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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2041-2052
Apoptotic Regulation in Primitive Hematopoietic Precursors
By
Rowayda Peters,
Serge Leyvraz, and
Lucien Perey
From the Centre Pluridisciplinaire d'Oncologie, University Hospital
(CHUV), Lausanne, Switzerland.
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ABSTRACT |
Bcl-2 and bcl-xL function as suppressors of programmed
cell death. The expression of bcl-2 protein in vivo is associated with long-lived hematopoietic cells such as mature lymphocytes and early
myeloid progenitors. Bcl-xL, a homologue of bcl-2, is also expressed in lymphocytes and thymocytes. In contrast, the bcl-2-related proteins (bax, bad, and bak) act by promoting apoptotic cell death as
shown from their expression in hematopoietic cell lines. We analyzed
the expression of bcl-2 and bcl-x proteins in hematopoietic precursors
obtained from various cell sources in adult mobilized peripheral blood
collected from 13 patients with solid tumors, 8 adult bone marrow, and
12 umbilical cord blood. The analysis was based on the expression of
the proliferation and activation specific antigens, CD38 and class II
(HLA-DR). Similarly, we analyzed the expression of bcl-2-related
proteins bcl-xL, bax, bad, and bak before and during
ex-vivo expansion. Hematopoietic precursors expressing strongly the
CD34 antigen (CD34s+) and lacking CD38 or HLA-DR
expression were analyzed by using three-color immunofluorescence
staining. The majority of CD34+ cells expressed bcl-2 and
unexpectedly showed a bimodal distribution of low and high expression.
More cells that lacked or expressed low density CD38 expressed low
bcl-2 than the more differentiated counterparts (those with high
density CD38). Immaturity (ie, little or no HLA-DR) is associated with
the expression of low bcl-2 compared with HLA-DR+.
However, HLA-DR /low population contained a lower number
of cells expressing low bcl-2 (30% to 40%) than
CD38/low in comparable samples. The hematopoietic
precursors with bcl-2low and bcl-2high formed a
homogeneous population of undifferentiated lymphoid-like cells having a
similar forward scatter. These cells expressed strongly the
bcl-xL protein (>95%) but were bax low (4% to 12%), bad low (0% to 0.8%), and bak low (0% to 3%). The expression of apoptosis specific protein (ASP) was also low (3.4% ± 3.1%) as was
Annexin V. In addition, the CD34+/CD38
showed low cell cycle activity (<2.2%). Induction of apoptosis by
overnight incubation of CD34 cells in serum-deprived medium resulted in
the upregulation of bcl-2 as a single population histogram. Thus, these
results suggest that in quiescent hematopoietic precursors, the bcl-2
protein plays a less prominent role as a survival promoter than
bcl-xL and that the low bcl-2 expression did not promote apoptosis. During day 10 of ex vivo expansion of CD34+
cells in liquid culture containing stem cell factor, interleukin-3 (IL-3), IL-6, IL-1 , and erythropoietin, the
CD34+/CD38 cells expressed high bcl-2 as a
single population histogram, and greater than 90% were
bcl-xL high. However, the expression of pro- and apoptotic
antigens increased: bax (10% to 15%), bad (5% to 8%), bak (6% to
14%), and ASP (6% to 10%). These results show the importance of
monitoring the expression of these proteins when defining the culture
conditions for ex vivo expansion.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE REGULATION of cell survival plays an
important role in embryogenesis, development, and regulation of the
immune system as well as maintenance of adult tissue
homeostasis.1 The hematopoietic stem cell is defined by its
ability to restore hematopoiesis after marrow ablation. Unlike the
differentiated hematopoietic cells, this cell must be long-lived and
possess the capability of self-renewal throughout the lifetime of its
host.2-5
Apoptosis, the active physiological form of programmed cell death
(PCD), is the normal fate for terminally differentiated hematopoietic
cells resulting in their finite and characteristic life span. The
apoptotic process can be induced by a variety of stimuli leading to the
activation of a specific series of metabolic and morphological changes
and by the activation of endogenous endonucleases that ultimately
produce the typical DNA fragmentation at the internucleosomal
level.1,6,7 There is mounting evidence that the apoptotic
death process can be controlled by endogenous factors such as
proto-oncogenes, eg, c-myc, and p53,8 and by exogenous
factors such as cytokines and other growth factors, eg, interleukin-3
(IL-3), IL-10, granulocyte-macrophage colony-stimulating factor, stem
cell factor (SCF), and erythropoietin (EPO),9-13 cytotoxic
drugs,14 and x-ray irradiation.15
To date, the bcl-2 is known to belong to a growing family of
apoptosis-regulatory gene products, which may serve either as death
antagonists (Bcl-2, Bcl-xL, Bcl-w, Bfl-1, Brag-1, Mcl-1, and A1) or death agonists (Bax, Bak, Bad, Bid, Bik, Hrk, and the alternatively spliced bcl-xs).16,17 These
proteins share some sequence homology with bcl-2 but differ in their
tissue and activation dependent expression pattern as well as in their
structural features.17 Bcl-2 and bcl-xL
dimerize with several members of bcl-2 family of proteins thereby
changing the ratio of these members and altering the threshold of cell
death.18-21
Both bcl-2 and bcl-x function as suppressors of PCD upon growth factor
withdrawal in cytokine-dependent hematopoietic cell lines.22-25 The expression of bcl-2 protein in vivo is
associated with long-lived cells in many lineages such as mature
lymphocytes and neurons.16 The pattern of bcl-2 expression
and its functional importance during lymphopoiesis and during
differentiation of early myeloid progenitors have been well
studied.26-29
Although bcl-2 protein expression has been well studied in early and
more mature hematopoietic cells, comparatively little data exist on its
expression in undifferentiated precursors.30-32 As yet, no
data are available with regard to peripheral blood and, in particular,
in cytokine mobilized precursors. In common with bcl-2, bcl-x protein
expression has been shown in undifferentiated bone marrow (BM)
precursors as well as lymphocytes and thymocytes.16,31 Similarly, the expression of bax, bad, and bak proteins has been shown
in hematopoietic cell lines on growth factor
withdrawal.16,19,20,33
To provide a better insight into the maintenance of hematopoietic
precursor cells reflected by their long-term survival and during their
cellular activation and differentiation, we have evaluated the bcl-2
expression from three cell sources, in granulocyte colony-stimulating
factor (G-CSF) mobilized peripheral blood (MPB), umbilical cord blood
(CB), and adult BM. Immunophenotypically, these precursors were
previously identified as strongly CD34+,34
lacking the expression of lineage specific antigens,35,36 and CD38 or human leukocyte antigen D-related (HLA-DR).34
Therefore, we analyzed bcl-2 expression in CD34+ cells
based on the intensity of CD38 antigen expression or
HLA-DR. We performed similar analyses to evaluate the
expression of bcl-x and bcl-xL. We investigated the
expression of bax, bad, bak, and apoptosis-specific proteins (ASP and
Annexin V). We also studied the cell cycle status of purified
CD34+/CD38 and compared them with
CD34+/CD38+ in MPB and CB. We used a short-term
incubation program to test the susceptibility of these precursors to
apoptosis after 1 hour, 2 hours, and 24 hours incubation in serum- and
growth factor-deprived medium. Finally, we expanded the
CD34+ cells in liquid culture containing SCF, IL-3, IL-6,
IL-1, and EPO37 and examined the expression of the bcl-2
family members (bcl-2, bcl-xL, bax, bad, and bak) in
primitive hematopoietic precursors.
We show that the undifferentiated precursors express bcl-2. Most
interestingly, the bcl-2 expression shows a bimodal distribution (low
and high). These precursors express high density bcl-xL. We
also show that hematopoietic precursors have low apoptotic activity
(bad 0% to 0.8%, bak 0% to 3%, bax 4% to 12%, and ASP 3% to
7%), low DNA synthesizing activity (0.5% to 2.2%), and under deprived conditions upregulated bcl-2. Similarly, the upregulation of
bcl-2 also occurred during ex vivo expansion and was accompanied by an
increase in the expression of bad (5% to 8%), bak (6% to 14%), bax
(10% to 15%), and ASP (6% to 10%).
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MATERIALS AND METHODS |
Patients, mobilization procedure, and controls.
A total of 26 MPB samples were collected from 13 patients with solid
tumors aged 39 to 58 years (median, 46 years). These were 5 with breast
cancer, 7 with small-cell lung cancer, and 1 with germ-cell cancer.
Hematopoietic progenitor cells were mobilized by high-dose chemotherapy
(epirubicine 150 mg/m2) (Farmorubicine; Pharmacia, Milan,
Italy) followed by 5 µg/kg subcutaneous G-CSF daily, for 10 days
(Filgrastim; Roche, Basel, Switzerland).
BM samples were taken from 8 normal donors aged 38 to 63 years (median,
50 years). CB samples were obtained from 12 full-term deliveries. All
patients and controls gave informed consent and the protocols were
approved by the ethical committee of the Medical Faculty, University of
Lausanne, Switzerland. Mononuclear cells (MNCs) were collected after
centrifugation on a density gradient (Lymphoprep; Nycomed, Oslo,
Norway) and washed twice in phosphate-buffered saline (PBS). Residual
erythrocytes were depleted by lysis with isomolar ammonium chloride
buffer for 10 minutes at room temperature (RT) and were subsequently
washed in PBS. The cell line KG-1A, a CD34+ tumor cell line
derived from a human acute myelogenous leukemia was obtained from the
American Type Culture Collection (Rockville, MD).
Determination of membrane and cytoplasmic antigen expression by flow
cytometry.
Three-color immunofluorescence (IF) staining was performed first by
labeling membrane antigens with 2-color IF (PE plus streptavidin tri-color) and intracellular antigens with fluorescein isothiocyanate (FITC). Cell suspensions (0.5-1 × 106) were incubated
at RT with anti-CD34 conjugated to biotin (QBEND 10; Inotech, Dottikon,
Switzerland). After incubation and washing, a mixture of anti-CD38
phycoerythrin (PE; Leu-17; Becton-Dickinson Immunocytometry Systems
[BDIS], San Jose, CA) or anti-HLA-DR-PE (class II; BDIS) and
tri-color conjugated streptavidin (Caltag Laboratories Inc, Burlingame,
CA) were added and cells were incubated and washed. Fixation and
permeabilization of the prelabeled cells were performed as described by
Pizzolo et al38 and Francis et al39 by using a
commercially available reagent, Ortho Permeafix (OPF; Ortho Diagnostic
Systems, Inc, Raritan, NJ). Of this fixative, 1 mL diluted in a 1:1
ratio with distilled water was incubated for 40 minutes at RT and cells
were washed. These permeabilized cells were then stained for
intracellular antigens.
For direct staining, cells were stained with anti-bcl-2 conjugated to
FITC (Dako Diagnostic AG, Zug, Switzerland) for 30 minutes at RT and
washed. For indirect staining with anti-bcl-xL, anti-bcl-x, anti-bad (Transduction Laboratories, Lexington, KY), anti-bak, anti-bax
(Santa Cruz Biotechnology, Inc, Basel, Switzerland), and anti-ASP
(c-jun/AP-1 [Ab-2] Oncogene Science Inc, Uniondale, NY), the cells
were first incubated with the primary antibody for 30 minutes at RT
followed by washing and further incubation for 30 minutes with
species-specific second layer antisera conjugated to FITC. Purified
goat antimouse Ig-FITC (GAM IgG-FITC; Southern Biotechnology
Associates, Birmingham, AL) was used for bcl-xL and bad;
affinity-purified goat antirabbit IgG (F(ab )2-FITC (Ready-System AG,
Zürich, Switzerland) for bcl-x, bax, and ASP and affinity purified donkey antigoat IgG-FITC was used for bak (Santa Cruz Biotechnology Inc, Basel, Switzerland). We also used Annexin V-FITC (Nexins Research BV, Maastricht, The Netherlands) in a 3-color IF
staining of surface antigens with CD34-Tri-color and CD38-PE. The KG-1A
cell line was analyzed for bcl-2 expression. Isotype-matched, irrelevant antibodies served as controls.
Flow cytometric analysis was performed on the FACScan equipped with an
argon laser tuned to 488 nm. Data acquisition was performed with lysis
II software (BDIS). A total of 50,000 to 500,000 events were acquired
in listmode data file depending on the CD34 count. Analysis of
intracellular antigen expression was strictly performed on high
positive CD34 cells based on the intensity level of CD38 antigen. High
positive (CD383+), intermediate positive
(CD382+), and low positive or negative
(CD38/low) populations were identified after (1) gating
the lymphoid population on the basis of their forward light scatter
(FSC)/side light scatter (SSC) features, followed by (2) identifying
the CD34+ population by their SSC and CD34 antigen
expression, and finally (3) by combining both the expression of CD34
and CD38 antigens.
To define the regions for CD38 negative from CD38 positive
hematopoietic cells, we used the biological control to set the level
for CD38 antigen expression by staining peripheral blood cells with
CD45RO-FITC and CD38-PE. Mature resting T cells are CD45RO+
and CD38 low or negative.40
The logarithmic scale for CD34+/CD38/low
region was higher by 20% to 25% than the maximum PE fluorescence of
the isotype control (R3 in Fig 1). R4 is
used for gating CD34 cells with moderate expression of CD38 antigen
(CD382+) and R5 for cells with high intensity of CD38
(CD383+; Fig 1). Once determined, the selected gates were
used identically for analyzing all samples. These were constant, and
were stored and recalled systematically for sample analysis. We used a
similar pattern of gating to divide the CD34 strong positive cells into negative/low, intermediate, and high positive by using HLA-DR intensity
of expression. Further analysis was performed on the total CD34 cell
population expressing the antigen weakly (w) and strongly (s) by gating
on the lymphoid cell population and dividing the CD34 cells by forward
light scatter properties into small, intermediate, and large. Quadrants
and histograms were defined by using isotype controls.
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| Fig 1.
FACS analysis of MPB mononuclear cells. (A) Forward
scatter (size) and side scatter (density) of MPB MNCs showing region R1
in which CD34+ cells are located. (B) Scattogram of
CD34-tri-color (Fl 3) versus side scatter (SSC) showing only
CD34+ cells in R2 that were also gated in R1. (C and D)
CD34-tri-color (Fl 3) and CD38-PE (Fl 2) expression from R2 showing the
gating of CD34+ cells based on CD38 antigen density level
(R3, R4, and R5). Region R3 used to define
CD34+/CD38/low cells matching the
biological control (see Materials and Methods). Region R3 include
CD34+ cells with PE-CD38 fluorescence more by 20% to
25% than the maximum PE fluorescence of irrelevant isotype-control
(log 101). Region R4 defines
CD34+/CD382+ and region R5 defines
CD34+/CD383+ cells. Regions R3, R4, and R5
were stored and constantly used for all samples to analyze the third
antigen and for cell sorting experiments. (C) The gating of only CD34
strong positive cells are shown, where greater than 98% of
CD38/low cells are located in region R3. (D) The gating
of CD34 weak positive cells are shown, in which region R3 lack the
CD38 cells.
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Cell sorting.
The cells from the three gates (CD38/low,
CD382+, and CD383+) were sorted separately by
using the FACSvantage (BDIS) and were examined for morphology. In these
sorting experiments, 2 to 4 × 106 MNCs were
stained with anti-CD34 and anti-CD38 (see above). The CD34+
cells were gated according to their scatter features, then by using the
same gating system as above, only the CD34s+ population was
isolated expressing different intensity of CD38 antigen.
Cytospins were prepared and stained with Wright Giemsa's stain and
visualized by light microscopy. Photographs were taken at ×1,000
magnification.
Cell selection.
The CD34+ cells contained in the MPB and CB were positively
selected by using the miniMACS CD34 isolation kit (Miltenvi Biotect, Bergisch Gladbach, Germany). The separation technique was performed according to the manufacturer's instructions. Ninety-five percent of
the cells identified by flow cytometry were
CD34+/CD38+.
For the separation of CD34+/CD38 cells, we
enriched for immature precursors by one round of immunomagnetic
depletion of more mature CD34+ cells. Briefly,
CD34+ cells (see above) selected from MNCs (1 × 108/mL) were incubated with stem cell
enrichment antibody cocktail containing glycophorin-A, CD3, CD2, CD56,
CD24, CD19, CD14, CD16, CD66b, CD36, CD45RA, and CD38 (Stem Cell
Technologies Inc, Vancouver, BC, Canada) for 30 minutes at 4°C
followed by 30 minutes incubation with magnetic beads. The magnetic
beads together with bound cells were then retained by using a strong
magnet (eg, VarioMACS). The remaining cells were washed and identified
by flow cytometry as CD34+/CD38.
Cell cycle analysis.
To analyze the cell cycle status of CD34+ subsets, MNCs
from MPB and CB were initially selected and phenotyped as
CD34+/CD38+ and
CD34+/CD38 as described above. The cells
were first stained for surface CD34 (CD34-FITC; BDIS) followed by 1 mL
of a hypotonic cocktail of propidium iodide (50 µg/mL PI plus 0.1%
Triton X-100, Sigma, Basel, Switzerland) and 0.1% sodium citrate at a
cell concentration of 0.5 to 1 × 106/mL and incubated
at 4°C until analyzed. Cell cycle analysis was performed by using a
FACScan equipped with the Cell Fit or Lysis II software. Twenty
thousand events were collected. The regions defining G0/G1 phase, S,
and G2+M phase were set by using total MNCs as an internal control.
Chicken erythrocytes nuclei (CEN DNA QC particle kit, BDIS) were used
for assessing the optical performance of the FACScan. DNA from KG-1A
cell line was also stained with CD34-FITC and PI and used as positive
control.
Short-term incubation.
We performed a short-term incubation in medium deprived of serum and
growth factors to induce apoptosis. MNCs from MPB and CB were incubated
in RPMI 1640 at a cellular density of 106/mL (Seromed,
Fakola, Basel, Switzerland). After 1 hour, 2 hours, and overnight
incubation, the expression of bcl- 2, ASP, and DNA cell cycle was
analyzed. Cell count and cell viability were determined before and
after incubation. The cells were buffered in a humidified atmosphere of
5% CO2 in air at 37°C for 30 minutes by using Falcon tubes (BDIS). The tubes were sealed and incubated at RT. Three-color IF
staining was performed as described above, the cells were labeled for
membrane CD34-Tri-color and CD38-PE before fixation and
permeabilization, and then stained for intracellular antigens
(bcl-2-FITC and ASP-FITC). PI was used for the staining of DNA after
24-hour incubation. Cells were analyzed on the FACScan with Lysis II
and Cell Fit software.
Ex vivo expansion.
CD34+ cells selected from four MPBs used at a cellular
density (3 × 104/mL) were grown as described
previously.37 With exceptions, the autologous plasma was
replaced by human AB plasma and 100 ng/mL of SCF was used
instead of 10 ng/mL (Amgen Inc, Thousand Oaks, CA). All other growth
factors were used as described, and IL-6 (Serono, Geneva, Switzerland),
IL-3 (Sandoz Pharma Ltd, Basel, Switzerland), IL-1, and EPO were
purchased from R&D Systems Inc (Oxon, UK).
Cultures were incubated at 37°C in a humidified atmosphere
containing 5% CO2 and were demidepopulated weekly for 3 weeks by removal of one half of the culture volume, which was replaced with fresh medium and growth factors.
On day 10, nonadherent cells were removed and were assessed for
cellular enrichment, differentiation, and apoptotic activity by flow
cytometry using three-color IF. The cells were labeled for membrane
CD34-Tri-color and CD38-PE in combination with (1) an antigen specific
for the bcl-2 family of proteins such as bcl-2, bcl-xL,
bax, bad, and bak and (2) the ASP antigen (see above).
Evaluation of CD34+ cells by flow cytometry was performed
on day 0 and day 10 of culture. Isotype-matched irrelevant antibodies served as controls.
Peptide neutralization.
The specificity of the pro-apoptotic antibodies (bax, bad and bak) was
tested by flow cytometry on KG-1A cell line before and after the
induction of apoptosis. The growing KG-1A cells (>98% viable) were
divided into two parts. Cells (0.5-1 × 106) were
washed twice and one part was incubated in Iscove's modified Dulbecco's medium (IMDM) containing 20% fetal bovine serum and 2 mmol/L L-Glutamine. Apoptosis in the second part was induced by serum
deprivation. The cells were incubated in IMDM and L-Glutamine alone.
Cells were incubated for 24 hours at 37°C in a humidified atmosphere of 5% CO2. Cell viability was assessed by
trypan blue staining and analysis by flow cytometry. For FACScan
analysis of bax, bad, and bak proteins, cells were fixed and
permeabilized for 40 minutes with Ortho Permeafix, stained for 30 minutes with the primary antibody and 30 minutes with the secondary
antibody as described above.
For neutralization, the anti-bak antibody (1:200 dilution) was
incubated overnight at 4°C with a 20- to 80-fold excess specific blocking peptide antigen in PBS (bak; N-20; Santa Cruz Biotechnology Inc). KG-1A cells induced for apoptosis were first permeabilized and
then incubated in the peptide/antibody mixture for 30 minutes followed
by incubation with the secondary antibody. Cells were then analyzed by
flow cytometry.
Statistics.
For analysis, the simple regression and the paired t-test with
two tailed P values were used.
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RESULTS |
Bcl-2 expression in primitive hematopoietic cells.
To assess the expression pattern of bcl-2 in hematopoietic cells with
high-density CD34 antigen (CD34s+), in particular in those
lacking CD38 antigen, we identified three populations based on the
differential expression of CD38 (CD38/low,
CD382+, and CD383+).
CD34+/CD38/low cells were defined as those
CD34+ cells with PE-CD38 fluorescence similar to
CD45RO+/ CD38/low T cells (region R3 in
Fig 1C). Using this stringent definition, a consistent frequency of
CD34+/CD38/low cells was found in MPB, CB,
and BM. The frequency was 7.1% ± 3.06% in MPB, 10% ± 4.77% in CB, and 5.74% ± 5.62% in BM.
In the three populations (CD38/low, CD382+,
and CD383+) from MPB, CB, and BM, greater than 95% of
cells showed bcl-2 staining higher than the highest intensity observed
with isotype-matched negative control antibody and higher than the
level of bcl-2 staining in the granulocytes obtained from four whole,
fresh MPB samples. Interestingly, the bcl-2 expression in these cells
yielded a bimodal histogram corresponding to two populations (low and
high; Fig 2). The mean fluorescent
intensity (MFI) in cells with low bcl-2 and cells with high bcl-2
expression between CD38/low and CD383+ is
shown in Table 1. The histograms for low
bcl-2 and high bcl-2 were reproducibly distributed within a range of 52 to 164 immunofluorescent channel and 153 to 464, respectively. The
ratio for the cells with low bcl-2 to the cells with high bcl-2 in
CD38/low (R3) was high and decreased significantly as
the intensity of CD38 antigen increased
(Table 2). The ratio was the highest in MPB
(1.2 ± 0.85) and the lowest in BM (0.67 ± 0.5). Forward light scatter (FSC) characteristics of cells with bcl-2low and
bcl-2high in the CD38/low from 22 MPB
samples showed no statistical differences in size (MFI-FSC for
bcl-2low 106 ± 12.6 and bcl-2high 111 ± 12.3, P < .289). These results were confirmed by light microscopy of CD38/low sorted cells from six
experiments. The cells showed a homogeneous population with regard to
size and an undifferentiated lymphoid-like appearance with regard to
morphology (Fig 3A).
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| Fig 2.
Bimodal distribution of bcl-2 protein expression
(bcl-2low and bcl-2high) in primitive
hematopoietic precursors and their progeny. Mononuclear cells (MNCs)
were stained with membrane antigens (CD34-tri-color and CD38-PE), then
permeabilized with Ortho Permeafix and stained for anti-bcl-2 FITC.
Cells were analyzed according to CD38 intensity level,
CD38/low (R3), CD382+ (R4), and
CD383+ (R5) by using only the strong CD34 positive cells
(Fig 1C). These selected gates were identically used for analyzing all
samples. MNCs were also permeabilized and stained with mIgG to define
the threshold between bcl-2-positive and bcl-2-negative cells.
Similarly, the granulocytes from whole, fresh MPB samples were also
analyzed. The bcl-2 gates (bcl-2low and
bcl-2high) were decided by the histograms distribution. (A)
The bimodal distribution of bcl-2 in MPB, CB, and BM. (B) The
comparison in bcl-2 fluorescent intensity between the mature
granulocytes and the hematopoietic precursors
(CD34s+/CD38/low) seen in Fig 2A.
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Table 1.
Mean Fluorescent Intensity of Bcl-2 Bimodal Distribution
(bcl-2low and bcl-2high) Based on CD38 Antigen
Expression Using CD34 Strong Positive Cells Obtained From MPB, CB, and
BM
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Table 2.
The Ratio of Bcl-2low and
Bcl-2high Calculated for CD34+ Hematopoietic
Precursors (CD38/low and HLA-DR/low) and
Compared With the Ratio for More Mature Cells (CD383+ and
HLA-DR3+) in MPB, CB, and BM
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Similarly, a bimodal distribution of bcl-2 expression was obtained when
high density CD34+ cells were gated according to HLA-DR
intensity level. The ratio in the cells with low bcl-2 to the cells
with high bcl-2 in MPB, CB, and BM was high in
HLA-DR/low cells and decreased significantly as the
intensity of HLA-DR antigen increased (Table 2). However,
HLA-DR/low cells had 30% to 40% less
bcl-2low expression than their counterparts
CD38/low in parallel samples (12 MPB, 6 CB, and 3 BM).
Only one histogram with high intensity of bcl-2 expression was seen in
the cell line (KG-1A).
Because we obtained two populations of bcl-2 within the
CD34s+ cells, we were interested in examining the
morphology of these cells. We went on to sort CD34s+ cells
from the three gates (CD38/low, CD382+, and
CD383+). Morphological examination of cytospins prepared
from R3, R4, and R5 showed progressively larger cells with distinct
morphologies (Fig 3). Among the
CD34+/CD38/low cells, 87% to 91% were
lymphoid-like with scanty cytoplasm, large homogeneous nuclei, and rare
nucleoli (Fig 3A). The contaminating cells were mainly lymphocytes (3%
to 5%) and mature myeloid cells (6% to 8%). CD382+ cells
were lymphoblast-like with more cytoplasm, large nuclei, and one or
more nucleoli (Fig 3B). CD383+ cells were myeloblast-like
with large amounts of cytoplasm containing granules, large granular
nuclei, and many nucleoli (Fig 3C).
The relative size of the three cell populations
(CD38/low, CD382+, and CD383+)
were confirmed by light scatter. The average sizes for the three populations as determined by FSC significantly showed the progressive increase in cell size with increasing CD38 intensity of expression, eg,
in MPB the MFI increased from 105.28 ± 18 in
CD38/low to 129.94 ± 8.09 in CD383+
(P < .0001). However, the cells showed low levels of
granularity in all of the three populations. Similarly a significant
increase in cell size was shown with increasing HLA-DR expression, eg, in MPB the MFI increased from 116.69 ± 10.2 to 131.62 ± 7.9, (P < .0001) in HLA-DR/low and
HLA-DR3+, respectively. A more homogeneous population of
small cells with primitive morphology was found within the
CD38/low sorted cells as compared with those of
HLA-DR/low. The results suggest the presence of two
subpopulations of hematopoietic precursors, CD38 and
HLA-DR, that vary in bcl-2 expression and in cell size.
Bcl-2 expression in CD34+ cells.
We then analyzed the bcl-2 expression by using the total CD34
population with weak and strong expression of the antigen. The average
frequency of cells expressed in the CD34 antigen increased with
increasing cell size: only 7.02% ± 4.06% were small, 26.9% ± 11.60% were intermediate, and 66.0% ± 12.90% were large cells in
MPB. The CD34s+ cells formed 45% to 55% of the total
CD34+ population. A bimodal expression of bcl-2 was
obtained in CD34s+ cells similar to those gated according
to CD38 intensity level. However, no bimodal distribution was seen
within the CD34w+ cells. The CD34w+ cells
showed a heterogeneous pattern of bcl-2 expression. These results show
variable levels of bcl-2 expression throughout hematopoietic cell
differentiation.
Expression of bcl-x.
The expression of bcl-x protein was also evaluated in seven MPB, two
CB, and two BM samples. Similar to bcl-2, greater than 95% of
hematopoietic cells expressed bcl-x based on CD38 antigen level and
cell size. The intensity of bcl-x was high in the majority of
CD34+ cells. However, greater than 90% of
CD34s+ cells expressed high density bcl-xL
presented as a narrow histogram. Similar intensity of
bcl-xL expression was also obtained for
CD34+/CD38 cells selected from three MPB
samples (Fig 4).
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| Fig 4.
Histograms of bcl-x-FITC and bcl-xL-FITC
staining after the immunomagnetic selection of CD34+ and
CD34+/CD38 cells from MPB. The left-hand
histogram represents the negative control (mIgG), the middle empty
histogram is the CD34+ cells expressing bcl-x and the
filled-up histogram is the CD34+/CD38
hematopoietic precursors expressing high intensity bcl-xL
protein. Cells stained for bcl-x and bcl-xL were first
stained with anti-CD34-PE antibody then permeabilized with Ortho
Permeafix. MNCs stained with mIgG were also permeabilized.
|
|
Expression of bax, bad, and bak.
The expression of pro-apoptotic proteins bax, bad, and bak was
evaluated in four MPB samples. The
CD34s+/CD38/low expressed low-density bax
(range, 4% to 12%), low bad (range, 0% to 0.8%), and low bak
(range, 0% to 3%) suggesting the characteristics associated with the
pluripotent stem cells. Based on CD38 antigen intensity, the expression
of bax, bad, and bak increased with increasing CD38 antigen expression.
In CD38+ population the percentage of cells expressing bax,
bad, and bak increased to (15% to 20%), (0.5% to 2%), and (2% to
11%), respectively. However, based on the intensity of CD34 antigen
expression, bax, bad, and bak further increased in cells expressing
low-density CD34 compared with those with high density. Bax ranged from
7% to 27%, bad ranged from 2% to 14%, and bak ranged from 7% to
30%. These findings suggest that the increase in pro-apoptotic protein expression is related to the process of maturation unlike control cells
such as the KG-1A cell line, in which a considerable increase in bax
and bak expression was obtained after induction of apoptosis (Fig 5A and B).
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| Fig 5.
Flow cytometric analysis of pro-apoptotic proteins
expressed by KG-1A cell line. Cells were induced for apoptosis after
overnight incubation in medium deprived of serum. For flow cytometric
analysis of bak, bad, and bax expression in KG-1A cells, cells were
permeabilized with Ortho Permeafix and stained with goat anti-bak,
mouse anti-bad, and rabbit anti-bax antibodies followed by staining
with FITC-conjugated antigoat, antimouse, and antirabbit IgG
antibodies, respectively. For neutralization, the anti-bak antibody
(goat polyclonal) was incubated overnight at 4°C with excess
peptide antigen. Cells were stained with the peptide/antibody mixture
followed by staining with FITC-conjugated antigoat IgG. (A) bax, bak,
and bad expression before induction of apoptosis. Only bad protein was
expressed by the majority of KG-1A cells. (B) Considerable increase in
bax and bak expression following induction of apoptosis. (C) The effect
of bak- blocking peptide (PEP) on bak expression in apoptotic cells.
Bak expression reduced from 78% before to 11% after neutralization
with bak peptide.
|
|
To determine whether the increase in percentage bak protein was caused
by antigen expression, we incubated anti-bak antibody with increasing
concentrations of the bak-peptide. The results clearly show a gradual
decrease in bak expression: bak decreased from 78% in the absence of
peptide to 11% in the presence of excess peptide (80:1; Fig 5C),
showing that the binding of the peptide to the antibody is highly
specific. The overall results show the reliability in analyzing the
pro- and anti-apoptotic proteins by flow cytometry.
Expression of apoptotic proteins by CD34 cells.
We aimed at identifying the CD34 cells undergoing apoptosis by studying
the expression of apoptosis-specific proteins such as ASP and Annexin V
and also analyzing their DNA profile (see below). Because the
expression of bcl-2low and bcl-2high within the
CD34s+ population was consistent, we decided to examine
cells expressing low intensity bcl-2 to find out if low bcl-2 triggers
apoptosis in hematopoietic precursors. ASP is an intracellular protein
of 45 kD that has been shown in mammalian cells induced for
apoptosis.8 The CD34s+/CD38/low
expressed low percentage of the ASP protein 3.4% ± 3.1%. The expression of ASP increased with increasing CD38 antigen density (5.8% ± 5.25% and 7.9% ± 6.7% for CD382+ and
CD383+). However, the percentage of ASP also increased in
cells expressing the CD34 antigen weakly and in large cells, (7.05% ± 4.32%, 13.2% ± 4.69%, and 29.5% ± 10.43%, small,
intermediate, and large cells, respectively,
Fig 6). In total CD34s+ cells,
low intensity and lower percentage of ASP (6.4% ± 2.98% in 9 MPB
and 0.5% and 1% in 2 CB) were obtained compared with the whole
CD34+ cell compartment (20.6% ± 7.4% for MPB and
4.9% and 7.7% for CB). CD34+ cells were also stained with
Annexin V, a calcium-dependent phospholipid protein that has high
affinity for binding phosphatidyl serine expressed by apoptotic
cells.41,42 CD34s+ cells showed low intensity
and lower percentage positive Annexin (5.9% and 6.9%) compared with
the total CD34+ population (17.7% and 12.7%) in two MPB
samples. The expression of Annexin V by CD34 cells was similar to ASP
as was compared in the two above samples (5% and 7% in
CD34s+ and 17.7% and 18.2% in total CD34+
cells). These results show that the expression of apoptotic proteins occurred mainly among low-density CD34 cells and large cells and is
directly related to the increase in cell size and differentiation.
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| Fig 6.
ASP expression in CD34 positive cells obtained from MPB
samples gated according to cell size into small, intermediate, and
large. Region R6 identifies weak CD34 positive cells expressing the
ASP-protein, and region R7 identifies strong CD34 positive cells
expressing the ASP-protein. Three-color immunofluorescence staining was
performed. After staining of membrane antigens with anti-CD34-tri-color
and anti-CD38-PE antibodies, cells were permeabilized with Ortho
Permeafix, stained with anti-ASP-FITC, and analyzed on the FACScan.
|
|
Cell cycle status of CD34-positive cells.
We then investigated the cell cycle status of
CD34+/CD38 cells and compared them with
CD34+/CD38+ cells. CD34+ cells were
positively selected from three MPB and three CB samples. CD34+/CD38 cells were obtained from
CD34+ cells by using immunomagnetic depletion of more
mature CD34 cells (Fig 7A). Similarly, the
CD34+ cells from cell line KG-1A were also used as a
positive control. In both MPB and CB, a lower percentage of primitive
CD34+/CD38 cells was cycling (mean = 0.92%
of 3 MPB and mean = 1.63% of 3 CB; Fig 7B). In contrast, an increase
in the proportion of DNA synthesizing cells was obtained with
increasing CD38 antigen expression (mean = 4.4% in MPB and mean = 2.8% in CB). However, among the KG-1A cells, significantly higher
proportions were in the proliferative cell cycle (37%). These results
show that CD34+/CD38 cells from the two
blood sources (MPB and CB) have low cell-cycle activity and neither
cells showed the presence of an apoptotic peak.
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| Fig 7.
Flow cytometric analysis of propidium iodide stained
CD34+/CD38 cells obtained from MPB. (A)
The immunomagnetic separation of
CD34+/CD38 cells obtained from selected
CD34+ cells. Greater than 90% of CD34+
cells were CD38+. The MFI for CD38+ cells
was 675. After negative selection, the MFI for CD38
cells reduced to 15. (B) Histogram showing PI-fluorescence of selected
CD34+/CD38 cells stained with two-color
immunofluorescence (CD34-FITC and PI) and gated on CD34+
cells. M1 defines cells in G0/G1 phase, M2 defines cells in S phase,
and M3 defines cells in G2+M phase.
|
|
Induction of apoptosis.
We aimed at examining the susceptibility of CD34s+ cells
with low expression of bcl-2 for apoptosis at 1 hour, 2 hours, and after overnight incubation (24 hour) in medium deprived of serum and
growth factors. Mononuclear cells from six MPB samples were tested.
Cell numbers and cell viability were investigated at the beginning and
after 1 hour, 2 hours, and 24 hours of incubation, and the percentage
of cells recovered was 91% to 97% with percentage viability exceeding
90% of those originally counted. Phenotypic analysis showed that 95%
to 98% of gated CD34+ cells identified in the fluorescence
versus SSC dot plot were recovered after 24-hour incubation.
Interestingly a bimodal histogram of bcl-2 low and bcl-2 high obtained
in CD34s+ cells gated according to CD38 intensity was seen
before (Fig 8A) and after 1-hour and 2- hour incubation but disappeared after 24-hour incubation representing
only a single population histogram (Fig 8B). However, the disappearance
of the histogram representing cells with low bcl-2 expression did not
reduce the number of events collected, in particular those cells
lacking CD38 antigen expression (Fig 8B). Furthermore, ASP expression
in CD34s+/CD38/low increased from 2.1% to
4.3% after overnight incubation in comparison with the more mature
cells (CD34+/CD38+) in which ASP expression
increased from 3.4% to 9.8%.
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| Fig 8.
Changes in bcl-2 expression of
CD34+/CD38/low hematopoietic precursors
during overnight incubation of MPB in serum-free RPMI. Cells were
analyzed using three-color immunofluorescence of CD34-tri- color,
CD38-PE, and bcl-2-FITC. After membrane antigen staining with anti-CD34
and anti-CD38 antibodies, cells were permeabilized with Ortho Permeafix
and stained for anti-bcl-2. Bcl-2 expression was analyzed according to
CD38 antigen intensity level (regions R3, R4, and R5 in Fig 1C). Data
show one representative experiment of six. (A) Bcl-2 expression before
incubation, representing a bimodal histogram of bcl-2low
(MFI = 95) and bcl- 2high (MFI = 259). (B) Bcl-2
expression in the same cells after incubation showing a single
histogram of bcl- 2high (MFI = 212).
|
|
No apoptotic peak was obtained after 24-hour incubation: only a slight
increase of cells in S/G2 + M phase ranging from 1.0% to 2.0%
compared with 0.8% to 1.4% before incubation. These results may show
that under deprived conditions CD34s+ cells with low bcl-2
upregulated their bcl-2 to escape apoptosis.
Ex vivo expansion of CD34+ cells.
Cultures were maintained for 3 weeks. Cellular proliferation peaked on
day 10 with a cell output greater than 75-fold the input number (range,
58-200) and a viability exceeding 90%.
On day 0, the starting percentage of CD34+ ranged from 85%
to 95%, and on day 10 of culture, 6% to 10% were CD34+
representing a threefold increase. Among the CD34+ cells a
mean of 12% were CD38, giving a ninefold increase in
the CD34+/CD38 cell population. On day 10 of
culture, CD34+/CD38 expressed bcl-2 as a
single population histogram and greater than 90% of cells were
bcl-xL high. The expression of bax increased from 12%,
8%, and 6% to 15%, 10%, and 15%; bad increased from 0%, 0%, and
0.8% to 5%, 5%, and 8%; bak increased from 3%, 0%, and 2% to
6%, 14%, and 12%; and ASP increased from 7%, 5%, 3%, and 3% to
6%, 10%, 10%, and 7%.
| |
DISCUSSION |
In this study, we analyzed the expression of bcl-2 and its related
proteins in hematopoietic precursors and progenitors from adult MPB,
CB, and adult BM. To our knowledge, this is the first time that bcl-2
analysis was performed according to cellular differentiation by using
the expression of CD38 or HLA-DR (class II) as activation markers. We
showed that variable levels of bcl-2 are expressed by the most
primitive hematopoietic cells
(CD34s+/CD38/low or
CD34s+/HLADR/low) as well as the more
mature ones
(CD34s+/CD383+/HLA-DR3+). We also
showed a progressive conversion of bcl-2low to
bcl-2high and a stable expression for cells with
bcl-2high at steady state and during stimulation.
During cellular differentiation/maturation the increase in bcl-2
intensity is associated with the transition into intermediate and large
cells. The expression of bcl-2 in primitive hematopoietic cells has
also been shown by others in fetal liver and during ontogenesis.32,43 Primitive BM precursors
(CD34+/CD33/HLA-DR+) have also
been shown to express bcl-2.30 In contrast, these primitive
hematopoietic precursors were found to be bcl-2 negative by Park et
al31 by using a different technique.
Unexpectedly, we identified a small subpopulation of hematopoietic
precursors with a low bcl-2 expression but strong CD34+ and
lacking CD38 antigen. These cells are different from other numerous
CD34s+/CD383+ progenitors on the basis of cell
size, cell morphology, bcl-2 expression, cell cycle status, and
expression of apoptotic proteins. The
CD34s+/CD38/low subpopulation is composed of
undifferentiated lymphoid-like cells that have low DNA synthesizing
activity, low apoptotic activity, and resist serum and growth factors
deprivation by upregulating their bcl-2 expression. Furthermore, these
cells form morphologically a homogeneous population of cells (87% to
91%) expressing two levels of bcl-2 (low and high). These were present
in MPB, CB, and BM, although the cells with low bcl-2 are more present
in blood than in BM. The differences in bcl-2 expression suggest two
functionally distinct subpopulations existing within the
CD34s+/CD38/low cells. We propose that cells
with low bcl-2 are quiescent and reside within the resting stem cell
compartment in the G00 phase of the cell
cycle as shown in the model of stem cell kinetics described by Gordon
and Blackett.44 These cells are capable of upregulating
their bcl-2 expression. This hypothesis is shown when
CD34s+/CD38/low cells upregulated their
bcl-2 not after 1 or 2 hours of incubation but after overnight
incubation (results from 6 experiments). Further evidence for this
hypothesis comes from our data on ex vivo expansion of hematopoietic
cells showing the cells that were characterized on day 10 of culture as
CD34+/CD38 expressed high-density bcl-2 as
single population histogram. The presence of two functionally different
subpopulations within the CD34+/CD38 was
shown previously in vitro in hematopoietic precursors found in BM and
CB.45 The authors identified cells producing colony-forming unit-cells (CFU-C) within the standard long-term culture-initiating cells (LTC-IC) assay period (5 to 8 weeks), and those who went beyond
the 8-week period started proliferating in extended LTC-IC. Similarly
in murine studies, functional distinction has been described between
day-12 CFU-spleen cells and the more quiescent and primitive long-term
repopulating cells.46
The results in the present study as well as two previous
reports31,32 suggest that bcl-2 expression in
CD34+ cells undergoing differentiation is important for
maintaining the colony-forming potential. High bcl-2 expression is
associated with high-intensity CD38 and increase in cell size
suggesting that bcl-2 may play an important role in proliferating
cells. These findings were further supported by a recent report showing that the exposure of hematopoietic progenitors to bcl-2 antisense decreased cell survival and inhibited the outgrowth of
granulocyte-macrophage colony-forming cells.9
As with CD38 expression, the intensity of bcl-2 increased with
increasing HLA-DR antigen expression. Fewer cells expressing low bcl-2
were found within the CD34s+/HLADR/low
when compared with CD38/low cells. In addition,
CD34s+/HLA-DR/low cells showed more
heterogeneity when isolated by using 2-color IF cell sorting (data not
shown).
Similar results were obtained by Rusten et al34 using
immunomagnetic selection of normal adult BM precursors. A more
homogeneous population of CD34+/CD38 cells
was obtained compared with CD34+/HLA-DR
cells. The variation in the expression of bcl-2 within the
CD38/low and HLA-DR/low on
CD34s+ cells may reflect subpopulations with different
functions and or maturity. In this regard, Rusten et al34
showed that the two subpopulations differ in their primitive progenitor
cell content. The CD34+/38 contained higher
frequency of HPP-CFCS and LTC-ICS, whereas the CD34+/DR cells contained more committed
erythroid progenitors. Furthermore, Huang and Terstappen47
reported that CD34+/CD38/DR+,
but not CD34+/CD38+/DR fetal
human BM cells have features of human hematopoietic stem cells in that
they can give rise to each of the hematopoietic cell lineages in vitro.
However, primitive hematopoietic stem cells have also been shown within
the CD34+/HLA-DR subset.48
Furthermore, because of their low bcl-2 expression and small cell size,
it is highly unlikely that the
CD34s+/CD38/low we have identified might be
mistaken for lymphoid precursors.28 The CD34+
subset of CD10+ lymphoid precursor cells have also been
shown to express low bcl-2.28,29 Not only are these cells
almost devoid of the tested lineage associated surface and cytoplasmic
markers (<4% CD10+, <1% CD19+, <2%
CD7+, <5%TdT+, and <2% MPO+,
Perey et al, in press), but also lack the expression of CD38 antigen
and reside in the CD34s+ compartment (Fig 1C). The majority
of CD10+ lymphoid precursors in our hands were found within
a population either expressing lower density CD34 antigen or lacking
its expression. This is in agreement with those results reported for
normal BM, showing the absence of B-lymphoid committed cells within
CD34s+/CD38/low defined on the basis of CD10
expression.49
The expression of bcl-2low and bcl-2high within
the CD34s+ population is unlikely to be related to the
permeabilization technique that we adopted for the measurement of
intracellular proteins. The fixative we used (Ortho Permeafix)
permeabilizes cells without altering their scatter features (Fig 1A)
and their membrane or cytoplasmic staining.38,39
Furthermore, this fixative has been used in a recent study in a
quantitative flow cytometry assay to evaluate the bcl-2 level in normal
BM and acute myeloid leukemia cells.50 Similarly, the
expression of low and high bcl-2 within the CD34s+ is a
stable phenomenon and unlikely to have been caused by postharvesting induction of bcl-2. The presence of double peaks was shown in all types
of cells (MPB, CB, and BM) and after short-term incubation (1 hour and
2 hours).
It has been suggested that bcl-2 plays a potential role in maintaining
long-term survival in a variety of cell types, in particular hematopoietic stem cells. However, recent data by Park et
al31 provide evidence that bcl-xL, a
functionally homologous protein16 may be essential for
long-term survival in the early stages of hematopoiesis. The authors
show that the immature quiescent
CD34+/Lin/CD38 cells are
bcl-2 negative. The later cell subpopulation relies on
bcl-xL for survival.
Our results show that bcl-x (in particular bcl-xL) is
expressed in all subpopulations of hematopoietic precursors including the bcl-2low/CD38/low/CD34s+
cells. These cells were selected and shown to strongly express the
bcl-xL protein as a single population histogram. The strong bcl-xL expression in the later cell subpopulation might be
required to compensate for their low bcl-2 expression during early
hematopoiesis by protecting them from apoptosis. Hematopoietic cells in
fetal liver were shown to express both bcl-2 and
bcl-x.43,51 However, the data provided by these studies
support bcl-x as the predominant regulator of cell survival during
embryonic hematopoiesis. Also, bcl-x in the fetus protects the
hematopoietic cells as well as the nervous system against
apoptosis.51 The nonlymphoid hematopoietic lineage remains
unaffected in bcl-2-deficient mice.52 Our results and those
previously reported are in favor of the bcl-xL potential role in maintaining the survival of hematopoietic stem cell
populations.
We and others have shown that these cells have no or very low cell
cycle activity as well as low apoptotic activity. We show that
hematopoietic precursors (CD34s+/CD38/low)
are the lowest in expressing apoptotic proteins, ASP, Annexin V (data
not shown), and bax, bad, and bak, in agreement with two recent reports
showing that a small percentage of mobilized CD34 cells are entering
apoptosis.53,54 So this study provides evidence for full
viability and complete integrity of the quiescent precursors with low
bcl-2 expression.
Nevertheless, the results on day 10 of ex vivo expansion show an
upregulation of bax, bad, bak, and ASP within the
CD34+/CD38 cell population. The culture
condition that we have used was shown previously to expand
hematopoietic cells that were successfully reinfused as an adequate
alternative to MPB.37 Therefore, during ex vivo expansion,
the expression of pro- and apoptotic proteins needs to be monitored to
define the best culture condition. These conditions should be chosen in
such a way as to keep the primitive precursors from entering apoptosis.
These results highlight the need for understanding the complex process
that regulates apoptosis.
| |
FOOTNOTES |
Submitted June 24, 1997;
accepted May 11, 1998.
Supported by grants from La Recherche Suisse contre le Cancer (KFS
170-9-1995), La Ligue Vaudoise contre le Cancer, and La Société de la Loterie de la Suisse Romande.
Address reprints requests to Rowayda Peters, MSc, PhD, Centre
Pluridisciplinaire d'Oncologie, CHUV, BH 06, Rue du Bugnon, 1011 Lausanne, Switzerland; e-mail: Serge.Leyvraz{at}chuv.hospvd.ch.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are very grateful to Prof George Janossy (Department of Clinical
Immunology, Royal Free Hospital School of Medicine, London, UK) for
helpful advice and critical suggestions. We thank Dr Curzio Ruegg for
helpful suggestions and comments and Dr Philippe Jaunin and Mrs Eveline
Faes for superb technical assistance. We thank the department of
Gynecology for providing umbilical cord blood for use in this study. We
gratefully acknowledge the following pharmaceutical companies: Serono
for kindly providing us with IL-6, Amgen for SCF, and Sandoz for IL-3.
Special thanks is given to Mrs Martine van Overloop for typing the
manuscript.
| |
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Abstract
Introduction
Abstract
