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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2829-2837
HEMATOPOIESIS
Effects of cell cycle activation on the short-term engraftment
properties of ex vivo expanded murine hematopoietic cells
Stephen J. Szilvassy,
Todd E. Meyerrose, and
Barry Grimes
From the Blood and Marrow Transplant Program, the Division of
Hematology/Oncology, Lucille P. Markey Cancer Center,
University of Kentucky, Lexington, KY.
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Abstract |
Loss of long-term hematopoietic stem cell function in vitro is
associated with cell cycle progression. To determine whether cytokine-induced proliferation also limits the rate of short-term engraftment and potential clinical utility of ex vivo expanded hematopoietic cells, murine
Sca-1+c-kit+Lin cells were
cultured in interleukin-6 (IL-6), IL-11, granulocyte colony-stimulating
factor (G-CSF), stem cell factor, flk-2 ligand, and
thrombopoietin for 7 days. Cells amplified 2000-fold were then stained
with Hoechst 33342, separated into G0/G1 (72% ± 3%) or S/G2/M (27% ± 3%) fractions by flow
sorting, and injected into lethally irradiated mice. Although long-term
(more than 6 months) engraftment of lymphoid and myeloid lineages was
greater in primary and secondary recipients of expanded cells residing
in G0/G1 at the time of transplantation, there
were no noted differences in the short-term (less than 6 weeks)
recovery kinetics of circulating blood cells. When hematopoietic cells
were expanded in cultures containing the tetrapeptide stem cell
inhibitor N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) to reduce progenitor
cycling prior to transplantation, again there were no differences
observed in short-term reconstitution by inhibited or uninhibited
cells. Interestingly, AcSDKP significantly accelerated engraftment by
expanded hematopoietic cells when administered in vivo at the time of
transplantation. Leukocytes recovered to 20% of normal levels
approximately 1 week faster, and thrombocytopenia was largely abrogated
in AcSDKP-treated versus untreated mice. Therefore, while AcSDKP can
accelerate the engraftment of ex vivo expanded hematopoietic
progenitors, which suggests a relatively simple approach to improve
their clinical utility, its effects appear unrelated to cell cycle arrest.
(Blood. 2000;95:2829-2837)
© 2000 by The American Society of Hematology.
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Introduction |
Cancer patients receiving high-dose chemotherapy and/or
radiotherapy followed by hematopoietic stem cell (HSC) transplantation typically endure between 10 days and 5 weeks of profound neutropenia and thrombocytopenia, depending on the number and source of HSCs infused.1-4 During this time, infections and clotting
disorders can be controlled by the administration of antibiotics,
hematopoietic growth factors, and platelet transfusions, but none of
these treatments completely abrogate the depression in blood cell
counts or usefully hasten hematopoietic recovery. In recent years,
several clinical studies have examined the possibility that HSCs
stimulated with recombinant cytokines in vitro may generate partially
differentiated progeny able to mediate more rapid hematopoietic
rescue.5-10 Although moderately efficacious in
mice,11-13 this cell-based therapy has not yet been proven
to be consistently beneficial in humans and underscores our currently
limited understanding of the role of different classes of hematopoietic
cells in early engraftment and potential changes in their
transplantation potential during ex vivo culture. For example, although
expanded cells can accelerate hematological recovery by 1-2 weeks when
transplanted into lethally irradiated mice, engraftment is slower than
predicted from the large number of in vitro colony-forming cells (CFCs)
and spleen colony-forming units (CFU-S) infused.12,14 One
possible explanation for this discrepancy is provided by our recent
demonstration that CFCs generated in vitro acquire a homing defect that
reduces by 10-fold their ability to localize to hematopoietic organs
after intravenous infusion.15 It will be critical to
resolve this issue before further clinical testing of expanded
hematopoietic cells because their functional potency may be severely
compromised even if large numbers of primitive cells can be generated
in culture.
Several recent studies indicate that the entry of cultured HSCs into
active cell cycle dramatically reduces their homing ability and
subsequent long-term repopulating potential. For example, a 2-hour
preincubation of murine bone marrow (BM) cells with either interleukin-3 (IL-3) or IL-3 plus IL-12 and stem cell factor (SCF) substantially decreases the seeding of all subsets of cobblestone area-forming cells (CAFCs) to both the BM and spleen.16 A
similar decline in the seeding efficiency of murine CFU-S isolated from regenerating marrow after hydroxyurea treatment was noted 20 years ago.17 Human CD34+-mobilized peripheral blood
cells that traverse from G0 to G1 over a
36-hour incubation with IL-3, SCF, and flk-2 ligand also lose their
capacity to regenerate hematopoiesis in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.18 Of critical
importance to the successful clinical application of cultured
hematopoietic cells, these effects on engraftment are not irreversible.
Rather, they constitute a plastic feature associated with
the cyclic oscillation of expanding stem/progenitor cells between
proliferation and quiescence.19 It is not known whether
similar deleterious effects of cell cycling also extend to cells that
mediate early hematopoietic reconstitution. If so, the seeding and
early engraftment kinetics of vigorously proliferating stem and
progenitor cells generated in culture might be improved by
transiently inhibiting their cycling immediately prior to transplantation.
In the present study, we tested this hypothesis using
N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) as a candidate stem cell inhibitor that may mediate such effects on short-term engraftment. This tetrapeptide, which is naturally present in serum, can protect mice
against lethal doses of cytosine arabinoside (Ara-C) by
preventing the entry of CFU-S into S phase.20 In
Ara-C-treated patients, administration of AcSDKP also decreases the
depth of granulocyte and platelet nadirs.21 Recently,
Suzuki et al22 showed that AcSDKP increased the survival
and engraftment of lethally irradiated mice transplanted with a
normally limiting dose of unfractionated BM cells. This
effect was mediated by an increased localization of CFU-S and
granulocyte-macrophage colony-forming units (CFU-GM) to the
marrow as early as 1 day after transplantation,22
suggesting an effect on the homing of primitive hematopoietic cells.
Therefore, we stimulated highly enriched
Sca-1+c-kit+Lin murine HSCs
in serum-free suspension cultures optimized for maximal generation of
clonogenic progeny. Ex vivo expanded CFCs (almost entirely in S phase)
were then inhibited by in vitro exposure to AcSDKP. When we compared
the rate of short-term hematopoietic reconstitution in mice that were
transplanted with arrested or unmanipulated expanded cells, no
beneficial effects of cell cycle down-modulation were noted. This lack
of effect of cell cycle activation on short-term engraftment potential
was supported by additional experiments in which ex vivo expanded cells
in G0/G1 or S/G2/M were physically
separated before transplantation by cell sorting. Both fractions
exhibited identical short-term engraftment kinetics, but only expanded
cells in G0/G1 retained high levels of
long-term multilineage repopulating ability. Interestingly, and in
marked contrast to the inability of AcSDKP to improve engraftment of in
vitro-treated cells, in vivo administration of the tetrapeptide significantly accelerated leukocyte and platelet recovery in lethally irradiated mice transplanted with ex vivo expanded cells. Our data
indicate that the potential clinical utility of expanded hematopoietic
cells for mediating rapid hematological recovery after myeloablative
cancer therapy can be improved by the relatively simple treatment of
recipients with a bioactive peptide. Although the mechanism of AcSDKP
action is currently undefined, it appears unrelated to its ability to
inhibit cell cycle entry.
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Materials and methods |
Animals
Marrow donors were 5- to 8-week-old C57BL/6 (B6) mice
(Ptprca [Ly-5.2]).
Age-matched B6 or B6.SJL (Ptprcb
[Ly-5.1]) mice were used as recipients as indicated. Mice were purchased via the National Cancer Institute Animal Program from Charles
River Laboratories (Frederick, MD) and maintained under specific
pathogen-free conditions in the animal facility of the Chandler Medical
Center (University of Kentucky, Lexington, KY).
Enrichment of hematopoietic stem cells
Sca-1+c-kit+Lin cells
were isolated from the BM of B6 mice injected 1 day earlier with 150 mg/kg 5-fluorouracil (5-FU; Roche Laboratories, Nutley, NJ) as
previously described15 using a dual-laser
fluorescence-activated cell sorter (FACSVantage; Becton Dickinson
Immunocytometry Systems, San Jose, CA). Reanalysis of enriched cells
immediately after sorting indicated a purity typically greater
than 90%.
Competitive repopulation assays
The frequency of competitive long-term repopulating units (CRU)
among freshly isolated
Sca-1+c-kit+Lin marrow cells
was determined by limiting-dilution analysis in vivo (3, 10, 30, or 100 cells/mouse) as previously described.15,16 A level of at
least 5% donor-derived (Ly-5.2+) peripheral blood cells
detectable among B (B220+) and T (Thy-1+)
lymphoid compartments and myeloid
(Mac-1/Gr-1+) cells 10 weeks after transplantation of
Ly-5.1 recipients was defined as the threshold for positive engraftment
for maximum likelihood analysis.
Ex vivo expansion of sorted stem cells
Sorted Sca-1+c-kit+Lin
cells were cultured (2 × 103/mL) in serum-free
medium (StemPro-34; Life Technologies, Gaithersburg, MD) containing 50 U/mL penicillin, 50 µg/mL streptomycin, 2 mmol/L L-glutamine, and
0.05 mmol/L 2-mercaptoethanol (2-ME). The medium was supplemented with
the following recombinant cytokines: 10 ng/mL murine IL-6
(Peprotech, Rocky Hill, NJ) and human G-CSF (Amgen, Thousand Oaks, CA);
50 ng/mL murine IL-11; and 100 ng/mL each of murine SCF, murine
thrombopoietin (TPO) (R&D Systems, Minneapolis, MN), and human flk-2
ligand (FLK) (Peprotech). Cultures were incubated undisturbed at
37°C and harvested after 7 days. In some experiments AcSDKP
(purchased initially from Sigma [St Louis, MO] and later generously
provided by Biomeasure [Milford, MA]) was added daily to duplicate
cultures to a final concentration of 108 to
1018 mol/L (with 100-fold serial dilutions between)
beginning on day 1.
CFC assays
Unseparated, sorted, or ex vivo expanded BM cells were assayed for
CFCs as previously described.12 The proportion of
progenitors in S phase was determined by incubating 2 aliquots of cells
for 1 hour at 37°C in Dulbecco's modified Eagle's medium (DMEM)
containing 2% fetal bovine serum (FBS) with or without 200 µg/mL
hydroxyurea (HU)(Sigma). Cells were then washed and plated at
104 or 2 × 103 cells/dish,
respectively. Colonies were counted 12 days later, and the percent of
CFCs in S phase (%S) was calculated using the following formula:
where
x = colony counts after exposure to HU, and y = colony counts after
incubation without HU.
Cell cycle fractionation by FACS
Ex vivo expanded BM cells were separated into fractions in
G0/G1 or S/G2/M phases of the cell
cycle as follows. Cells were resuspended at approximately
5 × 106/mL in Hank's balanced salt solution (HBSS)
containing 10% FBS, 1 mg/mL glucose, 20 mmol/L HEPES
(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid), and 5 µg/mL
Hoechst 33342 (HO) (Molecular Probes, Eugene, OR) and
incubated in the dark at 37°C for 30 minutes. Cells were then
washed twice in the same buffer without HO dye and analyzed on a
FACSVantage flow cytometer equipped with an Enterprise II laser
providing the 351-nm excitation wavelength for HO. HO fluorescence was
detected with a 424/44-nm bandpass filter, and sort windows were
established using linear gates. Cells were kept on ice during sorting
and protected from light. In preliminary experiments, 50 µmol/L
Verapamil (Sigma) was included in the medium during HO staining and
sorting to prevent contamination of the G0/G1 fraction by cycling cells that have effluxed the dye. No differences were noted in the cloning efficiency or engraftment of the sorted fractions with or without Verapamil; so it was not routinely included in this method. Finally, cells were assayed before and after HO staining to verify that these procedures were not toxic to progenitors able to generate colonies of mature myeloid and erythroid cells in
semisolid medium.
Measurement of short-term hematopoietic reconstitution kinetics and
long-term repopulating ability
Just prior to transplantation, B6.SJL mice were exposed to 9 Gy
total body  -irradiation administered in 2 doses of 4.5 Gy approximately 3 hours apart. Ablated animals were injected
intravenously with either 103 freshly sorted
Sca-1+c-kit+Lin BM cells,
the entire product of 103 sorted cells obtained after ex
vivo expansion as described, or 106 expanded BM cells that
had been separated into G0/G1 or
S/G2/M populations by FACS. When AcSDKP was administered,
it was mixed with the cells to be injected immediately prior to
transplantation, and each animal received 10 or 100 µg. After
transplantation, mice were bled from the retro-orbital sinus on days 6, 9, 12, 15, 18, 25, 32, 42, 56, and 120. Until day 25, only half of the mice in each cohort were analyzed alternately 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 blood using a System 9118+
Hematology Series Cell Counter (BioChem ImmunoSystems, Allentown, PA).
At selected times, blood samples were also stained with a donor-specific anti-Ly-5.2+ monoclonal antibody (mAb)
(clone ALI4A2) conjugated with fluorescein isothiocyanate (FITC) and
phycoerythrin-conjugated (PE-conjugated) mAbs specific for B
lymphocytes (anti-CD45R/B220; clone RA3-6B2); 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 and/or progenitor cells were quantitated
using a FACScan instrument (Becton Dickinson). After 120 days, BM cells
were assayed for CFCs and injected into lethally irradiated B6.SJL mice
(0.5 femurs/mouse) for secondary repopulation. Secondary mice were
analyzed 10 weeks later for donor-derived peripheral blood leukocytes
and BM CFCs as described above.
Statistical analysis
The statistical significance of differences between means was
assessed using the 2-tailed Student t test and assuming unequal variances.
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Results |
Delayed hematopoietic reconstitution by enriched
Sca-1+c-kit+Lin stem
cells
In previous studies, we established the efficacy of using large
numbers of ex vivo expanded murine hematopoietic cells as a cellular
therapy to support the normally delayed engraftment observed after
transplantation of highly enriched HSCs.12 Toward optimization of this preclinical model of stem cell expansion and to
further define the parameters that influence the engraftment of
cultured hematopoietic cells, we initiated the present study with minor
modifications to our earlier method. First, we used a different stem
cell sorting procedure to isolate
Sca-1+c-kit+Lin cells
(instead of Thy-1loSca-1+H-2Khi
cells used previously12) that are highly enriched in
competitive repopulating units. Limiting-dilution assays established
that Sca-1+c-kit+Lin cells
contain 1 CRU per 15 cells (95% confidence limits, 1 per 12 to 1 per
23 cells)16 (data not shown). More mature progenitors such
as CFCs (approximately 60 per 103 cells) and CFU-S
(approximately 3 per 103 cells on day 12 and
0 per 103 cells on day 8) are largely
depleted.15
To determine if
Sca-1+c-kit+Lin cells
exhibit delayed hematopoietic reconstitution kinetics, as we have
demonstrated previously with other enriched stem cell
populations,12 103 sorted cells (containing at
least 67 CRU) were transplanted into lethally irradiated Ly-5 congenic
hosts. Circulating blood cells were counted once or twice per week from
6 days to 4 months after transplantation to measure early hematopoietic
reconstitution kinetics. Transplanted mice required approximately 13 days to recover 20% normal levels of leukocytes and platelets (Figure 1). Leukocyte recovery was biphasic,
characterized by a transient fall in counts to a nadir just above 20%
of normal on day 32, but reached normal levels by day 56. Erythrocytes
declined to a nadir just above 50% of normal values on day 15, but
they also recovered slowly thereafter. Notably, normal blood counts
were not achieved for any compartment until after approximately 7 weeks.
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| Fig 1.
Hematopoietic reconstitution kinetics of enriched stem
cells and their ex vivo expanded progeny.
Lethally irradiated B6.SJL mice were injected with 103 Ly-5
congenic Sca-1+c-kit+Lin+ BM cells
(open circles; n = 7 mice from 3 experiments), or their entire
expanded progeny (approximately 2 × 106 cells)
generated after 7 days of culture in IL-6, IL-11, G-CSF, SCF, FLK, and
TPO (closed circles; n = 37 mice from 5 experiments). Shown are the
mean ± SEM (standard error of the mean) number of (A) peripheral
blood leukocytes, (B) erythrocytes, and (C) platelets counted on the
indicated days after transplantation. The ranges of blood counts in
normal B6.SJL mice are defined by the upper shaded areas; the upper
bounds for leukocytes (9.2 × 103/µL) and
erythrocytes (11.4 × 106/µL) are off scale. The
radiation-induced decline of circulating cells in controls that did not
receive a transplant is denoted, until their deaths, by the hatched
lines. The lower shaded areas indicate minimal engraftment thresholds
for leukocytes, 2 × 103/µL (20% of normal);
erythrocytes, 4.7 × 106/µL (50% of normal); and
platelets, 2 × 105/µL (20% of normal). Standard
errors not shown are too small for the scale used.
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Four months after transplantation, virtually all of the circulating B
(98%) and T (77%) lymphocytes and myeloid cells (91%) in these mice were derived from donor stem cells (Table
1). Femoral cellularity and CFC content had
also recovered slightly above normal. When BM from reconstituted
animals was transplanted into secondary lethally irradiated B6.SJL
mice, high levels of engraftment were again achieved in both lymphoid
(approximately 50%) and myeloid (66%) compartments after 10 weeks,
and marrow progenitors were regenerated to 90% of normal numbers.
Thus, Sca-1+c-kit+Lin cells
represent very primitive HSCs with multipotent, albeit delayed,
repopulating ability.
Ex vivo expansion of enriched hematopoietic stem cells generates
progeny with more rapid engraftment kinetics
To promote the generation from enriched stem cells of
hematopoietic cells with potentially more rapid engraftment
kinetics, Sca-1+c-kit+Lin
cells were cultured in StemPro-34 serum-free medium containing a more
extensive cocktail of cytokines (IL-6, IL-11, G-CSF, SCF, FLK, and TPO)
than used in our previous studies.12 This combination was
selected on the basis of preliminary experiments in which 10 different
cytokine combinations, added to 5 different commercially available
serum-free media, were compared for their ability to support a maximal
generation of total nucleated cells and CFCs from enriched HSCs (data
not shown). After 7 days, the entire expanded progeny of
103
Sca-1+c-kit+Lin cells
(approximately 2 × 106 cells containing 91 600 ± 11 600 CFCs) were transplanted into 37 lethally irradiated mice (5 experiments). Compared to freshly isolated HSCs, posttransplant anemia
was now completely eliminated, and the duration of thrombocytopenia was
reduced by approximately 4 days (Figure 1). Leukocyte counts were also
moderately elevated prior to day 12. However, the transient burst in
leukocyte production which peaked on day 18 after transplantation of
fresh stem cells was lost (presumably due to differentiation of myeloid
progenitors in vitro), so the overall time to sustained leukocyte
recovery was extended by approximately 1 week.
Four months after transplantation, 85% of leukocytes were derived from
expanded cells and comprised the majority of B (93%) and T (72%)
lymphocytes and granulocytes and/or monocytes (76%) (Table 1). The
levels of B (P < .05) and myeloid (P < .005)
engraftment were significantly lower than observed with uncultured
Sca-1+c-kit+Lin cells,
indicating that expanded cells were somewhat compromised in long-term
hematopoietic potential. Marrow cellularity recovered to only 70% of
normal, but each femur contained 2.3-fold more CFCs than were present
in either normal animals or recipients of uncultured HSCs. Marrow from
recipients of expanded cells also retained the ability to regenerate
multilineage progeny and CFCs 10 weeks after retransplantation into
secondary hosts, but total leukocyte and myeloid engraftment was again
impaired (P < .007 and P < .02, respectively)
compared with marrow from recipients of fresh
Sca-1+c-kit+Lin cells (Table
1). Thus, expansion of enriched stem cells under these optimized
conditions rendered progeny able to mediate moderately earlier
hematopoietic reconstitution, albeit with some reduction in long-term potential.
Cell cycle activation compromises the long-term, but not short-term,
hematopoietic reconstitution potential of ex vivo expanded progenitors
Although ex vivo expanded hematopoietic cells engrafted moderately
faster than freshly isolated HSCs in these and our earlier studies,
reconstitution was still slower than expected considering their high progenitor content. Several recent studies indicate that
cycling stem cells exhibit diminished long-term repopulating potential.18,19,23 To examine the possibility that
cytokine-induced proliferation of hematopoietic cells may compromise
the short-term repopulating ability and potential clinical utility of
ex vivo expanded cells, we initially measured the number of expanded
CFCs in S phase. In normal BM, 53% ± 1% of clonogenic progenitors
were killed by exposure to HU (5 experiments). In striking contrast, 90% ± 1% of CFCs generated from
Sca-1+c-kit+Lin cells after
7 days of culture in IL-6, IL-11, G-CSF, SCF, FLK, and TPO were killed
by HU (15 experiments). To compare the functional properties of
expanded hematopoietic cells that were either
proliferating or not proliferating at the time of transplantation,
cultured cells were stained with HO dye and separated into
G0/G1 and S/G2/M fractions. In
normal BM, approximately 82% and 16% of all nucleated cells (n = 9)
were in G0/G1 and S/G2/M,
respectively (Figure 2). As expected,
virtually all
Sca-1+c-kit+Lin cells (98%)
isolated from day 1 post-5-FU BM were in
G0/G1. S/G2/M cells increased
significantly during culture and represented almost one-third of all
expanded cells (Figure 2). Note that because HO staining measures the
cycling (DNA content) of all nucleated cells in these cultures, many of
which are nearing terminal differentiation (entering
G0/G1) and only approximately 5% of which are
detectable as CFCs, this measurement is not directly comparable to the
functional assessment of CFC cycling (90%) by HU exposure.
Nevertheless, both types of experiments provide evidence of dramatic
cycle activation of ex vivo expanded hematopoietic cells.
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| Fig 2.
Ex vivo expanded hematopoietic cells are actively
cycling.
The proportion of nucleated cells in G0/G1 or
S/G2/M phases of the cell cycle was determined by HO
staining of (A) normal murine BM, (B)
Sca-1+c-kit+Lin+ cells isolated
from day 1 post-5-FU BM, or (C) these cells' progeny
generated after ex vivo expansion. Numbers in each panel represent the
mean ± SEM of 3-9 measurements per population. The horizontal bars
(C) represent the gates used for sorting expanded cells into
G0/G1 or S/G2/M fractions prior to
transplantation.
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Lethally irradiated Ly-5.1 mice were transplanted with 106
(Ly-5.2+) G0/G1 or
S/G2/M expanded cells, and the rate of short-term
hematopoietic reconstitution was measured by temporal analysis of
circulating blood counts, as described above. Figure
3 shows that these fractions did not differ
significantly from each other with respect to their short-term
engraftment kinetics. However, as demonstrated previously by others in
different culture systems, proliferating and nonproliferating expanded
cells did differ significantly in their contribution to
lympho-hematopoiesis at later times. Four months after transplantation, both populations contributed measurably to B, T, and myeloid cells, but
recipients of S/G2/M cells exhibited significantly lower
engraftment that was particularly pronounced in the myeloid lineage
(Table 2). Similar defects in repopulating
ability were noted in secondary animals transplanted 10 weeks
previously with primary marrow regenerated from S/G2/M
cells. These data demonstrate that the deleterious effects of cycle
activation on hematopoietic cell function after ex vivo expansion are
limited only to their long-term repopulating ability. The short-term
hematopoietic reconstitution potential of cycling expanded cells is
not reduced.
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| Fig 3.
Lack of cell cycle effects on short-term reconstitution
by ex vivo expanded hematopoietic cells.
Lethally irradiated B6.SJL mice were injected with either
106 G0/G1 expanded cells (open
squares; n = 23 mice from 4 experiments); 106
S/G2/M expanded cells (closed squares; n = 10 mice from 4 experiments); or 2 × 106 unfractionated expanded
cells that had been subjected to cell cycle arrest by in vitro exposure
to AcSDKP (open circles; n = 13 mice from 2 experiments). Shown are
the mean ± SEM number of (A) peripheral blood leukocytes, (B)
erythrocytes, and (C) platelets counted on the indicated days after
transplantation. Details of normal counts and thresholds for
engraftment, as denoted by the shaded areas, are described in the
Figure 1 legend.
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Table 2.
Reduced long-term hematopoietic reconstitution potential
of expanded cells that are cycling at transplantation
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AcSDKP accelerates the engraftment of ex vivo expanded hematopoietic
cells through a mechanism unrelated to cell cycle inhibition
To further examine the effects of cell cycling on the short-term
repopulating ability of ex vivo expanded hematopoietic cells, we used
the tetrapeptide AcSDKP to inhibit the cells' proliferation in
culture. AcSDKP was selected for these experiments because of its
ability to block proliferation of murine and human CFCs in
vitro24,25 and its demonstrated ability to enhance the
engraftment of normally limiting numbers of whole mouse BM cells in
irradiated hosts.22
Sca-1+c-kit+Lin cells were
expanded in IL-6, IL-11, G-CSF, SCF, FLK, and TPO, as described above.
Beginning 1 day after culture initiation, AcSDKP was added daily to
duplicate cultures to a final concentration of 1018
to 108 mol/L. Nucleated cells were counted after a
total of 7 days, and the proportion of expanded CFCs in S phase was
measured after exposure to HU. Because each of the 3 experiments
performed yielded slightly different overall expansion, results were
pooled and are expressed as a fraction of control values (Figure
4). Maximal inhibition of CFC cycling was
observed with 1012 mol/L AcSDKP, resulting in a
reduction from 90% in S phase, as observed in control cultures
(without AcSDKP), to approximately 50% (P = .03). Net cell
expansion was also reduced by approximately 40% (P = .009)
compared with controls (Figure 4). As reported previously by
others,25 the effective dose range was narrow, and this
inhibitory effect was lost above or below 1012
mol/L.
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| Fig 4.
AcSDKP inhibits the cycling of hematopoietic progenitors
generated in vitro.
Sca-1+c-kit+Lin cells were
cultured (2 × 103/mL) in serum-free medium
containing IL-6, IL-11, G-CSF, SCF, FLK, and TPO. Except for control
cultures, AcSDKP was added daily to duplicate wells on days 1-6. Shown
are (A) the mean ± SEM number of nucleated cells counted after 7 days
and (B) the proportion of cycling CFCs in each culture assayed by
plating cells in methylcellulose-based medium before or after exposure
to HU, as described in the "Materials and methods." The reduction
in total nucleated cell production and CFC cycling in cultures
containing 1012 mol/L AcSDKP was statistically
significant. (*indicates P < .01, and **indicates
P < .03.) The data given are pooled from 3 experiments.
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To determine whether expanded cells that had been subjected to cycle
inhibition exhibited any differences in their early hematopoietic reconstitution kinetics, 2 × 106
cells (ie, the average number of cells generated from 103
Sca-1+c-kit+Lin cells in
cultures without AcSDKP) were transplanted into individual lethally
irradiated mice. The rate of leukocyte recovery in 13 mice (2 experiments) was essentially identical to that of mice transplanted
with an equal number of hematopoietic cells expanded without AcSDKP
(compare Figures 1A and 3A). Thus, cell cycle inhibition was certainly
of no benefit and, in fact, appeared to be moderately deleterious to
erythrocyte and platelet recovery (compare Figure 3B with Figure 1B,
and Figure 3C with Figure 1C). These results were not unexpected given
the lack of effect of cell cycle status on short-term engraftment, as
determined from the HO sorting experiments above.
Suzuki et al22 demonstrated an improved rate of survival
and recovery of circulating blood cells in mice injected simultaneously with normal BM cells and AcSDKP. This in vivo treatment was thus evaluated in a final attempt to hasten reconstitution by purified HSCs
or their ex vivo expanded progeny. In 10 myeloablated animals injected
with 103 sorted cells together with 10 µg AcSDKP (3 experiments), we did not observe any improvement in leukocyte,
erythrocyte, or platelet regeneration kinetics; the results, identical
to Sca-1+c-kit+Lin cells in
Figure 1, are not shown. An additional 20 mice (2 experiments) each
received the entire expanded progeny of 103
Sca-1+c-kit+Lin cells
(approximately 2 × 106 cells) and either 10 or 100 µg AcSDKP. The recovery of circulating blood cells was monitored as
above and is compared in Figure 5 to the
rate of hematopoietic reconstitution observed with fresh HSCs or
expanded cells injected without the peptide. The 10 and 100 µg AcSDKP
groups gave identical results and were thus pooled. Engraftment was
clearly enhanced in mice that were treated with AcSDKP. Leukocyte and
erythrocyte counts were significantly higher (P < .005) in
treated versus untreated recipients of expanded cells on every day they
were measured, from 9 days to 4 months after transplantation.
As a result, white blood cells recovered permanently to
20% of normal counts within approximately 13 days. This recovery was
approximately 9 days faster than in mice receiving a transplant of
expanded cells without AcSDKP and contemporaneously with uncultured
stem cells, but with the added benefit of elevated and sustained counts
prior to reaching this threshold (Figure 5A). Indeed, when last
analyzed on day 120, leukocyte counts in mice injected with expanded
cells and AcSDKP were almost 2-fold higher than leukocyte counts in
mice that received a transplant of expanded cells alone. Platelets also
never declined below 20% of normal at any time after transplantation
in mice injected with ex vivo expanded cells and AcSDKP. The
improvement over untreated controls was significant on every time
between day 9 and day 42 (P < .005) (Figure 5C).
View larger version (34K):
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| Fig 5.
In vivo treatment of transplant recipients with AcSDKP
promotes the engraftment of ex vivo expanded hematopoietic cells.
Lethally irradiated mice were injected with the expanded progeny of
Sca-1+c-kit+Lin BM cells
administered simultaneously with a single dose of 10 or 100 µg/mouse
of AcSDKP. Recovery kinetics of (A) peripheral blood leukocytes, (B)
erythrocytes, and (C) platelets were measured as described in Figure 1
and are depicted by the closed circles (mean ± SEM values for 20 mice from 2 experiments). Hematopoietic reconstitution by freshly
isolated Sca-1+c-kit+Lin cells
(hatched lines) or their expanded progeny transplanted without AcSDKP
(open circles) is duplicated from Figure 1 for comparison (note scale
change in panel A). Details of normal counts and thresholds for
engraftment, as denoted by the shaded areas, are described in the
Figure 1 legend.
|
|
AcSDKP also improved the ultimate level of engraftment by ex vivo
expanded hematopoietic cells (Table 1). Four months after transplantation, these animals exhibited significantly higher levels of
Ly-5.2+ leukocytes in peripheral blood than recipients of
cultured cells not treated with the peptide (93% vs 85%;
P < .03). Compared to the latter, a greater
proportion of circulating B lymphocytes (98% vs 93%;
P < .03) and T lymphocytes (81% vs 72%;
P < .04) were also derived from cultured cells, but the
slight improvement in myeloid engraftment (87% vs 76%) did not quite
reach statistical significance (P = .06). As in recipients of
expanded cells alone, BM CFCs recovered to higher levels in animals
transplanted with expanded cells plus AcSDKP than in animals
transplanted with uncultured HSCs (Table 1). Stem cells with secondary
transplantation potential were also regenerated in the BM of these
mice. Although the level of secondary engraftment in all lineages was
slightly reduced compared with BM from recipients of expanded cells
alone, this difference was not statistically significant
(P = .09 to .24). Overall, these data support the feasibility
of enhancing the clinical utility of cultured hematopoietic cells using
AcSDKP. The mechanism of this action is yet to be defined, but it
appears unrelated to the activity of AcSDKP as a cell cycle inhibitor.
| |
Discussion |
The ability of many cytokines to stimulate the
proliferation and differentiation of primitive HSCs into progenitors of
multiple lineages has been exploited in various in vitro systems to
generate grafts of tailored composition, with the hope that such cells will support rapid hematopoietic rescue when transplanted into myeloablated patients. Unfortunately, despite significant advances in
identifying culture conditions that support impressive amplification of
progenitor cell numbers and, to a lesser degree, stem
cells,26-29 the comparative lack of efficacy of ex vivo
expanded human hematopoietic cells in numerous clinical trials has been
disappointing.5,7,10 Nevertheless, the success of such cell
therapies in mice provides reason for optimism that this strategy is
sound11-13 and validates a preclinical model for detailed
study of the effect of in vitro manipulation on transplantable
hematopoietic cells.
Using such animal models, several recent reports have focused attention
on the role of the cell cycle in regulating engraftment potential.18,19 Notably, analysis of the long-term
repopulating ability of murine BM cells cultured in IL-3, IL-6, IL-11,
and SCF have revealed marked cyclic fluctuations in stem cell activity over 24-48 hours, with nadirs in engraftment potential occurring in
late S and early G2 phases of the cell cycle.19
Taken together with the empirical finding that the most rapid
hematopoietic reconstitution observed clinically is rendered by
cytokine-mobilized peripheral blood cells1 in which
stem/progenitor cells are largely quiescent,30 these data
suggest a basis for experimental manipulation of engraftment kinetics
by inducing cell cycle arrest in hematopoietic cells prior to their infusion.
In the present study we examined the hypothesis that cycle activation
of short-term repopulating cells generated in vitro might compromise
their ability to rapidly alleviate posttransplant cytopenia in a manner
analogous to the loss of long-term function observed with stem cell
proliferation. Two approaches were used to compare the short-term
hematopoietic reconstitution potential of actively cycling versus
quiescent expanded cells. In the first, the progeny of highly enriched
Sca-1+c-kit+Lin stem cells
generated after 1 week of serum-free culture in IL-6, IL-11, G-CSF,
SCF, FLK, and TPO were separated into G0/G1 and S/G2/M fractions by FACS. During the first several weeks
after transplantation of equal numbers of cells from these populations, circulating blood cells regenerated at a similar rate. In contrast and
as confirmation of the efficiency of cell separation in these experiments, expanded cells residing in G0/G1
at the time of transplantation gave rise to significantly higher levels
of donor lymphoid and myeloid cells after a cumulative 6.5 months in
primary and secondary recipients. This latter finding is consistent
with numerous published studies indicating a deleterious effect of
cycle activation on long-term stem cell function.18,19,23
Next we inhibited the cycling of progenitor cells during ex vivo
expansion by simultaneous exposure to the tetrapeptide AcSDKP. Although
we were able to achieve an approximately 50% reduction in the number
of CFCs in S phase, expanded progenitors that had been subjected to
cycle inhibition did not exhibit any improvement in short-term
reconstitution kinetics. Wiesmann et al31
obtained similar results with long-term repopulating
RhloThy-1.1loSca-1+c-kit+Lin
BM cells cultured for 48 hours in SCF, IL-6, and FLK and then subjected
to cycle arrest by exposure to SCF and transforming growth factor- 1
(TGF-1) for an additional 24 hours. The percent of cells in
S/G2/M decreased 2-fold after TGF-1 exposure (cycling was not measured functionally), but no significant effects on peripheral white blood cell recovery were observed between 2 and 12 weeks after transplantation. Taken together with our present findings,
these data suggest that, in contrast to more primitive stem cells that
maintain long-term hematopoiesis in vivo, cell cycle activation does
not compromise the functional activity of hematopoietic cells necessary
for early reconstitution. Despite the correlation between quiescence
and rapid reconstitution potential as exemplified by mobilized
peripheral blood progenitors,32 cell cycle down-modulation
thus does not appear to be an effective strategy to hasten engraftment
by cultured hematopoietic cells.
These results are somewhat surprising given the cell cycle-dependent
expression of several integrins that direct the homing of stem and
progenitor cells to the bone marrow.33-35 Efficient homing
of intravenously transplanted cells presumably represents the first
step in their timely engraftment. We have recently shown that CFCs
generated in vitro under the conditions reported here exhibit a 10-fold
reduction in their ability to home to the BM and spleen of irradiated
mice compared with their freshly isolated counterparts.15
This defect correlates with a 10-fold decrease in 1 integrin
expression on ex vivo expanded cells that retain the
Sca-1+c-kit+ phenotype (S.J.S, unpublished
data, February 1999). Presumably, reduced levels of 1 would result
in a decreased expression of all 6 adhesion molecules that use this
common chain. One of these molecules,  41 (or very late activation
antigen-4; VLA-4), is known to play a critical role in
stem cell homing.36-38 VLA-4 expression has been observed
to decrease during S phase.35 As a result, one might expect
that cycling expanded cells should home less efficiently and engraft
more slowly than quiescent cells expressing higher levels of this
integrin. This was not observed, which suggests that down-regulated
VLA-4 is partly compensated for by other integrins that may also be
down-regulated during expansion, but whose expression is not cell cycle
dependent. Potential candidates include CD43, CD44, and L-selectin, for
which a correlation between rapid hematopoietic reconstitution
potential and long-term marrow repopulating ability has previously been
noted.39,40 Our current studies are directed toward
addressing this issue in more detail.
Clearly the most interesting finding of this study was the ability of a
single dose of AcSDKP to enhance early reconstitution when administered
in vivo simultaneously with hematopoietic cells that had been expanded
without the inhibitor. The mechanism of this action is not currently
known but appears from our present data to be unrelated to AcSDKP's
activity as a stem cell cycle inhibitor.20 More likely,
AcSDKP improves the efficiency with which expanded cells home to bone
marrow. AcSDKP has been reported to increase (1) the adherence of CFU-S
to a cloned stromal cell line41 and (2) the number of
CFU-GM and day 12 CFU-S that can be recovered from the BM
of mice transplanted only 1 day earlier with whole normal marrow
cells.22 To our knowledge, the potential effects of AcSDKP
on the expression by mesenchymal cells of integrin receptors which may
mediate these cellular associations have not been investigated. AcSDKP
does not induce the secretion of cytokines, such as tumor necrosis
factor- (TNF-), IL-1, IL-3, IL-6, or G-CSF, when added daily for
several weeks to human long-term BM cultures (LTBMCs).42
Nevertheless, prevailing evidence suggests that AcSDKP exerts its
effects on hematopoietic progenitors indirectly through its action on
stromal cells. Cashman et al24 reported that adherent cells
were required for the inhibitory effects of AcSDKP on colony formation
by normal marrow progenitors. AcSDKP-mediated inhibition of CFC cycling
in LTBMCs was also blocked by the addition of macrophage inflammatory
protein-1 (MIP-1), an antagonist of stroma-derived MIP-1
which inhibits cycling of CFCs in the adherent layers of such
cultures.24,43 Our present finding that AcSDKP in vivo does
not hasten engraftment by highly enriched Sca-1+c-kit+Lin stem cells
also suggests that mature cells, which are depleted by stem cell
sorting but then regenerated during ex vivo culture, may play an
indirect role in the AcSDKP-mediated engraftment of expanded cells. Of
course, the extended time required for small numbers of purified stem
cells to generate detectable numbers of differentiated progeny in vivo
may also be an intrinsic characteristic of their peak position in the
hematopoietic cell hierarchy. Engraftment of these very primitive cells
may thus not be amenable to further acceleration by AcSDKP,
irrespective of the presence of accessory cells.
In summary, our studies indicate that cell cycle activation of
hematopoietic cells during ex vivo expansion does not deleteriously affect their ability to mediate rapid engraftment in vivo, as demonstrated here and elsewhere for more primitive long-term
repopulating stem cells. Engraftment kinetics are nevertheless amenable
to manipulation by relatively simple means, exemplified here using the
tetrapeptide AcSDKP. It is hoped that continued systematic analysis of
the parameters influencing early engraftment will culminate in the
successful clinical application of expanded human hematopoietic cells.
| |
Acknowledgments |
The authors thank Mr Michael Bass for technical assistance; Dr Pieter
Wierenga, University of Groningen, Groningen, The
Netherlands, for advice on in vitro studies of AcSDKP; Drs Edward Srour
and Christie Traycoff, Indiana University, IN, for advice on Hoechst staining; Dr Sylviane Moreau, Biomeasure, Milford, MA, for generously providing AcSDKP in the later stages of this study; and Drs Gary Van
Zant and Craig Jordan, the Blood and Marrow Transplant Program, University of Kentucky, Lexington, KY, for many stimulating
discussions and for critically reviewing the manuscript.
| |
Footnotes |
Submitted November 5, 1999; accepted January 6, 2000.
Supported by the Department of Internal Medicine and the
University of Kentucky Hospital, University of Kentucky Medical Center, Lexington, KY, and by grant R01-HL61392 to S.J.S. from the National Institutes of Health, Bethesda, MD. S.J.S. is supported by a Junior Faculty Scholar Award from The American Society of Hematology, Washington, D.C.
Reprints: Stephen J. Szilvassy, Hematology/Oncology-BMT,
Lucille P. Markey Cancer Center, Room CC414, 800 Rose Street, Lexington, KY 40536-0093; e-mail: szilvas{at}pop.uky.edu.
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.
| |
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October 1, 2001;
98(7):
2108 - 2115.
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Abstract
Introduction