrow Into Infarcted Myocardium: A Word of Caution
Circulation,
September 9, 2003;
108(90101):
II-247 - 252.
[Abstract]
[Full Text]
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C. S. McCauslin, J. Wine, L. Cheng, K. D. Klarmann, F. Candotti, P. A. Clausen, S. E. Spence, and J. R. Keller
In vivo retroviral gene transfer by direct intrafemoral injection results in correction of the SCID phenotype in Jak3 knock-out animals
Blood,
August 1, 2003;
102(3):
843 - 848.
[Abstract]
[Full Text]
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M. Nagano, B.-Y. Ryu, C. J. Brinster, M. R. Avarbock, and R. L. Brinster
Maintenance of Mouse Male Germ Line Stem Cells In Vitro
Biol Reprod,
June 1, 2003;
68(6):
2207 - 2214.
[Abstract]
[Full Text]
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P. Denning-Kendall, S. Singha, B. Bradley, and J. Hows
Cytokine Expansion Culture of Cord Blood CD34+ Cells Induces Marked and Sustained Changes in Adhesion Receptor and CXCR4 Expressions
Stem Cells,
January 1, 2003;
21(1):
61 - 70.
[Abstract]
[Full Text]
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P. A. Plett, S. M. Frankovitz, and C. M. Orschell-Traycoff
In vivo trafficking, cell cycle activity, and engraftment potential of phenotypically defined primitive hematopoietic cells after transplantation into irradiated or nonirradiated recipients
Blood,
November 15, 2002;
100(10):
3545 - 3552.
[Abstract]
[Full Text]
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J. F. Zhong, Y. Zhan, W. F. Anderson, and Y. Zhao
Murine hematopoietic stem cell distribution and proliferation in ablated and nonablated bone marrow transplantation
Blood,
November 15, 2002;
100(10):
3521 - 3526.
[Abstract]
[Full Text]
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N. Askenasy and D. L. Farkas
Optical Imaging of PKH-Labeled Hematopoietic Cells in Recipient Bone Marrow In Vivo
Stem Cells,
November 1, 2002;
20(6):
501 - 513.
[Abstract]
[Full Text]
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B. E. Strauer, M. Brehm, T. Zeus, M. Kostering, A. Hernandez, R. V. Sorg, G. Kogler, and P. Wernet
Repair of Infarcted Myocardium by Autologous Intracoronary Mononuclear Bone Marrow Cell Transplantation in Humans
Circulation,
October 8, 2002;
106(15):
1913 - 1918.
[Abstract]
[Full Text]
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O. Kollet, I. Petit, J. Kahn, S. Samira, A. Dar, A. Peled, V. Deutsch, M. Gunetti, W. Piacibello, A. Nagler, et al.
Human CD34+CXCR4- sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation
Blood,
September 26, 2002;
100(8):
2778 - 2786.
[Abstract]
[Full Text]
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N. Askenasy, T. Zorina, D. L. Farkas, and I. Shalit
Transplanted Hematopoietic Cells Seed in Clusters in Recipient Bone Marrow In Vivo
Stem Cells,
July 1, 2002;
20(4):
301 - 310.
[Abstract]
[Full Text]
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J. Wilpshaar, M. Bhatia, H. H. H. Kanhai, R. Breese, D. K. Heilman, C. S. Johnson, J. H. F. Falkenburg, and E. F. Srour
Engraftment potential of human fetal hematopoietic cells in NOD/SCID mice is not restricted to mitotically quiescent cells
Blood,
June 17, 2002;
100(1):
120 - 127.
[Abstract]
[Full Text]
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E. J. K. Noach, A. Ausema, J. H. Dillingh, B. Dontje, E. Weersing, I. Akkerman, E. Vellenga, and G. de Haan
Growth factor treatment prior to low-dose total body irradiation increases donor cell engraftment after bone marrow transplantation in mice
Blood,
June 17, 2002;
100(1):
312 - 317.
[Abstract]
[Full Text]
[PDF]
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A. Jetmore, P. A. Plett, X. Tong, F. M. Wolber, R. Breese, R. Abonour, C. M. Orschell-Traycoff, and E. F. Srour
Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients
Blood,
March 1, 2002;
99(5):
1585 - 1593.
[Abstract]
[Full Text]
[PDF]
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M. Takeuchi, T. Sekiguchi, T. Hara, T. Kinoshita, and A. Miyajima
Cultivation of aorta-gonad-mesonephros-derived hematopoietic stem cells in the fetal liver microenvironment amplifies long-term repopulating activity and enhances engraftment to the bone marrow
Blood,
February 15, 2002;
99(4):
1190 - 1196.
[Abstract]
[Full Text]
[PDF]
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T. Papayannopoulou, G. V. Priestley, B. Nakamoto, V. Zafiropoulos, and L. M. Scott
Molecular pathways in bone marrow homing: dominant role of {alpha}4{beta}1 over {beta}2-integrins and selectins
Blood,
October 15, 2001;
98(8):
2403 - 2411.
[Abstract]
[Full Text]
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T. C. C. Kerre, G. De Smet, M. De Smedt, F. Offner, J. De Bosscher, J. Plum, and B. Vandekerckhove
Both CD34+38+ and CD34+38- Cells Home Specifically to the Bone Marrow of NOD/LtSZ scid/scid Mice but Show Different Kinetics in Expansion
J. Immunol.,
October 1, 2001;
167(7):
3692 - 3698.
[Abstract]
[Full Text]
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S. J. Szilvassy, T. E. Meyerrose, P. L. Ragland, and B. Grimes
Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver
Blood,
October 1, 2001;
98(7):
2108 - 2115.
[Abstract]
[Full Text]
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O. Kollet, A. Spiegel, A. Peled, I. Petit, T. Byk, R. Hershkoviz, E. Guetta, G. Barkai, A. Nagler, and T. Lapidot
Rapid and efficient homing of human CD34+CD38{-}/lowCXCR4+ stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2mnull mice
Blood,
May 15, 2001;
97(10):
3283 - 3291.
[Abstract]
[Full Text]
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G. de Haan, S. J. Szilvassy, T. E. Meyerrose, B. Dontje, B. Grimes, and G. Van Zant
Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers
Blood,
August 15, 2000;
96(4):
1374 - 1379.
[Abstract]
[Full Text]
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S. J. Szilvassy, T. E. Meyerrose, and B. Grimes
Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells
Blood,
May 1, 2000;
95(9):
2829 - 2837.
[Abstract]
[Full Text]
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Top
Abstract
Introduction