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Cell Cycle-Related Changes in Repopulating Capacity of
Human Mobilized Peripheral Blood CD34+ Cells in Non-Obese
Diabetic/Severe Combined Immune-Deficient Mice

André
Gothot,

Johannes
C.M. van der Loo,

D.
Wade Clapp, and

Edward
F. Srour

1 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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Section

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.

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.16G0CD34+
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 G0compartment 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.

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
21The
selectivity of the assay for human progenitors was confirmed by polymerase chain
reaction (PCR) detection of human β-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 × 10−2 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 × 10−2 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
24mice
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 × 105NOD/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 β-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 GCamplifying a 345-bp fragment of human β-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.
AllP 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 G1were 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 PYhighcells, 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 G1by
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.

As for more primitive cells (Fig 2B),
the G0 compartment was significantly enriched in LTC-ICs compared
with the G1compartment (3.1% for G0CD34+ cells
v0.8% 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,
pairedt-test) than in mice injected with G1CD34+
cells (Fig4A).
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,
pairedt-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).

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,
pairedt-test). The selectivity of the assay for human clonogenic cells
was confirmed by PCR amplification of the human β-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.

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Table 1.

Phenotype of Chimeric Human Cells Harvested From
NOD/SCID Mice Transplanted With G0CD34+Cells

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

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
30whereas
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
G1subcompartments (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.

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Table 3.

Effect of Cell Cycle Progression Under TPO-SCF-FL
Stimulation on the Repopulating Activity of CD34+ Cells Initially
Isolated in G0

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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.

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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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SectionNext
Section

Footnotes

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.

Submitted April 30, 1998.

Accepted July 16, 1998.

Copyright © 1998 American Society of
Hematology

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Section

REFERENCES

↵

EmersonSGEx vivo expansion of hematopoietic
precursors, progenitors, and stem cells: The next generation of cellular
therapeutics [Review].Blood8719963082

FREE Full
Text

↵

MillerDGAdamMAMillerADGene transfer by
retrovirus vectors occurs only in cells that are actively replicating at the
time of infection.Mol Cell Biol1019904239

Abstract/FREE Full
Text

↵

HajihosseiniMIavachevLPriceJEvidence that
retroviruses integrate into post-replication host DNA.EMBO
J1219934969

Medline

↵

LearyAGZengHQClarkSCOgawaMGrowth factor
requirements for survival in G0 and entry into the cell cycle of primitive
human hemopoietic progenitors.Proc Natl Acad Sci USA8919924013

Abstract/FREE Full
Text

↵

OgawaMDifferentiation and proliferation of
hematopoietic stem cells [Review].Blood8119932844

Abstract/FREE Full
Text

↵

TraycoffCMKosakSTGrigsbySSrourEFEvaluation of ex
vivo expansion potential of cord blood and bone marrow hematopoietic
progenitor cells using cell tracking and limiting dilution
analysis.Blood8519952059

Abstract/FREE Full
Text

↵

BerardiACWangALevineJDLopezPScaddenDTFunctional
isolation and characterization of human hematopoietic stem
cells.Science2671995104

Abstract/FREE Full
Text

↵

van der LooJCPloemacherREMarrow- and
spleen-seeding efficiencies of all murine hematopoietic stem cell subsets are
decreased by preincubation with hematopoietic growth
factors.Blood8519952598

Abstract/FREE Full
Text

↵

Peters SO,

Kittler EL,

Ramshaw HS,

Quesenberry
PJ

Murine marrow cells expanded in culture with IL-3,
IL-6, IL-11, and SCF acquire an engraftment defect in normal
hosts.Exp Hematol231995461(erratum 23:568, 1995)

Medline

↵

PetersSOKittlerELRamshawHSQuesenberryPJEx vivo
expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and
stem cell factor leads to impaired engraftment in irradiated
hosts.Blood87199630

Abstract/FREE Full
Text

↵

TraycoffCMCornettaKYoderMCDavidsonASrourEFEx
vivo expansion of murine hematopoietic progenitor cells generates classes of
expanded cells possessing different levels of bone marrow repopulating
potential.Exp Hematol241996299

Medline

↵

RamshawHSRaoSSCrittendenRBPetersSOWeierHUQuesenberryPJEngraftment
of bone marrow cells into normal unprepared hosts: Effects of 5-fluorouracil
and cell cycle status.Blood861995924

Abstract/FREE Full
Text

↵

WangJCYDoedensMDickJEPrimitive human
hematopoietic cells are enriched in cord blood compared with adult bone marrow
or mobilized peripheral blood as measured by the quantitative in vivo
SCID-repopulating cell assay.Blood8919973319

↵

CashmanJDLapidotTWangJCDoedensMShultzLDLansdorpPDickJEEavesCJKinetic
evidence of the regeneration of multilineage hematopoiesis from primitive
cells in normal human bone marrow transplanted into immunodeficient
mice.Blood8919974307

Abstract/FREE Full
Text

↵

PflumioFIzacBKatzAShultzLDVainchenkerWCoulombelLPhenotype
and function of human hematopoietic cells engrafting immune-deficient
CB17-severe combined immunodeficiency mice and nonobese diabetic-severe
combined immunodeficiency mice after transplantation of human cord blood
mononuclear cells.Blood8819963731

Abstract/FREE Full
Text

↵

LaddACPyattRGothotAGrigsbySMcMahelJTraycoffCMSrourEFOrderly
process of sequential cytokine stimulation is required for activation and
maximaI proliferation of primitive human bone marrow CD34+ hematopoietic
progenitor cells residing in G0.Blood901997658

Abstract/FREE Full
Text

↵

GothotAPyattRMcMahelJRiceSSrourEFFunctional
heterogeneity of human CD34+ cells isolated in subcompartments of the G0/G1
phase of the cell cycle.Blood9019974384

Abstract/FREE Full
Text

↵

GothotAPyattRMcMahelJRiceSSrourEFAssessment of
proliferative and colony-forming capacity after successive in vitro divisions
of single human CD34+ cells initially isolated in G0.Exp
Hematol261998562

Medline

↵

ShultzLDSchweitzerPAChristiansonSWGottBSchweitzerIBTennentBMcKennaSMobraatenLRajanTVGreinerDLLeiterEHMultiple
defects in innate and adaptive immunologic function in NOD/LTSz-scid mice.J
Immunol1541995180

Abstract

↵

JordanCTYamasakiGMinamotoDHigh-resolution cell
cycle analysis of defined phenotypic subsets within primitive human
hematopoietic cell populations.Exp Hematol2419961347

Medline

↵

LapidotTPflumioFDoedensMMurdochBWilliamsDEDickJECytokine
stimulation of multilineage hematopoiesis from immature human cells engrafted
in SCID mice.Science25519921137

Abstract/FREE Full
Text

↵

SutherlandHJEavesCJLansdorpPMThackerJDHoggeDEDifferential
regulation of primitive human hematopoietic cells in long-term cultures
maintained on genetically engineered murine stromal
cells.Blood781991666

Abstract/FREE Full
Text

↵

TaswellCLimiting dilution assays for the
determination of immunocompetent cell frequencies.J
Immunol12619811614

Abstract

↵

ConneallyECashmanJPetzerAEavesCExpansion in
vitro of transplantable human cord blood stem cells demonstrated using a
quantitative assay of their lympho-myeloid repopulating activity in nonobese
diabetic-scid/scid mice.Proc Natl Acad Sci USA9419979836

Abstract/FREE Full
Text

↵

SrourEBregniMTraycoffCAgueroBKosakSHoffmanRSienaSGianniALong-term
hematopoietic culture-initiating cells are more abundant in mobilized
peripheral blood grafts than in bone marrow but have a more limited ex vivo
expansion potential.Blood Cells Mol Dis22199668

CrossRefMedline

↵

UchidaNHeDFrieraAReitsmaMSasakiDChenBTsukamotoAThe
unexpected G0/G1 cell cycle status of mobilized hematopoietic stem cells from
the peripheral blood.Blood891997465

Abstract/FREE Full
Text

↵

RobertsAWMetcalfDNoncycling state of peripheral
blood progenitor cells mobilized by granulocyte colony-stimulating factor and
other cytokines.Blood8619951600

Abstract/FREE Full
Text

↵

PetzerALZandstraPWPiretJMEavesCJDifferential
cytokine effects on primitive (CD34+CD38−) human hematopoietic cells: Novel
responses to Flt3-ligand and thrombopoietin.J Exp Med18319962551

Abstract/FREE Full
Text

↵

YonemuraYHsunKHirayamaFSouzaLMOgawaMInterleukin
3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem
cells.Proc Natl Acad Sci USA9319964040

Abstract/FREE Full
Text

↵

ZandstraPWConneallyEPetzerALPiretJMEavesCJCytokine
manipulation of primitive human hematopoietic cell self-renewal.Proc Natl Acad
Sci USA9419974698

Abstract/FREE Full
Text

↵

KobayashiMLaverJHKatoTMiyazakiHOgawaMThrombopoietin
supports proliferation of human primitive hematopoietic cells in synergy with
Steel factor and/or interleukin-3.Blood881996429

Abstract/FREE Full
Text

↵

GanOIMurdochBLarochelleADickJEDifferential
maintenance of primitive human SCID-repopulating cells, clonogenic
progenitors, and long term culture-initiating cells after incubation on human
bone marrow stromal cells.Blood901997641

Abstract/FREE Full
Text

↵

LarochelleAVormoorJHanenbergHWangJCBhatiaMLapidotTMoritzTMurdochBXiaoXLKatoIWilliamsDADickJEIdentification
of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse
bone marrow: Implications for gene therapy.Nat Med219961329

CrossRefMedline

↵

BhatiaMBonnetDKappUWangJCMurdochBDickJEQuantitative
analysis reveals expansion of human hematopoietic repopulating cells after
short-term ex vivo culture.J Exp Med1861997619

Abstract/FREE Full
Text

↵

LevesqueJPLeavesleyDINiuttaSVadasMSimmonsPJCytokines
increase human hematopoietic cell adhesiveness by activation of very late
antigen (VLA)-4 and VLA-5 integrins.J Exp Med18119951805

Abstract/FREE Full
Text

↵

LevesqueJPHaylockDNSimmonsPJCytokine regulation
of proliferation and cell adhesion are correlated events in human CD34+
hemopoietic progenitors.Blood8819961168

Abstract/FREE Full
Text

↵

LegrasSLevesqueJPCharradRMoromotoKLe
BousseCClayDJasminCSmadja-JoffeFCD44-mediated adhesiveness of human
hematopoieitc progenitors to hyaluronan is modulated by
cytokines.Blood8919971905

Abstract/FREE Full
Text

↵

ProsperFStroncekDMcCarthyJBVerfaillieCMMobilization
and homing of peripheral blood progenitors is related to reversible
downregulation of alpha4beta1 integrin expression and function.J Clin
Invest10119982456

Medline

↵

Traycoff CM,

Yoder M,

Hiatt K,

Srour EF

Functional association between expression of adhesion
molecules and marrow repopulating potential of primitive murine hematopoietic
progenitor cells.Exp Hematol251997736a(abstr)

↵

VerfaillieCMMc CarthyJBMc GlavePBDifferentiation
of primitive human multipotent hematopoietic progenitors into single lineage
clonogenic progenitors is accompanied by alterations in their interaction with
fibronectin.J Exp Med1741991693

Abstract/FREE Full
Text

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Blood October 15, 1998
vol. 92 no. 8
2641-2649

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