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Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2641-2649
RAPID COMMUNICATION
By
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.
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.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.
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).
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 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 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 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 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.
PCR detection of human 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.
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).
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).
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.
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).
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.
Submitted April 30, 1998;
accepted July 16, 1998.
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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Abstract
Introduction
Abstract
-globin gene in
individual colonies (see below). Hematopoietic colonies were scored
after 2 weeks according to standard criteria.
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
primer GAA TCC AGA TGC TCA
AGG CC and a 20-bp 3
) human hematopoietic cells: Novel responses to Flt3-ligand and thrombopoietin.
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