From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Quantitative Cell-Cycle Progression Analysis of the First Three Successive
Cell Cycles of Granulocyte Colony-Stimulating Factor and/or GranulocyteMacrophage Colony-Stimulating Factor-Stimulated Human C D 3 4 + Bone
Marrow Cells in Relation to Their Colony Formation
By Filip Lardon, Dirk R. Van Bockstaele, Hans-W. Snoeck, and Marc E. Peetermans
The bromodeoxyuridine (BrdU)-Hoechst flow cytometric
technique was applied to study the immediate cell kinetic
response of highly purified human (h) bone marrow progenitor cells (CD34+-sorted fraction) to h granulocyte colony-stimulating factor (G-CSF) and/or h granulocytemacrophage colony-stimulating factor (GM-CSF). The
technique permits us t o differentiate cycling from noncycling cells and to make a quantitative assessment of cellcycle progression during the first three consecutive cell cycles after stimulation. Semisolid agar and single-cell liquid
cultures were also performed to compare these initial
events to the effects observed after 14 days of culture. The
combination of G-CSF plus GM-CSF, acting synergistically
in day 14 cultures, was found t o have a subadditive effect
in the first cell cycles, thereby indicating partial overlap of
the different target cells. However, this combination accelerated transit through the cell cycle, as could be seen from
the higher number of cells in the third cell cycle after 72
hours of stimulation. We conclude that, apart from the
unresponsive cells, the CD34+ compartment consists of
cells responsive to both G-CSF and GM-CSF, and cells responsive to either one of the CSFs alone, and that the combination of the two CSFs speeds up the cell cycle traverse
rate for a significant fraction of the target cells that are
initially responsive for both G-CSF and GM-CSF. The latter
supports the hypothesis of an overlapping signalling pathway of G-CSF and GM-CSF.
0 1993 by The American Society of Hematology.
P
ROLIFERATION and differentiation of hematopoiconsent according to the regulations of the Ethics Committee of the
University of Antwerp, in tubes containing 2 mL lscove’s modified
etic progenitor cells are dependent on the continuous
Dulbecco’s Medium (IMDM) (GIBCO, Paisley, UK) and 5 U/mL
or intermittent supply of highly specific glycoproteins (GPs)
preservative-free heparin (Novo Industries, Bagsvaerd, Denmark).
termed colony-stimulating factors (CSFs), which act as reguCells were separated on a Lymphocyte Separation Medium (Boehlators of hematopoiesis. The interaction between these CSFs
ringer Mannheim GmbH, Mannheim, Germany) density gradient
and inhibitory factors is highly complex and their physioand washed twice. Remaining red blood cells (RBCs) were lysed
logic role involves proliferative changes of early stem cells
using an NH,CI-containing lysing solution.
and progenitors.’-4The in vitro systems used to study their
or
effects are CSF-supplemented semisolid colony
Progenitor (CD34) Labeling
long-term culture^,*^^ and based on the evaluation of the
Supernatant ofthe 43AI hybridoma (kindly donated by Dr H.J.
progeny at later stages of differentiation; thus, it represents
Biihring, University of Tubingen, Germany”) was used as a source
final outcome information. These assays do not give inforof anti-CD34. Mononuclear BM cells were incubated with 43A1
mation on the proliferative history of initially responding
supernatant in a 1/10 dilution at IO’ cells/mL for 20 minutes at
cells.
4”C, washed twice in IMDM, incubated with fluorescein isothioThe objective of the present study is to gain such early
cyanate (F1TC)-conjugated rabbit antimouse Igs ([Fab’], fragevent information to elucidate the well-known synergy of
ments) (RAM; Dako, Glostrup, Denmark) in a 1/50 dilution for 20
granulocyte-CSF (G-CSF) and granulocyte-macrophageminutes at 4°C and washed twice again before sorting.
CSF (GM-CSF)5,6in supporting myeloid progenitor-cell colony formation. We used the bromodeoxyuridine (BrdU)Hoechst flow cytometric technique, which provides a
From the Laboratory of Experimental Hematology, University of
unique source of cell cycle information, including accurate
Antwerp (UIA/UZA), Belgium.
estimates of the growth fraction, a separate visualization of
Submitted July 10, 1992; accepted January 22, 1993.
the first three consecutive cell cycles, discrimination beSupported by Grant No. 3.0133.91 ofthe Fund for Medical Scientween “fast” and “slow” responders, and discrimination betific Research (FG WO). F.L. is a holder ofthe Grant NFWO (Natween synchronous and asynchronous p r ~ l i f e r a t i o n . ’ ~ ’ ~tional Fund for Scient& Research of Belgium), “HippocrateInternational 1990” and of a “Belgisch Werk tegen Kanker”
Thus, it presents major advantages over the “classical” tech(1992-1993) grant. D. V.B. is holder of an SFO (special research
niques such as [3H]-thymidine pulse labeling and flow cytofund) grant (1992-1994) of the University of Antwerp (UIA).
metric DNA staining procedures, which only detect the
H.- W.S. is a research assistant ofthe NFWO. Cell-sorting equipglobal S-phase at the moment of pulse labeling or staining.
ment wasjinanced by the “Kom op tegen kanker” campaign (1989)
We have recently adapted some of the experimental conunder the auspices ofthe NFWO and by grants from the “Sportverditions to make the technique more suitable to analyze in
eniging tegen de Kanker”(l991) and the “United Fund ofBelgium”
greater detail growth factor-induced proliferative response
(1991).
of hematopoietic progenitors, and illustrated the power of
Address reprint requests to Dirk R. Van Bockstaele, PhD, Laboratory of Experimental Hematology, University of Antwerp (UIA/
the technique for analysis of hematopoietic cell-cycle kiUZA), Wilrijkstraat I O , B-2650 Edegem, Belgium.
netic~.’~-’~
MATERIALS AND METHODS
Bone Marrow (BM) Cells
BM samples were aspirated by sternal puncture, from hematologically normal patients undergoing cardiac surgery after informed
Blood, Vol 81, No 12 (June 15), 1993: pp3211-3216
The publication costs ofthis article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1993 by The American Society 0fHematolog.v.
0006-4971/93/8212-0024$3.00/0
321 1
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
LARDON ET AL
3212
Flow Cytometric Cell Sorting
Hoechst-Ethidium Bromide (EB) Staining Procedure
The CD34-labeled cells were sorted on a FACStaPLUScell sorter
(Becton Dickinson, Erembodegem, Belgium) equipped with an aircooled argon ion laser ILT model 5500A (Ion Laser Technology,
Salt Lake City, UT), tuned to 488 nm at 30 mW power. Sort windows were set to include cells with low side scatter (lymphocytes
and blastlike cells) and with positive green fluorescence (CD34+).
Side-scatter gating resulted in the omission of most mature myeloid
cells (that give a higher aspecific fluorescence background), thus
reducing the amount of aspecific fluorescing cells (false positives) in
the sorted fraction.
For bulk liquid and clonal semisolid cultures, normal R mode
was used and purities of greater than 95% were routinely obtained.
For single-cell liquid cultures, CD34+ cells were sorted in counter
mode at 1 cell/well using the Automatic Cell Deposition Unit
(ACDU; Becton Dickinson). Test sorts were performed before each
experiment using fluorescent microbeads (Polysciences, Wamngton, PA). On average, more than 97% of the wells contained only
one bead, less than 1% contained two beads, and 2% of the wells did
not contain any bead.
Fixed cells from the bulk cultures were washed twice in PBS and
resuspended in a solution of 0. I54 mol/L NaCI, 0. I mol/L Tris (pH
7.4) (P-L Biochemicals Inc, Milwaukee, WI), 0.5 mmol/L MgCI,,
0.2% bovine serum albumin (BSA) (Sigma), 0.1% Nonidet-P40
(Sigma), and 1.2 pg/mL Hoechst 33258 (Sigma). Thirty minutes
after staining with Hoechst dye, 1.5 pg/mL EB (Sigma) was added
for another 15 minutes, after which cells were ready for flow cytometric analysis.
Culture Procedures
Clonal semisolid agar cultures. After sorting, the cells were
washed twice in IMDM. Agar cultures were set up in triplicate in 1
mL containing 1,000 cells, 0.3% agar (Bacto-Agar; GIBCO), 20%
fetal calf serum (FCS), IMDM, and growth factors. These consisted
of G-CSF (specific activity IO' U/mg) and/or GM-CSF (specific
activity IO' U/mg) (both growth factors kindly donated by Dr S.C.
Clark, Genetics Institute, Cambridge, MA). These growth factors
were used at concentrations giving optimal colony formation in
preliminary experiments (ie, 50 ng/mL for G-CSF and 100 ng/mL
for GM-CSF). Cultures were kept at 37°C in 5% C 0 2and 5% 0, for
14 days, after which colonies were scored. Colonies were defined as
aggregates containing more than 40 cells.
Single-cell liquid cultures. CD34+ cells were cultured at 1 cell/
well in round-bottom 96-well plates (Falcon; Becton Dickinson).
Each well contained 50 p L culture medium consisting of IMDM,
20% FCS and either G-CSF, GM-CSF, or G-CSF + GM-CSF at the
above-mentioned concentrations.
After 14 days incubation at 37°C in 5% COz and 5% OZ, the
cultures were scored twice using different criteria for positivity of
the wells; either wells containing at least four cells, or wells containing more than 40 cells were considered positive.
Bulk (BrdU-supplemented) liquid progenitor cultures. After
sorting, the CD34+ cells were washed twice in IMDM and 1.5 X lo5
progenitor cells per well were incubated in IMDM with 20% FCS, 5
pmol/L BrdU (Sigma) and either G-CSF, GM-CSF, or G-CSF +
GM-CSF at the above-mentioned concentrations. In positive-control experiments, the medium was supplemented with 100 ng/mL
recombinant human stem cell factor (rhSCF; kindly donated by Dr
K.M. Zsebo, Amgen Biologicals, Thousand Oaks, CA), lOa U/mL
recombinant human interleukin-6 (rhIL-6) (specific activity 10' U/
mg; Boehringer Mannheim GmbH), 100 ng/mL rhIL-3 (specific
activity IO' U/mg; Genetics Institute) and G- and GM-CSF (at the
above-mentioned concentrations), thus providing optimal stimulatory conditions.
The low BrdU concentration and high cell concentration was
chosen to reduce any potential cytotoxicity while still retaining
enough Hoechst fluorescence quenching to be able to distinguish
the successivecell cycles.16After 72 hours incubation at 37°C in 5%
COz and 5% 02,cells were harvested, pelleted, and fixed in phosphate-buffered saline (PBS) with 30% (vol/vol) Et-OH and stored at
4°C.
Flow Cytometric EB/Quenched Hoechst Fluorescence
Analysis
The Hoechst-EB-stained cells were analyzed on the FACStaPLUS
(Becton Dickinson) flow cytometer using an air-cooled helium cadmium (He-Cd) laser model 3074-20 M/UV (MM) (Omnichrome,
Chino, CA), tuned to 325 nm at 30 mW power. Blue Hoechst fluorescence was collected through a 470 nm bandpass (BP) (bandwith
50 nm) filter. Red EB fluorescence was collected through a 630 nm
BP (bandwith 22 nm) filter. The relative proportions of cells in
successive cell compartments were calculated as described in the
Results section.
Statistics
Statistical analysis was performed using either Student's t-test for
paired samples or chi square statistical analysis. Results are expressed as mean (A = observed - expected) & SD.
RESULTS
The response of CD34+ cells from I O normal BM samples
to G-CSF and/or GM-CSF was evaluated using three different culture techniques that all give different information:
direct cell kinetic effects were evaluated using 72 hours
BrdU-supplemented bulk cultures, colony formation was
evaluated using classical 14 days semisolid agar cultures,
and colony-forming capacity versus proliferation was evaluated using 14-day single-cell liquid cultures.
Direct Effects of G-CSF/GM-CSF on the First Three
Consecutive Cell Cycles
The BrdU-Hoechst assay distinguishes cycling from noncycling cells and resolves different cell cycle phases throughout three successive cell cycles after activation.
Figure I shows a representative EB/BrdU-Hoechst bivariate flow-cytometric analysis derived from sorted CD34+
progenitor cells grown for 7 2 hours in the presence of 5
pmol/L BrdU. Either G-CSF (Fig 1A), GM-CSF (Fig 1 B), or
G-CSF GM-CSF (Fig IC) were added to the culture medium at culture setup and were present until harvest. Parallel cultures, lacking growth factors, were evaluated for background proliferation.
Region RI consists of quiescent (Gophase) cells. Cells in
region R2 have entered the Sand G2/M phase ofthe first cell
cycle. These cells incorporated BrdU as a thymidine analogue in their DNA, resulting in a quenched Hoechst fluorescence. In contrast, the dye EB does not exhibit BrdU-dependent quenching. Thus, there is a concomitant shift of
replicating cells to lower fluorescence intensities on the
Hoechst axis and higher fluorescence intensities on the EB
axis.
The Hoechst quenching effect during the second (region
+
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
KINETIC RESPONSE OF CD34’ CELLS TO G/GM-CSF
3213
Fig 1. Flow cytometric bivariate Hoechst 33258 versus EB fluorescence cytograms from CD34’ BM progenitor cells cultured in the
presence of G-CSF (A). GM-CSF (B), and G-CSF
GM-CSF (C). Cells in region R 1 represent quiescent Go-phase cells. Cells in region R 2
represent S- and G,M-phase cells of the first cell cycle. Cells in regions R3 and R 4 represent G;/S‘/G,M’-phase cells of the second and
G;’/S”/G,”M“-phase cellsof the third cell cycle, respectively. Further explanation on the analysis of this type of cytograms may be found in the
Results section or one of the other r e p ~ r t s . ’ ~ - ’ ~
+
GM-CSF (=40%) is significantly less (A = -15 f 8, P <
.0025 Student’s t-test/P < .001 chi square) than the predicted values, ie, the sum of percentages of proliferating cells
responding to G-CSF alone and GM-CSF alone (32%
23% = 55%).
The responding cells are divided into three compartments: slow responders in the S/G2M phases of the first cell
cycle, intermediate rate responders in the G,/S’/G2M
phases of the second cell cycle, and fast responders in the
G/S”/G;Mphases of the third cell cycle. After 72 hours,
the number of cells that have already entered the third cell
cycle after stimulation with the combination of G-CSF +
GM-CSF (=18%) is significantly higher (A = 5 f 3, P <
.0025 Student’s t-test/P < .001 chi square) than the predicted value, ie, the sum of the number of equivalent cells
after stimulation with either G-CSF alone or GM-CSF
alone (12% 1% = 13%).
For comparative purposes (positive control), a number of
cultures were evaluated for maximal stimulation using optimal growth conditions, ie, cultures supplemented with IL-3,
IL-6, SCF, G-CSF, and GM-CSF. Mean (normalized) fraction of proliferating cells under these conditions was 8 1.6%
k 9.8% (n = 5 ) , with 55% f 16% of the cells reaching the
third cell cycle.
R3) and the third (region R4) round of replication diminishes compared with that of the first round, because of reduced Hoechst quenching by bifilarily substituted chromatin (compared with unifilarily substituted
Both EB and Hoechst fluorescence increase during these
replication rounds, resulting in two parallel running signal
distributions that show up in a mirror image fashion compared with the first cycle distribution.
Table 1 summarizes the results of a series of 10 such 72hour G-CSF/GM-CSF stimulation experiments. The presented data (percent of cells in different compartments) are
normalized to reflect the relative proportions within the initial cell suspension (at the start of the culture), keeping in
mind that every 4 cells in region R4, every 2 cells in region
R3, and every 1 cell in region R2 originated from 1 cell in
the initial suspension. Background proliferation (cells growing without any growth factor) is substracted from each proliferation compartment. This background proliferation,
suggestive for the residual presence of growth factors in the
serum source, was always less than lo%, and its magnitude
depended mainly on the nature of the used serum source
(unpublished observations).
As can be seen from Table I , in the first three cell cycles
the percentage of proliferating cells responding to G-CSF
+
+
+
Table 1. Calculations of Successive Cell Cycle Compartment Sizes From CD34+ Progenitor Cells Cultured in the Presence of G-CSF,
GM-CSF. or G-CSF
GM-CSF for 7 2 Hours
+
Third Cell Cycle
CSF
% Viable
Cells
G-CSF
GM-CSF
G-CSF GM-CSF
86 + 7
81 ? 7
86 ? 7
+
Quiescent
% Proliferating
Cells
% Proliferating
Cells
Cells
First
Cell Cycle
(observed)
(predicted)
% Go
% S/G,M
32
23
-
5 6 + 13
-
67 + 8
40
55
48+10
A=-15+8
3+2
6+3
3+3
Second
Cell Cycle
% G;/S“/G;M
% G;/S’/G;M
(observed)
17i7
16+4
19k7
% G;/S”/G;M”
(predicted)
-
12
1
13
18
A=5?3
The numbers shown are t h e mean + SD (n = 10). calculated from EB/BrdU-Hoechstbivariate cytograms a s displayed in Fig. 1 and are normalized to
reflect the relative proportions within the initial cell suspension (see Results section). Background proliferation is substracted from each proliferation
compartment. Differences ( A ) between observed and expected values are statistically validated using either Student’s r-test for paired samples or chi
square analysis (see Results section).
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
LARDON ET AL
3214
Effects of G-CSF/GM-CSF on the Colony Formation in
Semisolid Agar Cultures
The effect of G-CSF and/or GM-CSF on colony numbers
after 14 days of incubation is illustrated in Fig 2. The mean
colony numbers per well for six separate experiments were
62,26, and 118 for G-CSF, GM-CSF, and G-CSF GMCSF, respectively. When used alone. G-CSF was a better
stimulator ofcolony formation than was GM-CSF. Simultaneous G-CSF and GM-CSF addition resulted in a significant synergistic increase (A = 30.0 k 15.7, P < .005 Student’s t-test/P < .OO 1 chi square) in colony numbers when
compared with the predicted value, ie, the sum of the numbers of colonies formed with each factor alone. This combination of G-CSF GM-CSF consistently stimulated growth
of large colonies (on average > 100 cells).
To make a comparison between proliferation (at least
four cells) and colony formation (more than 40 cells), single-cell suspension cultures were performed, in which wells
containing less than 40 cells could also be scored.
+
+
Direct Effects of G-CSF/GM-CSF in Single-cell
Suspension Cultures
CD34+ cells were sorted at one cell per well and proliferation (wells containing at least four cells) was compared with
160
-W 140
3 120
r
1
-
L
a,
a
v)
W
.-
100
-
c
-0 8 0 -
8
Lc
0
L
60
-
a,
Ll
E 403
c
20
n
”
I
G-CSF
GM-CSF
-
% 3F
+ GhI CSF
Fig 2. Colony formation of CD34’ BM progenitor cells in semiGMsolid agar cultures containing G-CSF, GM-CSF, or G-CSF
CSF. Actual colony formation is shown by open histograms (0),
while the hatched histogram (H) represents predicted values, ie,
the sum of colonies obtained with each factor alone. Triplicate
wells were assayed in each experiment and the numbers shown
are the mean colony number f SD (n = 6). Statistical analysis
was performed comparing differences between the actual colony
numbers obtained with G-CSF
GM-CSF and the predicted values. There was a significant synergistic increase (A = 30.0 2
15.7, P < ,005 Student’s r-test/P < .001 chi square) in colony
numbers obtained with G-CSF
GM-CSF.
+
+
+
colony formation (wells containing more than 40 cells) after
14 days of liquid culture. As can be seen from Fig 3A (colonies of more than 40 cells), the addition ofG-CSF/GM-CSF
gave qualitatively similar results as those obtained from colony scoring in semisolid agar assays. The mean colony numbers per plate (96 wells) for six separate experiments were
12,6, and 24 for G-CSF, GM-CSF, and G-CSF + GM-CSF,
respectively, showing a significant synergistic increase (A =
5.5 f 3.3, P < .005 for both Student’s t-test and chi square)
in colony numbers formed by the combined action of GCSF + GM-CSF, compared with the predicted value, ie, the
sum of colonies formed by the action of each factor alone.
However, if also wells containing less than 40 cells were
considered positive, only a slight increase of positive wells
can be seen compared with the number of positive wells
with G-CSF alone or with GM-CSF alone (Fig 3B). Here the
mean numbers of positive wells per plate for six separate
experiments were 2 1, 19, and 26 for G-CSF, GM-CSF, and
G-CSF
GM-CSF supplemented cultures, respectively.
Thus, addition of G-CSF + GM-CSF resulted in a significantly lower number of positive wells (A = - 13.5 f 8.3, P <
.005 Student’s t-test/P < .OO 1 chi square) compared with the
predicted value, ie, the sum of positive wells with G-CSF
alone and GM-CSF alone.
+
DISCUSSION
Synergy between G-CSF and GM-CSF has been documented repeatedly in semisolid assays of hematopoietic colony-forming ~ e l l s . ~The
, ~ experiments
.~
described in this report were undertaken to investigate the mechanism of
synergy of G-CSF + GM-CSF. We used the BrdU-Hoechst
quenching technique to unravel the full kinetic history of
CD34+ progenitor cells within the first 72 hours after stimulation with these CSFs.
The BrdU/Hoechst-EB bivariate patterns were always
compatible with the picture of initially synchronous responding populations starting from the G,,/G,-phase,
thereby suggesting that the G- and/or GM-CSF-responsive
targets are initially mainly in a nonproliferative state, somewhat in contradiction with previous investigation^.^ However, most of the initially proliferating (S-phase) CD34+
cells were probably excluded from the experiments by the
sorting procedure: only CD34+ cells with low side scatter
were withheld, whereas we observed that the minority of
S-phase CD34+ cells exhibit a larger side scatter (unpublished observations, using CD34-labeled, viable Hoechststained BM cells).
If one focuses attention to the number of responders (Table l/column “percent proliferating cells”), then it is obvious that within the first 72 hours of stimulation, both GCSF and GM-CSF stimulate growth, but the effects are not
synergistic in contrast to the effects observed in colony assays; the number of cells responding to G-CSF + GM-CSF
(40%) is significantly lower (A of - 15% f 8%)than the sum
of responders to either G-CSF alone or GM-CSF alone (together 55%). This confirms the data of Cook et all9 on an
elutriated fraction of BM progenitor murine cells and of
Strauss et a12’ on human CD34’ BM progenitor cells, where
initiation of DNA synthesis by recombinant CSFs was as-
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
3215
KINETIC RESPONSE OF CD34+ CELLS TO G/GM-CSF
sessed using [3H]-thimidine pulse labeling; G-CSF + GMCSF induced DNA synthesis greater than GM-CSF alone,
but synergy was not observed in any experiment.
In a first approximation one can consider the CD34+
compartment to be composed of cells that are potentially
responsive to either ( 1 ) G-CSF alone, (2) GM-CSF alone, (3)
both G-CSF and GM-CSF, or (4) nonresponsive to one of
these growth factors. From our data (Table 1) one can calculate the respective abundancies of these subsets: 40-23 =
17%, 40-32 = 8%, 15% (A), and 48% (quiescent cells) for
subsets (l), ( 2 ) , (3), and (4), respectively.
These early effects cannot be observed in semisolid agar
cultures because these cultures can only be scored after the
colony-forming cell underwent multiple cell divisions. How-
W
.c.
”[ A
d 25
a
G-CSF
GM-CSF
G-CSF
+ GM-CSF
401
ever, in single-cell suspension cultures, where at start of culture only one progenitor cell per well is present, one can also
detect proliferation below the (colony scoring) threshold of
40 cells. Therefore, these single-cell experiments were performed and cultures were scored twice, using different criteria. If only wells containing more than 40 cells (= colonies)
were scored, the results were parallel to agar culture results,
showing a synergistic effect of the combination of G-CSF
GM-CSF over the separate growth factors. Because each
well contained only one CD34+ progenitor cell, any effects
of G-CSF GM-CSF must be direct and not mediated by
accessory cells. If all proliferation-positive wells were scored
(ie, wells containing more than four cells), similar results
were obtained as in the BrdU-supplemented bulk cultures,
ie, a subadditive effect of G-CSF GM-CSF during the first
cell cycles.
If one focuses attention to the number of cells in the different cell cycles after 72 hours of stimulation, it is evident
that the combination of G-CSF and GM-CSF speeds up the
cell-cycle traverse rate for a significant number of target
cells, thereby increasing the number of cells that already
have entered the third cell cycle after 72 hours of CSF stimulation.
From our results (summarized in Table 1, “third cell cycle”) one can argue that the majority of the colonies that
appear will originate from (initially) G-CSF-responsive
progenitors or (initially) G- and GM-CSF-responsive progenitors in response to either G-CSF or the combined action
of both G- and GM-CSF. Only a minority of the colonies
will originate from (initially) GM-CSF-responsive targets
and the synergy of both growth factors (for colony formation) is mainly caused by an increased cycling rate of a fraction of (initially) G-CSF + GM-CSF-responsive target cells
that would, on single-factor stimulation, not reach the criteria to be scored as a colony (after 14 days). These double-responsive cells will, on double occupance of both receptor
sites, experience a more profound signal for proliferation
because ofcooperative interaction of the postreceptor signalling p a t h ~ a y s . ’ ~ ~ ~ , * ~ , * ~
+
+
+
s 35
a
(d
Fig 3. Proliferationof CD34+ B M progenitorcells in single liquid
cultures containing G-CSF, GM-CSF. or G-CSF
GM-CSF.
Ninety-six-well plates were scored twice using different criteria
for positivity of the wells; either wells containing at least four cells
(presented in [B]) or wells containing more than 40 cells ( = colonies, presented in [A]) were considered positive. The number of
positive wells is shown by open histograms (0).while hatched histograms (FA) present predicted values, ie, the sum of positive wells
obtained with each factor alone. The numbers shown are the mean
number of positive wells 2 SD (n = 6). Statistical analysis was
performed comparing differences between the numbers of positive
GM-CSF and the predicted values,
wells obtained with G-CSF
ie, the sum of the numbers of positive wells obtained with G-CSF
alone and GM-CSF alone. In (A), there is a significant (P < .005
Student’s t-testlchi square) synergistic increase (A = 5.5 -C 3.3)
in positive wells with G-CSF
GM-CSF over the sum of positive
wells with each factor alone. In (B), a significantly lower number of
positive wells (A =
13.5 2 8.3, P < .005 Student‘s t-testJP <
.001 chi square) is seen with G-CSF
GM-CSF than the sum of
positive wells with G-CSF alone and GM-CSF alone.
+
+
+
G-CSF
GM-CSF
G-CSF
+ GM-CSF
-
+
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
LARDON ET AL
3216
ACKNOWLEDGMENT
We thank Dr Jean-Claude Van der Auwera (Social Medicine,
UIA) for advice on the statistical analysis. The excellent technical
assistance of Marc Lenjou and Griet Nijs is gratefully acknowledged, as is the secretarial assistance of Monique Hoste. We also
want to express our gratitude to the cardiac surgery team (Prof
Walter, et al) of the University Hospital Antwerp for the regular
supply of normal BM samples.
REFERENCES
I. Metcalf D: The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature
339:27, 1989
2. Clark SC, Kamen R: The human hematopoietic colony-stimulating factors. Science 236: 1229, 1987
3. SiefCA: Hematopoietic growth factors. J Clin Invest 79: 1549,
1987
4. Moore MAS: In vivo and in vitro action of hematopoietic
colony stimulating factors. Proc Am Assoc Cancer Res 28:466,
1987
5. Hara H, Namiki M: Mechanism of synergy between granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in colony formation from human marrow
cells in vitro. Exp Hematol 17916, 1989
6. McNiece I, Andrews R, Stewart M, Clark S, Boone T, Quesenberry P: Action of interleukin-3, G-CSF and GM-CSF on highly
enriched human hematopoietic progenitor cells: Synergistic interaction of GM-CSF plus G-CSF. Blood 74: 110, 1989
7. Ikebuchi K, Clark SC, lhle JN, Souza LM, Ogawa M: Granulocyte colony-stimulating factor enhances interleukin 3-dependent
proliferation of multipotential hemopoietic progenitors. Proc Natl
Acad Sci USA 85:3445, 1988
8. Coutinho LH, Will A, Radford J, Schiro R, Testa NG, Dexter
TM: Effects of recombinant human granulocyte colony-stimulating factor (CSF), human granulocyte macrophage-CSF, and gibbon
interleukin-3 on hematopoiesis in human long-term bone marrow
culture. Blood 75:2118, 1990
9. Hogge DE, Cashman JD, Humphries RK, Eaves CJ: Differential and synergistic effects of human granulocyte-macrophage colony-stimulating factor and human granulocyte colony-stimulating
factor on hematopoiesis in human long-term marrow cultures.
Blood 77:493, I99 1
10. Rabinovitch PS, Kubbies M, Chen YC, Schindler D, Hoehn
H: BrdU-Hoechst flow cytometry: A unique tool for quantitative
cell cycle analysis. Exp Cell Res 174:309, 1988
I 1. Kubbies M, Hoehn H, Schindler D, Chen YC, Rabinovitch
PS: Cell cycle analysis via BrdU-Hoechst flow cytometry-principles and applications, in Yen A (eds): Flow Cytometry: Advanced
Research and Clinical Applications. vol II. Boca Raton, F’L, CRC,
1989, p 5
12. Poot M, Hoehn H, Kubbies M, Grossman A, Chen Y, Rabinovitch PS:Cell cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33258-Ethidium Bromide bivariate flow
cytometry, in Darzynkiewicz Z, Crissman HA (eds): Methods in
Cell Biology, vol 33; Flow Cytometry. San Diego, CA, Academic,
1990, p 185
13. Ormerod MG, Kubbies M: Cell cycle analysis ofasynchronous cell populations by flow cytometry using bromodeoxyuridine
label and Hoechst-Propidium Iodide stain. Cytometry 13:678,
1992
14. Van Bockstaele DR, Lardon F, Snoeck H-W, Peetermans
ME: BrdU-Hoechst flow cytometry using a 20 mW He-Cd laser
light source: II/Evaluation of growth factor induced bone marrow
progenitor (CD34+) proliferation. Cytometry Suppl 554, 1991
(abstr)
15. Lardon F, Van Bockstaele DR, Snoeck H-W, Peetermans
ME: Growth factor induced proliferation of hematopoietic progenitor (CD34+) cells from adult bone marrow and cord blood: Evaluation of the first three consecutive cell cycles. J Cell Biochem Suppl
16C:78, 1992 (abstr)
16. Van Bockstaele DR, Lardon F, Snoeck H-W, Peetermans
ME, Kubbies M: BrdU-Hoechst-Ethidium Bromide (EB) quenching technique for studying kinetics of hematopoiesis (letter). Blood
80:289, 1992
17. Kubbies M, Goller B, Van Bockstaele DR: Improved
BrdUrd-Hoechst bivariate cell kinetic analysis by Helium-Cadmium single laser exitation. Cytometry 13:782, I992
18. Buhring HJ, Ullrich A, Schaudt K, Muller CA, Busch W:
The product ofthe proto-oncogene c-kit (P145””‘) is a human bone
marrow surface antigen of hemopoietic precursor cells which is expressed on a subset of acute non-lymphoblastic leukemic cells. Leukemia 5:854, I99 I
19. Cook N, Dexter TM, Lord B1, Cragoe EJ Jr, Whetton AD:
Identification of a common signal associated with cellular proliferation stimulated by four haemopoietic growth factors in a highly
enriched population of granulocyte/macrophage colony-forming
cells. EMBO J 8:2967, 1989
20. Strauss LC, Welsh SB, Civin CI: Initiation in DNA synthesis
by colony-stimulating factors in subsets of human CD34+ marrow
cells. Exp Hematol 19:734, I99 1
2 1. Demetri GD, Griffin JD: Granulocyte colony-stimulating
factor and its receptor. Blood 78:279 1, 199 1
22. Vairo G, Hamilton JA: Signalling through CSF receptors.
Immunol Today 12:362, I99 1
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1993 81: 3211-3216
Quantitative cell-cycle progression analysis of the first three
successive cell cycles of granulocyte colony-stimulating factor and/or
granulocyte-macrophage colony-stimulating factor-stimulated human
CD34+ bone marrow cells in relation to their colony formation
F Lardon, DR Van Bockstaele, HW Snoeck and ME Peetermans
Updated information and services can be found at:
http://www.bloodjournal.org/content/81/12/3211.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.