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Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2641-2649
RAPID COMMUNICATION
Cell Cycle-Related Changes in Repopulating Capacity of Human Mobilized
Peripheral Blood CD34+ Cells in Non-Obese Diabetic/Severe
Combined Immune-Deficient Mice
By
André Gothot,
Johannes C.M. van der Loo,
D. Wade Clapp, and
Edward F. Srour
From the Division of Hematology/Oncology and Indiana Elks Cancer
Research Center, Department of Medicine, and Department of Pediatrics,
Herman B Wells Center for Pediatric Research, Indiana School of
Medicine, Indianapolis, IN.
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ABSTRACT |
Most primitive hematopoietic progenitor cells reside in vivo within
the G0/G1 phase of the cell cycle. By
simultaneous DNA/RNA staining it is possible to distinguish
G0 and G1 states and to isolate cells in
defined phases of the cell cycle. We report here the use of cell cycle
fractionation to separate human mobilized peripheral blood (MPB)
CD34+ cells capable of repopulating the bone marrow (BM)
of non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice.
In freshly isolated MPB, repopulating cells were predominant within the
G0 phase, because transplantation of CD34+
cells residing in G0 (G0CD34+)
resulted on average in a 16.6- ± 3.2-fold higher BM chimerism than
infusion of equal numbers of CD34+ cells isolated in
G1. We then investigated the effect of ex vivo cell cycle
progression, in the absence of cell division, on engraftment capacity.
Freshly isolated G0CD34+ cells were activated
by interleukin-3 (IL-3), stem cell factor (SCF), and flt3-ligand (FL)
for a 36-hour incubation period during which a fraction of cells
progressed from G0 into G1 but did not complete
a cell cycle. The repopulating capacity of stimulated cells was
markedly diminished compared with that of unmanipulated G0CD34+ cells. Cells that remained in
G0 during the 36-hour incubation period and those that
traversed into G1 were sorted and assayed separately in
NOD/SCID recipients. The repopulating ability of cells remaining in
G0 was insignificantly reduced compared with that of
unstimulated G0CD34+ cells. On the contrary,
CD34+ cells traversing from G0 into
G1 were largely depleted of repopulating capacity. Similar
results were obtained when G0CD34+ cells were
activated by the combination of thrombopoietin-SCF-FL. These studies
provide direct evidence of the quiescent nature of cells capable of
repopulating the BM of NOD/SCID mice. Furthermore, these data also
demonstrate that G0-G1 progression in vitro is associated with a decrease in engraftment capacity.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
IN VITRO MANIPULATION of hematopoietic
stem cells (HSC) has been the focus of numerous studies. Two main
objectives have been under evaluation: HSC expansion and somatic gene
therapy. The use of expanded grafts would in theory speed up the
hematological recovery after HSC transplantation and/or allow
for the use of small HSC collections, such as cord blood
samples.1 Information collected from ex vivo expansion
studies is also critical to design gene transfer protocols, because
retroviral transduction requires in vitro cycling of the target
cells.2,3
In defining strategies for HSC expansion or gene therapy, a decisive
factor to consider is the responsiveness of the most primitive cells to
cytokine stimulation in vitro. This is largely dependent on the cell
cycle status of the target cells, as cycling cells will be able to
respond quickly to mitogenic factors, whereas more quiescent cells will
not.4-7 In addition, cell cycle activation may have a
negative effect on the engraftment capability. Studies in murine models
have documented an engraftment defect of stimulated HSC: in vitro HSC
activation by various combinations of hematopoietic growth factors
resulted in a decrease of in vivo repopulating ability, compared with
unmanipulated marrow.8-10 Consistent with these results
were data acquired in our laboratory by PKH membrane cell
tracking11 that demonstrated the superiority of quiescent cells over proliferating cells in rescuing lethally irradiated recipients. In another report, evidence was presented that not only in
vitro stimulation, but also in vivo HSC cycling, as observed after
5-fluorouracil (5-FU) treatment, resulted in transient alterations in the functional state of HSC, leading to an engraftment
defect.12
In humans, experimental studies examining the impact of cell cycle
status on engraftment capacity have been unattainable due to the
absence of an in vivo model of human HSC. Xenotransplant models have
been recently developed which can address this question, most notably
the in vivo severe combined immune-deficient (SCID) mouse repopulating
cell (SRC) assay.13 In this model, human HSC intravenously
injected into immunodeficient recipients home to the bone marrow and
are able to proliferate to a large extent and differentiate into
various lineages.14,15 High levels of chimerism are
especially achieved with the non-obese diabetic/SCID (NOD/SCID) strain,
which was used in the present study.
To investigate the relationship of cell cycle status with the
engraftment capability of human hematopoietic cells, we took advantage
of a new method of cell cycle analysis and fractionation recently
developed in our laboratory.16-18 Conventional cell cycle analysis using DNA dyes classifies cells in the
G0/G1 phase as noncycling cells and cells in
the S/G2+M phase as cycling cells. This approach is of
limited utility when applied to the characterization of the cell cycle
status of human hematopoietic cells, because more than 85% of bone
marrow (BM) CD34+ cells and almost all of mobilized
peripheral blood (MPB) CD34+ cells are in the
G0/G1 phase. Simultaneous DNA/RNA staining with Hoechst 33342 (Hst) and Pyronin Y (PY) allows for further fractionation of the G0/G1 phase. Using this procedure, we
have been able to isolate viable CD34+ cells in the
G0 or G1 phase of the cell cycle and examine
their properties in vitro.16
G0CD34+ cells respond slowly to cell cycle
activation in cytokine-driven liquid culture, whereas
G1CD34+ cells proliferate immediately when
exposed to the same conditions.18 In addition, we have
shown by in vitro assays that the functional heterogeneity of human
CD34+ cells is correlated with their position within the
G0/G1 phase, with the G0
compartment being enriched in primitive hematopoietic cells.17 In the present study, we have used Hst/PY staining to analyze the NOD/SCID repopulating ability of MPB human
CD34+ cells isolated in G0 or G1.
Two main questions were addressed. First, by determining the
repopulating activity of freshly isolated CD34+ cells in
G0 or in G1, we assessed the cell cycle status
of putative stem cells. Second, the effect of cell cycle activation on
engraftment capability was investigated by transplanting animals with
CD34+ cells initially isolated in G0 and then
stimulated to progress into G1 by in vitro exposure to
cytokines.
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MATERIALS AND METHODS |
Mice.
NOD/LtSz-scid/scid (NOD/SCID)19 animals were kindly
provided by Dr Leonard Shultz (Jackson Laboratory, Bar Harbor, ME). Mice were housed in microisolators under pathogen-free conditions and
received autoclaved food and acidified water ad libitum. Animal experiments were performed in accordance with institutional guidelines approved by the Animal Care Committee of the Indiana University School
of Medicine.
Human cells.
BM and MPB samples were obtained from healthy adult volunteers
according to the guidelines established by the Human Investigation Committee of the Indiana University School of Medicine. Mobilization was achieved by daily granulocyte colony-stimulating factor (G-CSF) administration at 5 µg/kg (maximum, 480 µg/d) for 4 consecutive days. MPB cells were collected by apheresis on day 5. MPB
CD34+ cells were isolated by immunomagnetic selection using
a large scale Isolex 300i system (Baxter Healthcare, Irvine, CA) as per the manufacturer's instructions. CD34+ cell purity in the
selected product always exceeded 95%. In one experiment, BM cells
instead of MPB cells were used. BM cells were pooled from 5 normal
donors and selected with the same procedure. CD34+ cell
purity of this BM collection was 86%.
Cell cycle fractionation with Hst and Pyronin Y.
Fresh or cultured CD34+ cells were resuspended at 5 × 106 cells/mL in a 1 µg/mL solution of Hst (Molecular
Probes, Eugene, OR) in Hst buffer. Hst buffer consisted of Hanks
Balanced Salt Solution (HBSS; Biowhittaker, Walkersville, MD), 20 mmol/L HEPES (Biowhittaker), 1 g/L glucose, and 10% fetal calf serum
(FCS; Hyclone, Logan, UT). After incubation at 37°C for 45 minutes,
PY (Sigma, St Louis, MO), prepared in Hst Buffer, was added at a final
concentration of 1 µg/mL and cells were further incubated for another
45 minutes at 37°C. Cells were washed once, resuspended in Hst
buffer, and sorted on a FACStar Plus equipped with an argon laser
providing the 488 nm excitation for PY and a krypton laser providing
the 350 nm excitation for Hst. PY signal was selected with a 575 ± 13 nm bandpass filter and Hst was detected with a 424 ± 22 nm bandpass filter. Sorting windows were constructed as depicted in
Fig 1. Cells were kept on ice during
sorting to minimize dye leaking and were protected from light.
Viability of sorted cells always exceeded 98%. No toxic effect of
Hst/PY staining on cell proliferation, colony formation, long-term
culture-initiating cell (LTC-IC) frequency and NOD/SCID
repopulating activity was detected (data not shown).
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| Fig 1.
Single- and dual-parameter cell cycle analysis of BM and
MPB CD34+ cells. DNA histogram of BM (A) and MPB (B)
CD34+ cells, respectively, showing the relative absence
of replicating progenitor cells in MPB. Simultaneous DNA/RNA staining
with Hst and PY, identifying G0 and G1 regions
in similar samples of BM (C) and MPB (D) CD34+ cells.
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High resolution cell cycle analysis.
Cells were analyzed for Ki-67 expression and DNA content as recently
described by Jordan et al,20 with minor modification. Cells
were washed and resuspended in 1 mL phosphate-buffered saline (PBS) + 0.4% formaldehyde. After 30 minutes at 4°C, 1 mL of PBS + 0.2%
Triton X-100 was added and cells were left overnight at 4°C. Cells
were then washed twice in PBS + 1% bovine serum albumin (BSA) and
stained with fluorescein isothiocyanate (FITC)-conjugated anti-Ki-67
(clone MIB-1; Immunotech, Westbrook, ME) for 60 minutes at 4°C.
Isotype controls were stained in parallel. Finally, cells were washed
and resuspended in PBS + 1% BSA containing 5 µg/mL 7-aminoactinomycin-D (7-AAD; Sigma). After 3 hours of incubation on
ice, samples were run on a FACScan flow cytometer (BDIS, San Jose,
CA) using FL-1 and FL-3 channels for Ki-67 and 7-AAD,
respectively.
Human progenitor cell assay.
Human CD34+ cells were assayed in 1.3% methylcellulose,
30% FCS, 100 ng/mL stem cell factor (SCF), 10 ng/mL interleukin-3
(IL-3), 10 ng/mL IL-6, 5 ng/mL granulocyte-macrophage
colony-stimulating factor (GM-CSF), and 2 U/mL erythropoietin (EPO)
suspended in Iscove's modified Dulbecco's medium (IMDM).
When unseparated BM cells from NOD/SCID recipient mice were used, IL-6
was omitted to avoid the growth of murine progenitors.14,21
The selectivity of the assay for human progenitors was confirmed by
polymerase chain reaction (PCR) detection of human  -globin gene in
individual colonies (see below). Hematopoietic colonies were scored
after 2 weeks according to standard criteria.
LTC-IC assay by limiting dilution analysis.
M2-10B4 cells,22 obtained from the American Type Culture
Collection (Rockville, MD), were used to establish stromal layers. M2-10B4 cells harvested from large-scale cultures in RPMI 1640 with
10% FCS were irradiated at 8,000 cGy and plated in 96-well plates at
15,000 cells/well in 100 µL long-term culture medium (LTCM). LTCM
consisted of Myelocult (Stem Cell Technologies, Vancouver, British
Columbia, Canada) containing 10 6 mmol/L
hydrocortisone (Sigma). Within 1 week, human test cells were plated in
limiting dilution at 24 wells/cell dose in another 100 µL LTCM and
maintained at 37°C in 100% humidified atmosphere containing 5%
CO2, with weekly half-medium change. After 5 to 6 weeks,
120 µL medium were removed from each well followed by the addition of
150 µL of a mixture consisting of 3 parts FCS and 4 parts 3.3%
methylcellulose and containing at final concentration 5 × 102 mmol/L 2-mercaptoethanol, 100 ng/mL SCF, 25 ng/mL IL-3, 25 ng/mL IL-6, 25 ng/mL GM-CSF, and 2 U/mL EPO. After an
additional 2 weeks, wells were scored for the presence or absence of
hematopoietic colonies and the frequency of LTC-IC was calculated using
the maximum likelihood estimator.23
Short-term culture.
CD34+ cells initially isolated in G0 were
plated in a serum-free medium consisting of IMDM supplemented with 25 mmol/L HEPES (Biowhittaker), 10 µg/mL BSA, 10 µg/mL bovine insulin,
200 µg/mL transferrin (all from Stem Cell Technologies), 2 mmol/L
alanyl-glutamine, 1% (vol/vol) lipids cholesterol-rich, 1 mmol/L
sodium pyruvate (all from Sigma), 100 U/mL penicillin, 100 µg/mL
streptomycin, and 5 × 102 mmol/L
2-mercaptoethanol. Cells were stimulated during 36 hours by a
combination of 50 ng/mL IL-3, 100 ng/mL SCF, and 100 ng/mL flt-3 ligand
(FL) or a combination of 50 ng/mL thrombopoietin (TPO), 100 ng/mL SCF,
and 100 ng/mL FL. Viability of cultured cells was always greater than
98% as judged by trypan blue exclusion.
Transplantation of human cells into NOD/SCID mice.
Twelve- to 14-week-old NOD/SCID mice were sublethally irradiated with
300 cGy from a 137Cs source (GammaCell 40; Nordion
International, Kanata, Ontario, Canada). Mice
received 10 × 106 nonadherent CD34
cells irradiated with 2,000 cGy, as accessory cells, by intravenous tail injection. Two hours later, mice were transplanted with fresh or
cultured CD34+ cells. Animals were not treated with
cytokines during the experiments. After 6 to 7 weeks,14,24
mice were killed by cervical dislocation and bone marrow cells were
harvested from femurs and tibias by flushing the bones with HBSS-5%
FCS. Nucleated cells were isolated by centrifugation over
Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ), washed, and
resuspended in IMDM with 10% FCS for further analyses.
Flow cytometric analysis of engraftment.
Between 1.0 × 105 and 2.5 × 105
NOD/SCID BM cells were pelletted, resuspended in 50 µL mouse serum
(Sigma), and incubated with various mouse antihuman monoclonals for 20 minutes at 4°C. FITC-conjugated antibodies included anti-CD45,
CD33, CD19, CD15 (BDIS), and CD38 (Immunotech). Phycoerythrin
(PE)-conjugated anti-CD34 was obtained from BDIS and anti-CD45-PE was
obtained from Pharmingen (San Diego, CA). Samples were analyzed on a
FACScan (BDIS). Positive cells were identified by comparison with
isotypic controls and with cells harvested from control (not
transplanted) NOD/SCID mice stained with the same antibodies.
In the case of highly engrafted mice (>10% CD45+ cells),
CD45+CD34+ cells were sorted on a FACStar Plus
(BDIS) and assayed for their progenitor and LTC-IC frequencies.
PCR detection of human -globin gene.
Briefly, individual colonies were plucked and transferred into
Eppendorf tubes, lyzed with 500 µL water, and pelletted. DNA was
extracted with InstaGene matrix (Bio-Rad, Hercules, CA) as per the
manufacturer's instructions. Positive controls consisted of individual
colonies grown from purified human CD34+ cells and negative
controls were samples of 103 and 10 × 103 murine BM cells harvested from a control animal. PCR
was performed using a 20-bp 5 primer GAA TCC AGA TGC TCA
AGG CC and a 20-bp 3 primer CAA TCC AGC TAC CAT TCT GC
amplifying a 345-bp fragment of human -globin gene. After an
initial denaturation step of 5 minutes at 94°C, a hot start at
85°C was used, followed by 40 cycles consisting of 4 minutes at
94°C, 2 minutes at 56°C, and 1.5 minutes at 72°C. The final
cycle had a 10-minute extension step. PCR products were analyzed on a
1.5% agarose gel after staining with ethidium bromide.
Growth factors.
Human recombinant IL-3, IL-6, SCF, TPO, and GM-CSF were kind gifts from
Amgen (Thousand Oaks, CA). Human recombinant EPO was obtained from
Amgen. Human recombinant FL was a kind gift from Immunex (Seattle, WA).
Statistical analysis.
Comparisons were made using the Student's t-test. All
P values are two-sided.
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RESULTS |
Cell cycle fractionation of MPB CD34+ cells by
staining with Hst and PY.
We17,25 and others26,27 have already reported
the homogeneous G0/G1 cell cycle status of MPB
CD34+ cells compared with their BM counterparts. When
analyzed with a standard DNA histogram, up to 15% of BM
CD34+ cells are in the S/G2+M phase, whereas
less than 1% of MPB CD34+ cells are in the same actively
cycling compartment (Fig 1A and B). Thus, the vast majority of MPB
CD34+ cells cannot be ascribed to a precisely defined
position in the cell cycle, because cells in G0 and in
G1 have the same DNA content. Simultaneous DNA/RNA staining
with Hst and PY, respectively, was used in the present study to
discriminate the G0 phase from the G1 phase of
the cell cycle (Fig 1C and D). Cells in G0 were identified by minimal RNA content, whereas cells traversing into G1
were defined as cells with maximal RNA staining. The validity of this approach has been demonstrated in previous reports by kinetic and
phenotypic data from this laboratory.16-18 Most notably,
expression of Ki-67, a nuclear antigen specifically expressed by
cycling cells,20 was restricted to PYhigh
cells, whereas PYlow cells were Ki-67 negative.
Differential cell cycle status of colony-forming cells
(CFC) and LTC-IC in MPB CD34+
cells.
The frequency of late and primitive progenitor cells was determined in
CD34+ cells isolated in G0 or in G1
by standard CFC and LTC-IC assays, respectively. The overall clonogenic
activity was very similar, approximately 30%, in G0 and
G1CD34+ cells (P > .05;
Fig 2A). However, as already reported for
BM CD34+ cells,17 consistent differences were
observed in the distribution of the different types of progenitors: the
G0 compartment was relatively enriched in multipotential
progenitors (colony-forming unit-mixed [CFU-Mix]; 15%
in G0 v 4% in G1; P < .05),
whereas there were more committed myeloid progenitors in G1
(colony-forming unit-granulocyte-macrophage [CFU-GM];
16% in G0 v 28% in G1; P < .05).
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| Fig 2.
In vitro functional assays of MPB CD34+
cells isolated in G0 or in G1 phase of the cell
cycle. (A) Progenitor cell assay. Numbers of hematopoietic colonies are
given per 100 cells plated. Data shown are the mean ± SEM of four
experiments performed in duplicate. Total indicates total number of
hematopoietic colonies; BFU-E, burst-forming unit-erythroid; CFU-GM,
colony-forming unit-granulocyte/macrophage; CFU-MIX, CFU-Mixed. (B)
Frequencies of LTC-IC among G0 and
G1CD34+ cells. Frequencies per 100 cells are
reported as the mean ± SEM, n = 4. *P < .05 versus
G1CD34+ cells, paired t-tests.
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As for more primitive cells (Fig 2B), the G0 compartment
was significantly enriched in LTC-ICs compared with the G1
compartment (3.1% for G0CD34+ cells v
0.8% for G1CD34+ cells; P < .05).
NOD/SCID repopulating ability of MPB CD34+
cells isolated in G0 or in G1.
Comparison of the repopulating ability of
G0CD34+ and G1CD34+
cell populations was assessed by transplanting equal numbers of either
cell fraction sorted from single-donor MPB samples. After 6 to 7 weeks,14,24 the extent of human cell engraftment was evaluated by flow cytometric determination of human CD45+
and/or CD34+ cells in suspensions of BM cells
harvested from recipient mice. High numbers of human CD45+
cells (up to 75% after injection of 2 × 106 cells)
were detected in the BM of animals transplanted with
G0CD34+ cells. Of interest is the fact that a
large fraction of chimeric human CD45+ cells was also
CD34+ (Fig 3B). In sharp
contrast, low numbers of CD45+ cells were demonstrated
after transplantation of G1CD34+ cells and
chimeric CD34+ cells were mostly undetectable (Fig 3D). In
four separate experiments in which MPB CD34+ cells sorted
in either G0 or G1 were transplanted at cell
doses ranging from 2 × 106 to 0.7 × 106, the repopulating activity of
G0CD34+ cells was consistently severalfold
higher than that of G1CD34+ cells. The
percentage of human CD45+ cells in mice receiving
G0CD34+ cells was on average 16-fold higher
(range, 12.3- to 19.1-fold; P < .05, paired
t-test) than in mice injected with
G1CD34+ cells (Fig
4A). In one additional experiment in which fractions of BM
CD34+ cells instead of MPB CD34+ cells were
used as grafts, similar results were obtained: at a cell dose of 0.8 × 106 cells transplanted, the average percentage of
chimeric human CD45+ cells was 3.6% in mice transplanted
with G0CD34+ cells (n = 2 mice) and only 0.2%
in animals receiving G1CD34+ cells (n = 3 mice).
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| Fig 3.
Comparison of FACScan analysis of BM cells harvested from
NOD/SCID mice transplanted 6 weeks previously with 1.0 × 106 MPB CD34+ cells isolated in
G0 or in G1 phase of the cell cycle. Cells were
stained either with isotypic control antibodies (upper panels) or with
antihuman CD45-FITC and antihuman CD34-PE (lower panels). (A and B)
Mouse transplanted with G0CD34+ cells; (C and
D) mouse transplanted with G1CD34+ cells.
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| Fig 4.
Comparison of engraftment capacity of MPB
CD34+ cells isolated in G0 or in
G1 phase of the cell cycle. Data from four experiments are
reported as the mean ± SEM. Each experiment was performed with MPB
CD34+ cells isolated from a different donor (n = 2 to 4 animals per experiment). (A) Percentages of chimeric human
CD45+ cells in the BM of mice transplanted 6 weeks
previously with indicated numbers of G0 or
G1CD34+ cells. Average chimerism was
statistically higher after transplantation of
G0CD34+ cells than after transplantation of
G1CD34+ cells (P < .05, paired
t-test). (B) Numbers of human progenitor cells per 50 × 103 BM cells 6 weeks posttransplantation in the same
recipient mice. Average human progenitor output was statistically
higher in G0- versus G1-transplanted mice
(P < .05, paired t-test).
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The presence of human clonogenic cells in BM low-density cells
harvested from recipient mice was also determined by a progenitor cell
assay selective for human cells.14,21 The numbers of
engrafted human progenitors were in good correlation with those of
CD45+ cells (Fig 4B), indicating that functional human
hematopoietic cells were present in chimeric mice. Human progenitors in
the marrow were on average 22-fold more numerous in mice transplanted with G0CD34+ cells than in those receiving
G1CD34+ cells (P < .05, paired
t-test). The selectivity of the assay for human clonogenic
cells was confirmed by PCR amplification of the human -globin gene
in DNA extracts prepared from individual colonies. In two experiments
in which DNA from individual colonies derived from the BM of 2 to 4 mice was analyzed by PCR, human sequences were detected in 23 of 24 progenitors.
Differentiative capacity of repopulating cells present in MPB
G0CD34+ cells.
The phenotypic profile of engrafted CD45+ cells in
G0-transplanted mice was determined by two-color staining
(Table 1). A mean of 19.9% of
CD45+ cells were also CD34+ 6 to 7 weeks after
transplantation. Expression of CD19 and CD33 antigens on 55.7% and
34.2%, respectively, of human CD45+ cells demonstrated the
presence of repopulating cells with lympho-myeloid potential. The same
phenotyping method was used to characterize the differentiation of
engrafted human CD34+ detected in
G0-transplanted mice (Table 1). Small but detectable numbers of CD34+ cells had low or no expression of the CD38
antigen, suggesting that primitive cells were maintained. In accordance
with the study of Pflumio et al,15 the majority of
engrafted CD34+ cells (64.6%) coexpressed CD19, whereas
only 18.2% were CD33+, showing the predominant
differentiation of human MPB CD34+ cells towards the
B-lymphoid lineage in the NOD/SCID model.
CD34+ cells were reisolated from the BM of highly positive
animals and assayed in vitro. Both granulo-monocytic (CFU-GM) and erythroid progenitors (burst-forming unit-erythroid
[BFU-E]) were demonstrated in progenitor cell assays at
frequencies of 1.36% ± 0.1% and 0.1% ± 0.02%, respectively.
Primitive hematopoietic progenitor cells assayed as LTC-ICs were also
present, although at a lower frequency than in the original graft
(0.1% ± 0.02% v 3.1% ± 0.8%).
Effect of G0-G1 progression in vitro on the
repopulating ability of MPB CD34+ cells.
Given the marked difference of repopulating activity between MPB
CD34+ cells residing in vivo (ie, in a fresh MPB sample) in
G0 or in G1, we were interested in examining
whether the same relationship existed for cells progressing from
G0 to G1 under in vitro cytokine stimulation.
The experimental design is shown in Fig 5.
A first Hst/PY fractionation was used to isolate CD34+
cells in G0 or in G1 (Fig 5A), and defined cell
doses were used for mice transplantation on the original day of cell
isolation. The remaining cells isolated in G0 were plated
in serum-free conditions and stimulated with a combination of 100 ng/mL
FL, 100 ng/mL SCF, and 50 ng/mL IL-3, a cytokine mixture previously
shown to promote the expansion of primitive cells detected as
LTC-ICs.28 After 36 hours in culture, a second cell cycle
fractionation step was used to separate cells remaining in
G0 from those traversing into G1 under these
conditions (Fig 5B). A short culture period was chosen to minimize cell
proliferation such that only the effect of progression to an activated
state was investigated and not the result of maturational cell
divisions potentially leading to stem cell exhaustion. At this time
point, the mean increase in total cells was 1.18- ± 0.17-fold (n = 3). Viability of cultured cells always exceeded 98%, as judged by
trypan blue exclusion. To assess the fidelity of the cell cycle
fractionation at 36 hours, aliquots of sorted cells were reanalyzed
with 7-AAD and Ki-67 (Fig 5C and D). In the representative experiment
shown in Fig 5, approximately 75% of PYlow cells were
still in G0 as defined by the absence of Ki-67 expression, whereas more than 80% of PYhigh cells were mitotically
activated, Ki-67-positive cells. Cells reisolated in these two
fractions were used in transplantation experiments at the same dose as
those used 36 hours before. All recipient mice were analyzed for the
presence of chimeric human CD45+ cells 6 weeks
posttransplantation (Table 2).
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| Fig 5.
Cell sorting strategy to isolate CD34+
cells during ex vivo G0-G1 progression. (A) MPB
CD34+ cells in G0 were isolated in a first
Hst/PY sort and either directly injected into recipient animals or
plated in ex vivo conditions. (B) After 36 hours, a second Hst/PY sort
was used to separate cultured cells transiting to G1 from
those remaining in G0. Both cell subsets were then assayed
in NOD/SCID mice. Cell-cycle status of cells isolated in G1
(C) or G0 (D) after culture was confirmed by restaining
samples of sorted cells with 7-AAD and Ki-67. Percentages of cells
detected in each quadrant are indicated.
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Table 2.
Effect of Cell Cycle Progression Under IL-3-SCF-FL
Stimulation on the Repopulating Activity of CD34+ Cells
Initially Isolated in G0
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As already shown, freshly isolated CD34+ cells residing in
G0 were able to produce a high level of engraftment. As for
cells remaining in G0 after 36 hours of cytokine
stimulation, the level of repopulating activity was comparable or
slightly reduced compared with that observed in noncultured
G0 cells (experiments 1 and 2, Table 2). Most strikingly,
CD34+ cells initially isolated in G0 and
traversing from G0 into G1 during in vitro
culture had virtually no repopulating ability.
This differential engraftment potential of cultured cells could be
explained by the presence of two different subsets of
G0CD34+ cells: a subset enriched in
repopulating cells, which responded poorly to mitogenic stimulation in
vitro and remained sequestrated in the G0 compartment after
36 hours in culture, and a highly responsive subset, transiting quickly
into G1, but initially devoid of repopulating activity.
Alternatively, these observations could be the result of a loss of
repopulating capacity resulting directly from the progression to an
active state in the cell cycle. To distinguish between these two
options, we performed two experiments in which fresh
G0CD34+ cells were cultured for 36 hours under
the same conditions as those described above, then directly
transplanted into NOD/SCID recipients without restaining with Hst/PY
and separation into quiescent and activated fractions (experiments 2 and 3, Table 2). In both experiments, the engraftment efficiency of
unfractionated cultured cells was massively reduced compared with that
of fresh G0CD34+ cells indicating that
repopulating activity was compromised after ex vivo culture, most
likely by progression into G1. Because no adjustment to the
number of cells remaining in G0 among total cells used as a
graft was made, it is difficult to estimate the magnitude of chimerism
lost due to decreased numbers of G0 cells in the graft.
A negative effect of IL-3 on the in vitro maintenance of primitive
hematopoietic cells has now been highly suspected,29,30 whereas TPO has emerged as a potent stimulator of putative stem cells.28,31 To ensure that our observations on the effect
of G0-G1 progression were not dependent on a
suboptimal combination of cytokines, we substituted IL-3 with TPO in an
additional experiment. MPB CD34+ cells initially isolated
in G0 were exposed to the combination of TPO-SCF-FL for 36 hours and then fractionated into G0 and G1 subcompartments (Fig 5). The same relationship between cell cycle progression and SRC activity as that determined after IL-3-SCF-FL stimulation was observed such that CD34+ cells traversing
into G1 were largely devoid of reconstituting ability
compared with cells remaining in G0
(Table 3). This finding suggests that in vitro mitotic activation is intrinsically detrimental to the maintenance of SRC activity, independently of the type of
cytokines used during ex vivo culture.
View this table:
[in this window]
[in a new window]
|
Table 3.
Effect of Cell Cycle Progression Under TPO-SCF-FL
Stimulation on the Repopulating Activity of CD34+ Cells
Initially Isolated in G0
|
|
| |
DISCUSSION |
The present report describes for the first time a strong relationship
between cell cycle status and BM repopulating potential, based on a
direct cell cycle fractionation method originally described by our
group.16-18 Using simultaneous DNA/RNA staining and various in vitro assays, we previously demonstrated that primitive
hematopoietic cells were mainly found in the G0 phase of
the cell cycle. We show herein that this relationship also applies to
NOD/SCID repopulating cells contained within freshly isolated human MPB
CD34+ cells. Although we did not use limiting dilution
analysis to determine the frequency of SRC in mitotically quiescent and
activated subsets of MPB CD34+ cells,13 we show
a significantly large difference in the readout of these two cell
populations in the NOD/SCID mouse model. This suggests that SRC are
highly enriched in the G0 compartment, that SRC present in
the G1 phase have a reduced proliferative capacity in the
murine microenvironment, or a combination of both
phenomena. This is further evidence of the ability of our
cell cycle fractionation method to isolate functionally defined
populations of human hematopoietic progenitor/stem cells based on their
cycling status. Although the endpoint analysis of engraftment at 6 to 7 weeks posttransplantation is in agreement with other
investigators,14,24 the exact nature and position of these
cells in the hierarchy of the pool of engrafting hematopoietic
progenitor cells remain to be fully elucidated.
Few studies have examined the outcome of ex vivo culture of putative
stem cells in the NOD/SCID model. Differences were pointed out in the
behavior of SRC compared with that of in vitro assayable human
hematopoietic cells, ie, CFC and LTC-IC. Gan et al32 showed that incubation of hematopoietic cells on stromal cells maintained or
even expanded CFC and LTC-IC, whereas SRC declined over the same period
of time. In gene marking experiments, after prestimulation over the
C-terminal fragment of fibronectin,33 high levels of transduction were observed in CFC and LTC-IC, but no transduced cells
were detected in the BM of recipient mice. Finally, using CB
CD34+CD38 cells, Bhatia et
al34 were able to demonstrate a twofold increase of SRC in
short-term culture, although in a similar study, Conneally et
al24 showed that ex vivo expanded SRC had a reduced
proliferative capacity compared with that of freshly isolated SRC. It
appears therefore that expansion of SRC is at best limited and achieved at the expense of a decrease in the generative capacity of individual repopulating cells.
The ability of the DNA/RNA staining method to sort cells in precise
phases of the cell cycle was used in the present study to determine the
effect of in vitro cell cycle activation on the repopulating ability of
putative stem cells, in the absence of cell division. A short cytokine
stimulation period of 36 hours was used to induce a fraction of
CD34+ cells initially isolated in G0 to
progress into G1 without executing a complete cell cycle,
while allowing almost an equal number of cells to remain in
G0. Thus, the only variable tested in these experiments was
the movement along the G0/G1 phase of the cell cycle and not the effect of cellular divisions. This was confirmed by
the absence of an increase in cell numbers during ex vivo culture. Our
data show that engagement in G1 after cytokine stimulation is associated with a decrease in repopulating capacity. Two mutually nonexclusive mechanisms may underlie these observations. It is possible
that SRC are sequestrated in the G0 compartment during culture, whereas more differentiated cells quickly traverse into G1. This is most likely, because we previously demonstrated
that CD34+ cells isolated in G0 by the Hst/PY
method were still heterogeneous in their ability to respond to cytokine
stimulation,16,18 with the most quiescent cells being the
most primitive. Another possible explanation is that ex vivo
G0-G1 transition may directly affect the
capacity of pre-existing SRC to repopulate the BM of the recipient animal. The decline in repopulating capacity of unfractionated stimulated cells demonstrates that G1 progression, under
culture conditions used in this study, does indeed reduce SRC activity.
This study provides evidence that ex vivo culture can modify primitive
hematopoietic functions of putative stem cells before any cellular
division actually occurs. This is compatible with at least two
hypotheses. First, commitment and/or differentiation may take
place during the G0-G1 transition. Second,
homing of primitive progenitor cells to the BM microenvironment is
possibly impaired after cell cycle activation. The latter hypothesis is also suggested by studies that have now established a direct
relationship between cytokine stimulation and affinity and/or
expression of integrins at the surface of human hematopoietic
progenitors35-38 or between cell cycle status, expression
of adhesion molecules, and engraftment of murine progenitor
cells.39 Because the BM microenvironment is thought to
comprise multiple loci supporting hematopoietic progenitors at
different levels of differentiation,40 ex vivo modulation
of adhesive properties by cycle activation may impair the ability for
stimulated primitive cells to home to adequate sites and express their
full generative and differentiative capacity. Further studies will be
needed to elucidate cell cycle-related changes in adhesion molecules
affinity and/or expression, potentially leading to impaired
engraftment.
| |
FOOTNOTES |
Submitted April 30, 1998;
accepted July 16, 1998.
Supported by National Institutes of Health Grant No. R01 HL55716 and a
research award from the Phi Beta Psi Sorority to E.F.S. Herman B Wells
Center for Pediatric Research is a Center of Excellence in Molecular
Hematology (NIDDK P50 DK 49218). A.G. is supported by a fellowship from
the Fonds National de la Recherche Scientifique (FNRS, Brussels,
Belgium) and by a travel grant from the Centre Anticancéreux
près l'ULg (University of Liège, Liège, Belgium).
Address reprint requests to Edward F. Srour, PhD, Indiana University
School of Medicine, 1044 W Walnut St, R4-202, Indianapolis, IN 46202.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr David A. Williams (Herman B Wells Center for
Pediatric Research, Indiana University School of Medicine) for the use
of the NOD/SCID mice facility, Susan Rice and Jon McMahel for technical
assistance in flow cytometric cell sorting, and Ryan Cooper for help in
immunomagnetic CD34+ cell selection. We are also grateful
to Amgen and Immunex for generous gifts of recombinant human cytokines.
| |
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Blood,
August 15, 2005;
106(4):
1479 - 1487.
[Abstract]
[Full Text]
[PDF]
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K. Leibundgut, N. M.R. Schmitz, and A. Hirt
Catalytic Activities of G1 Cyclin-Dependent Kinases and Phosphorylation of Retinoblastoma Protein in Mobilized Peripheral Blood CD34+ Hematopoietic Progenitor Cells
Stem Cells,
August 1, 2005;
23(7):
1002 - 1011.
[Abstract]
[Full Text]
[PDF]
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X. Li, M. M. Le Beau, S. Ciccone, F.-C. Yang, B. Freie, S. Chen, J. Yuan, P. Hong, A. Orazi, L. S. Haneline, et al.
Ex vivo culture of Fancc-/- stem/progenitor cells predisposes cells to undergo apoptosis, and surviving stem/progenitor cells display cytogenetic abnormalities and an increased risk of malignancy
Blood,
May 1, 2005;
105(9):
3465 - 3471.
[Abstract]
[Full Text]
[PDF]
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E. F. Srour, X. Tong, K. W. Sung, P. A. Plett, S. Rice, J. Daggy, C. T. Yiannoutsos, R. Abonour, and C. M. Orschell
Modulation of in vitro proliferation kinetics and primitive hematopoietic potential of individual human CD34+CD38-/lo cells in G0
Blood,
April 15, 2005;
105(8):
3109 - 3116.
[Abstract]
[Full Text]
[PDF]
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S. Fukuda, H. E. Broxmeyer, and L. M. Pelus
Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1{alpha}(CXCL12)/CXCR4 axis
Blood,
April 15, 2005;
105(8):
3117 - 3126.
[Abstract]
[Full Text]
[PDF]
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T. Byk, J. Kahn, O. Kollet, I. Petit, S. Samira, S. Shivtiel, H. Ben-Hur, A. Peled, W. Piacibello, and T. Lapidot
Cycling G1 CD34+/CD38+ Cells Potentiate the Motility and Engraftment of Quiescent G0 CD34+/CD38-/low Severe Combined Immunodeficiency Repopulating Cells
Stem Cells,
April 1, 2005;
23(4):
561 - 574.
[Abstract]
[Full Text]
[PDF]
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J. P. Chute, G. G. Muramoto, J. Fung, and C. Oxford
Soluble factors elaborated by human brain endothelial cells induce the concomitant expansion of purified human BM CD34+CD38- cells and SCID-repopulating cells
Blood,
January 15, 2005;
105(2):
576 - 583.
[Abstract]
[Full Text]
[PDF]
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Y. J. Summers, C. M. Heyworth, E. A. de Wynter, C. A. Hart, J. Chang, and N. G. Testa
AC133+ G0 Cells from Cord Blood Show a High Incidence of Long-Term Culture-Initiating Cells and a Capacity for More Than 100 Million-Fold Amplification of Colony-Forming Cells In Vitro
Stem Cells,
September 1, 2004;
22(5):
704 - 715.
[Abstract]
[Full Text]
[PDF]
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K. W. Christopherson II, G. Hangoc, C. R. Mantel, and H. E. Broxmeyer
Modulation of Hematopoietic Stem Cell Homing and Engraftment by CD26
Science,
August 13, 2004;
305(5686):
1000 - 1003.
[Abstract]
[Full Text]
[PDF]
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W. Wagner, A. Ansorge, U. Wirkner, V. Eckstein, C. Schwager, J. Blake, K. Miesala, J. Selig, R. Saffrich, W. Ansorge, et al.
Molecular evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis
Blood,
August 1, 2004;
104(3):
675 - 686.
[Abstract]
[Full Text]
[PDF]
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K. Z. Nagy, S. Laufs, B. Gentner, S. Naundorf, K. Kuehlcke, J. Topaly, E. C. Buss, W. J. Zeller, and S. Fruehauf
Clonal Analysis of Individual Marrow-Repopulating Cells after Experimental Peripheral Blood Progenitor Cell Transplantation
Stem Cells,
July 1, 2004;
22(4):
570 - 579.
[Abstract]
[Full Text]
[PDF]
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F. Mazurier, O. I. Gan, J. L. McKenzie, M. Doedens, and J. E. Dick
Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment
Blood,
January 15, 2004;
103(2):
545 - 552.
[Abstract]
[Full Text]
[PDF]
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D. N. Levasseur, T. M. Ryan, K. M. Pawlik, and T. M. Townes
Correction of a mouse model of sickle cell disease: lentiviral/antisickling {beta}-globin gene transduction of unmobilized, purified hematopoietic stem cells
Blood,
December 15, 2003;
102(13):
4312 - 4319.
[Abstract]
[Full Text]
[PDF]
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P. Kurre, J. Morris, B. Thomasson, D. B. Kohn, and H.-P. Kiem
Scaffold attachment region-containing retrovirus vectors improve long-term proviral expression after transplantation of GFP-modified CD34+ baboon repopulating cells
Blood,
November 1, 2003;
102(9):
3117 - 3119.
[Abstract]
[Full Text]
[PDF]
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G. N. Schwartz, B. A. Vance, B. M. Levine, M. Fukazawa, W. G. Telford, D. Cesar, M. Hellerstein, and R. E. Gress
Proliferation kinetics of subpopulations of human marrow cells determined by quantifying in vivo incorporation of [2H2]-glucose into DNA of S-phase cells
Blood,
September 15, 2003;
102(6):
2068 - 2073.
[Abstract]
[Full Text]
[PDF]
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S. Stier, T. Cheng, R. Forkert, C. Lutz, D. M. Dombkowski, J. L. Zhang, and D. T. Scadden
Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of human hematopoietic stem cells
Blood,
August 15, 2003;
102(4):
1260 - 1266.
[Abstract]
[Full Text]
[PDF]
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F. Geronimi, E. Richard, I. Redonnet-Vernhet, I. Lamrissi-Garcia, M. Lalanne, C. Ged, F. Moreau-Gaudry, and H. de Verneuil
Highly Efficient Lentiviral Gene Transfer in CD34+ and CD34+/38-/lin- Cells from Mobilized Peripheral Blood after Cytokine Prestimulation
Stem Cells,
July 1, 2003;
21(4):
472 - 480.
[Abstract]
[Full Text]
[PDF]
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Y. Guan, B. Gerhard, and D. E. Hogge
Detection, isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML)
Blood,
April 15, 2003;
101(8):
3142 - 3149.
[Abstract]
[Full Text]
[PDF]
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S. Laufs, B. Gentner, K. Z. Nagy, A. Jauch, A. Benner, S. Naundorf, K. Kuehlcke, B. Schiedlmeier, A. D. Ho, W. J. Zeller, et al.
Retroviral vector integration occurs in preferred genomic targets of human bone marrow-repopulating cells
Blood,
March 15, 2003;
101(6):
2191 - 2198.
[Abstract]
[Full Text]
[PDF]
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P. Hematti, S. E. Sellers, B. A. Agricola, M. E. Metzger, R. E. Donahue, and C. E. Dunbar
Retroviral transduction efficiency of G-CSF+SCF-mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L-mobilized cells in nonhuman primates
Blood,
March 15, 2003;
101(6):
2199 - 2205.
[Abstract]
[Full Text]
[PDF]
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P. J. Quesenberry, G. A. Colvin, and J.-F. Lambert
The chiaroscuro stem cell: a unified stem cell theory
Blood,
December 15, 2002;
100(13):
4266 - 4271.
[Abstract]
[Full Text]
[PDF]
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J. Roesler, S. Brenner, A. A. Bukovsky, N. Whiting-Theobald, T. Dull, M. Kelly, C. I. Civin, and H. L. Malech
Third-generation, self-inactivating gp91phox lentivector corrects the oxidase defect in NOD/SCID mouse-repopulating peripheral blood-mobilized CD34+ cells from patients with X-linked chronic granulomatous disease
Blood,
December 15, 2002;
100(13):
4381 - 4390.
[Abstract]
[Full Text]
[PDF]
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J. P. Chute, A. A. Saini, D. J. Chute, M. R. Wells, W. B. Clark, D. M. Harlan, J. Park, M. K. Stull, C. Civin, and T. A. Davis
Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow
Blood,
December 15, 2002;
100(13):
4433 - 4439.
[Abstract]
[Full Text]
[PDF]
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J. Cashman, B. Dykstra, I. Clark-Lewis, A. Eaves, and C. Eaves
Changes in the Proliferative Activity of Human Hematopoietic Stem Cells in NOD/SCID Mice and Enhancement of Their Transplantability after In Vivo Treatment with Cell Cycle Inhibitors
J. Exp. Med.,
November 4, 2002;
196(9):
1141 - 1150.
[Abstract]
[Full Text]
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S. Huygen, O. Giet, V. Artisien, I. Di Stefano, Y. Beguin, and A. Gothot
Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture
Blood,
September 26, 2002;
100(8):
2744 - 2752.
[Abstract]
[Full Text]
[PDF]
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S.-J. Lu, C. Quan, F. Li, L. Vida, and G. R. Honig
Hematopoietic Progenitor Cells Derived from Embryonic Stem Cells: Analysis of Gene Expression
Stem Cells,
September 1, 2002;
20(5):
428 - 437.
[Abstract]
[Full Text]
[PDF]
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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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N. C. Josephson, G. Vassilopoulos, G. D. Trobridge, G. V. Priestley, B. L. Wood, T. Papayannopoulou, and D. W. Russell
Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors
PNAS,
June 11, 2002;
99(12):
8295 - 8300.
[Abstract]
[Full Text]
[PDF]
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H. Glimm, P. Tang, I. Clark-Lewis, C. von Kalle, and C. Eaves
Ex vivo treatment of proliferating human cord blood stem cells with stroma-derived factor-1 enhances their ability to engraft NOD/SCID mice
Blood,
May 1, 2002;
99(9):
3454 - 3457.
[Abstract]
[Full Text]
[PDF]
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O. Giet, D. R. Van Bockstaele, I. Di Stefano, S. Huygen, R. Greimers, Y. Beguin, and A. Gothot
Increased binding and defective migration across fibronectin of cycling hematopoietic progenitor cells
Blood,
March 15, 2002;
99(6):
2023 - 2031.
[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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J.-J. Lataillade, D. Clay, P. Bourin, F. Herodin, C. Dupuy, C. Jasmin, and M.-C. Le Bousse-Kerdiles
Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G0/G1 transition in CD34+ cells: evidence for an autocrine/paracrine mechanism
Blood,
February 15, 2002;
99(4):
1117 - 1129.
[Abstract]
[Full Text]
[PDF]
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M. A. Dao, J. Hwa, and J. A. Nolta
Molecular mechanism of transforming growth factor beta -mediated cell-cycle modulation in primary human CD34+ progenitors
Blood,
January 15, 2002;
99(2):
499 - 506.
[Abstract]
[Full Text]
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M. Scherr, K. Battmer, U. Blomer, B. Schiedlmeier, A. Ganser, M. Grez, and M. Eder
Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells
Blood,
January 15, 2002;
99(2):
709 - 712.
[Abstract]
[Full Text]
[PDF]
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Y. J. Summers, C. M. Heyworth, E. A. de Wynter, J. Chang, and N. G. Testa
Cord Blood G0 CD34+ Cells Have A Thousand-Fold Higher Capacity For Generating Progenitors In Vitro Than G1 CD34+ Cells
Stem Cells,
November 1, 2001;
19(6):
505 - 513.
[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]
[PDF]
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M. Drouet, F. Herodin, F. Norol, F. Mourcin, and J.F. Mayol
Cell Cycle Activation of Peripheral Blood Stem and Progenitor Cells Expanded Ex Vivo with SCF, FLT-3 Ligand, TPO, and IL-3 Results in Accelerated Granulocyte Recovery in a Baboon Model of Autologous Transplantation but G0/G1 and S/G2/M Graft Cell Content Does Not Correlate with Tranplantability
Stem Cells,
September 1, 2001;
19(5):
436 - 442.
[Abstract]
[Full Text]
[PDF]
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J. J. Minguell, A. Erices, and P. Conget
Mesenchymal Stem Cells
Experimental Biology and Medicine,
June 1, 2001;
226(6):
507 - 520.
[Abstract]
[Full Text]
[PDF]
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L. E. Perez, H. M. Rinder, C. Wang, J. B. Tracey, N. Maun, and D. S. Krause
Xenotransplantation of immunodeficient mice with mobilized human blood CD34+ cells provides an in vivo model for human megakaryocytopoiesis and platelet production
Blood,
March 15, 2001;
97(6):
1635 - 1643.
[Abstract]
[Full Text]
[PDF]
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A. Sirven, F. Pflumio, V. Zennou, M. Titeux, W. Vainchenker, L. Coulombel, A. Dubart-Kupperschmitt, and P. Charneau
The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells
Blood,
December 15, 2000;
96(13):
4103 - 4110.
[Abstract]
[Full Text]
[PDF]
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H. Glimm, I.-H. Oh, and C. J. Eaves
Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0
Blood,
December 15, 2000;
96(13):
4185 - 4193.
[Abstract]
[Full Text]
[PDF]
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J. D. Cashman and C. J. Eaves
High marrow seeding efficiency of human lymphomyeloid repopulating cells in irradiated NOD/SCID mice
Blood,
December 1, 2000;
96(12):
3979 - 3981.
[Abstract]
[Full Text]
[PDF]
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S. Barrette, J. L. Douglas, N. E. Seidel, and D. M. Bodine
Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to Moloney murine leukemia virus-based vectors
Blood,
November 15, 2000;
96(10):
3385 - 3391.
[Abstract]
[Full Text]
[PDF]
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J. Wilpshaar, J. H. F. Falkenburg, X. Tong, W. A. Noort, R. Breese, D. Heilman, H. Kanhai, C. M. Orschell-Traycoff, and E. F. Srour
Similar repopulating capacity of mitotically active and resting umbilical cord blood CD34+ cells in NOD/SCID mice
Blood,
September 15, 2000;
96(6):
2100 - 2107.
[Abstract]
[Full Text]
[PDF]
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P. W. Zandstra, D. A. Lauffenburger, and C. J. Eaves
A ligand-receptor signaling threshold model of stem cell differentiation control: a biologically conserved mechanism applicable to hematopoiesis
Blood,
August 15, 2000;
96(4):
1215 - 1222.
[Abstract]
[Full Text]
[PDF]
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C. M. Orschell-Traycoff, K. Hiatt, R. N. Dagher, S. Rice, M. C. Yoder, and E. F. Srour
Homing and engraftment potential of Sca-1+lin- cells fractionated on the basis of adhesion molecule expression and position in cell cycle
Blood,
August 15, 2000;
96(4):
1380 - 1387.
[Abstract]
[Full Text]
[PDF]
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E. F. Srour;, C. Eaves, and H. Glimm
Proliferative history and hematopoietic function of ex vivo expanded human CD34+ cells
Blood,
August 15, 2000;
96(4):
1609 - 1612.
[Full Text]
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H. J. Kim, J. F. Tisdale, T. Wu, M. Takatoku, S. E. Sellers, P. Zickler, M. E. Metzger, B. A. Agricola, J. D. Malley, I. Kato, et al.
Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates
Blood,
July 1, 2000;
96(1):
1 - 8.
[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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B. Schiedlmeier, K. Kuhlcke, H. G. Eckert, C. Baum, W. J. Zeller, and S. Fruehauf
Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice
Blood,
February 15, 2000;
95(4):
1237 - 1248.
[Abstract]
[Full Text]
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R. A. J. Oostendorp, J. Audet, and C. J. Eaves
High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo
Blood,
February 1, 2000;
95(3):
855 - 862.
[Abstract]
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D. A. Williams, A. W. Nienhuis, R. G. Hawley, and F. O. Smith
Gene Therapy 2000
Hematology,
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2000(1):
376 - 393.
[Abstract]
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H. Glimm and C.J. Eaves
Direct Evidence for Multiple Self-Renewal Divisions of Human In Vivo Repopulating Hematopoietic Cells in Short-Term Culture
Blood,
October 1, 1999;
94(7):
2161 - 2168.
[Abstract]
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T. Holyoake, X. Jiang, C. Eaves, and A. Eaves
Isolation of a Highly Quiescent Subpopulation of Primitive Leukemic Cells in Chronic Myeloid Leukemia
Blood,
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94(6):
2056 - 2064.
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J. E. Brandt, A. M. Bartholomew, J. D. Fortman, M. C. Nelson, E. Bruno, L. M. Chen, J. V. Turian, T. A. Davis, J. P. Chute, and R. Hoffman
Ex Vivo Expansion of Autologous Bone Marrow CD34+ Cells With Porcine Microvascular Endothelial Cells Results in a Graft Capable of Rescuing Lethally Irradiated Baboons
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V. I. Rebel, M. Tanaka, J.-S. Lee, S. Hartnett, M. Pulsipher, D. G. Nathan, R. C. Mulligan, and C. A. Sieff
One-Day Ex Vivo Culture Allows Effective Gene Transfer Into Human Nonobese Diabetic/Severe Combined Immune-Deficient Repopulating Cells Using High-Titer Vesicular Stomatitis Virus G Protein Pseudotyped Retrovirus
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S. S. Case, M. A. Price, C. T. Jordan, X. J. Yu, L. Wang, G. Bauer, D. L. Haas, D. Xu, R. Stripecke, L. Naldini, et al.
Stable transduction of quiescent CD34+CD38- human hematopoietic cells by HIV-1-based lentiviral vectors
PNAS,
March 16, 1999;
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2988 - 2993.
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Abstract
Introduction
Abstract