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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 509-518
Neutrophilic Cell Production by Combination of Stem Cell Factor and
Thrombopoietin From CD34+ Cord Blood Cells in
Long-Term Serum-Deprived Liquid Culture
By
Nobukuni Sawai,
Kenichi Koike,
Susumu Ito,
Hadija Hemed Mwamtemi,
Yumi Kurokawa,
Tatsuya Kinoshita,
Kazuo Sakashita,
Tsukasa Higuchi,
Kouichi Takeuchi,
Masaaki Shiohara,
Hiroshi Miyazaki,
Takashi Kato, and
Atsushi Komiyama
From the Department of Pediatrics and the Blood Transfusion Service,
Shinshu University School of Medicine, Matsumoto, Japan; and the
Pharmaceutical Research Laboratory, Kirin Brewery Co, Ltd, Takasaki,
Japan.
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ABSTRACT |
In the present study, we investigated the effects of stem cell
factor (SCF) and/or thrombopoietin (TPO) on the cell production by cord blood CD34+ cells using a serum-deprived liquid
culture system. Although SCF alone supported a modest production of
neutrophilic cells and a remarkable generation of mast cells, the
addition of TPO to the culture containing SCF caused an apparent
generation of neutrophilic cells, identified by immunocytochemical
staining and flow cytometric analysis. The significant production of
neutrophilic cells by SCF and TPO was persistently observed from 2 weeks to 2 to 3 months of culture. The interaction between SCF and TPO on the neutrophilic cell generation was greater than the combined effects of SCF with granulocyte colony-stimulating factor (G-CSF) or
granulocyte-macrophage colony-stimulating factor (GM-CSF). The addition
of neutralizing antibody against G-CSF or GM-CSF did not influence the
SCF + TPO-dependent neutrophilic cell production. A single-cell
culture study showed that not only
CD34+CD38+ c-kit+ cells but
also CD34+CD38 c-kit+ cells
were responsible for the neutrophilic cell generation. In clonal cell
cultures, GM progenitors as well as erythroid progenitors and
multipotential progenitors expanded in the cultures supplemented with
SCF and TPO. The neutrophilic cells grown by SCF + TPO were at
myeloblast to band cell stages, and scarcely matured to segmented neutrophils. In addition, the cells generated by SCF + TPO were stained with monoclonal antibodies against myeloperoxidase, elastase, lactoferrin, and CD11b, but they had negligible levels of alkaline phosphatase (ALP) and CD35. The replating of the
CD34c-kit/low CD15+ cells
grown by SCF + TPO into a culture containing SCF + G-CSF permitted
both the terminal maturation into segmented cells and the appearance of
ALP and CD35. These results indicate the existence of a
G-CSF/GM-CSF-independent system of neutrophilic cell production.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
A HUGE NUMBER OF CIRCULATING neutrophils
are supplied daily from a small number of hematopoietic stem cells to
maintain homeostasis. In cases of infection this neutrophil production is rapidly increased. It is generally held that this differentiation pathway is regulated in part by growth factors including granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage
colony-stimulating factor (GM-CSF), interleukin (IL)-3, and stem cell
factor (SCF). Although there is increasing evidence that G-CSF plays an
important role as an endogenous regulator of neutrophil
production,1,2 the existence of G-CSF-independent
mechanisms of granulopoiesis is also shown on the basis of the results
obtained using G-CSF or G-CSF receptor-deficient mice.2,3
Thrombopoietin (TPO), c-mpl ligand, was cloned by several
independent groups as a growth factor for the megakaryocyte-platelet lineage.4-8 Treatment with TPO was shown to accelerate
platelet, red blood cell, and neutrophil recovery in myelosuppressed
mice, indicating the in vivo effects of TPO on multiple cell
lineages.9 Kobayashi et al10 reported that TPO
synergizes with SCF and/or IL-3 in support of the formation of
GM colonies as well as multilineage colonies and erythroid colonies. In
addition, TPO was shown to augment the expansion efficiency for various
types of hematopoietic progenitors including GM progenitors in the
presence of several cytokines including SCF in a liquid suspension
culture system.11,12 Our recent study showed that the
addition of TPO to cultures containing SCF or SCF + GM-CSF caused a
significant increase in the production of GM colony-forming cells by
primitive hematopoietic progenitors of patients with juvenile chronic
myelogenous leukemia.13 These lines of evidence prompted us
to examine the possibility of neutrophil production induced by SCF + TPO. For this purpose, serum-deprived liquid cultures were initiated
with CD34+ cord blood cells, and were maintained with the
repeated addition of TPO and/or SCF.
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MATERIALS AND METHODS |
Factors and antibodies.
Human recombinant TPO, SCF, IL-3, GM-CSF, and erythropoietin (EPO) were
provided by Kirin Brewery Co, Ltd (Takasaki, Japan). Human recombinant
G-CSF and rabbit antihuman G-CSF polyclonal antibody were generously
provided by Chugai Pharmaceutical Co (Tokyo, Japan). A 1:10,000
dilution of antihuman G-CSF antibody neutralized the activity of 100 pg
of G-CSF.14 A mouse monoclonal antibody (MoAb) for GM-CSF
(Ab-1; Oncogene Science, Uniondale, NY) was used at 2 µg/mL, as
reported previously.15
For the flow-cytometric analysis, MoAbs for CD34 (8G12, fluorescein
isothiocyanate, FITC; allophycocyanin, APC), CD2 (S5.2, FITC), CD14
(M P9, FITC), CD38 (HB7, APC), and c-kit (104D2, phycoerythrin, PE)
were purchased from Becton Dickinson Immunocytometry Systems (Mountain
View, CA); the MoAbs for CD15 (80H5, FITC) and CD19 (J4.119, FITC) were
from Immunotech S.A. (Marseilles, France), and the MoAb for CD41b
(TP80, FITC) was from Nichirei (Tokyo, Japan). The MoAb for glycophorin
A (GPA, JC159, FITC) was from Dako (Glostrup, Denmark).
For the immunocytochemical analysis, purified MoAbs for human
myeloperoxidase (MPO, CLB-MPO-1) and CD2 (T11) were purchased from
Immunotech S.A. The MoAb for human lactoferrin (2B8) was from Advanced
Immunochemical (Long Beach, CA); MoAbs for CD11b (2LPM19c), CD14
(Tük 4), CD15 (C3D-1), CD19 (HD37), GPA (JC159), CD35 (To5) and
human elastase (NP57) were from Dako; MoAbs for human eosinophil
peroxidase (MAB1087) and mast cell tryptase (MAB1222) were from
Chemicon International Inc (Temecula, CA).
Cell preparation.
Cord blood samples were aspirated in heparinized plastic syringes from
the umbilical vein at normal delivery. Fully informed consent was
obtained from the mothers of all neonates before harvesting the
specimens. Mononuclear cells (MNCs) were separated by density centrifugation over Ficoll-Plaque (Pharmacia, Fine Chemicals, Piscataway, NJ), washed twice, and suspended in Ca2+- and
Mg2+-free phosphate-buffered saline (PBS) containing 1 mmol/L EDTA-2Na, and 2.5% fetal bovine serum (FBS, Hyclone, Logan,
UT). After treatment with Silica (Immuno-Biological Laboratories,
Fujioka, Japan) for 30 minutes at 37°C, CD34+ cells
were enriched using a Dynal CD34 Progenitor Cell Selection System
(Dynal A.S., Oslo, Norway). In brief, 1.0 × 107 cells
were mixed with the same number of polystyrene beads coated with a MoAb
specific for CD34 (Dynabeads M-450 CD34), and incubated for 30 minutes
at 4°C. Bead-rosetted cells were separated by a magnet. For the
detachment of the beads from the cells, affinity-purified polyclonal
antibodies against the Fab portion of anti-CD34 antibody (Detach-a-Bead
CD34) were added, and incubation was performed for 45 minutes at room
temperature. The detached beads were removed by the magnet, and the
cells were collected as CD34+ cells. More than 90% of the
isolated cells were CD34+, as determined by FACScan flow
cytometry (Becton Dickinson).
Serum-deprived liquid culture.
Serum-deprived liquid cultures were performed in a 24-well culture
plate (#3047; Becton Dickinson) using a modification of the technique
described previously.16 Two × 104
CD34+ cells were cultured in each well containing 2 mL
 -medium (Flow Laboratories Inc, Rockville, MD) supplemented with 1%
deionized bovine serum albumin (BSA; Sigma Chemical Co, St Louis, MO),
600 µg/mL fully iron-saturated human transferrin (approximately 98% pure; Sigma), 16 µg/mL soybean lecithin (Sigma), 9.6 µg/mL
cholesterol (Nakalai Chemicals Ltd, Tokyo, Japan), and 10 ng/mL of TPO,
10 ng/mL of G-CSF, 10 ng/mL of GM-CSF, and 10 ng/mL of SCF, alone or in
combination. The plates were incubated at 37°C in a humidified atmosphere flushed with a mixture of 5% CO2, 5%
O2, and 90% N2. Half of the cells and culture
medium was replaced weekly with fresh medium containing growth
factor(s). The number of viable cells was determined by a trypan-blue
exclusion test using hemocytometers, and the cells were processed for
immunocytochemical staining and flow cytometric analysis.
Clonal cell culture.
Clonal cell cultures were performed in 35-mm Lux suspension culture
dishes (#171099; Nunc, Naperville, IL) by the technique described
previously.17 The culture consisted of 1 to 5 × 103/mL cells, -medium, 0.9% methylcellulose (Shinetsu
Chemical Co, Tokyo, Japan), 1% deionized BSA, 30% FBS, 100 U/mL of
IL-3, 10 ng/mL of GM-CSF, 10 ng/mL of G-CSF, 10 ng/mL of SCF, and 2 U/mL of EPO. Dishes were incubated at 37°C in a humidified
atmosphere flushed with a 5% CO2. On day 14, GM colonies,
erythroid bursts, and mixed erythroid colonies consisting of erythroid
cells and cells of other than erythroid lineage were scored in situ on
an inverted microscope according to the criteria described
previously.18,19
Serum-deprived single-cell culture.
To elucidate which subpopulations of CD34+ cells generated
neutrophilic cells under stimulation with TPO + SCF, two-step sorting was performed, as described previously.13 Cord blood MNCs
were incubated with 20 µL FITC-conjugated anti-CD34 MoAb, 5 µL
APC-conjugated anti-CD38 MoAb and 20 µL PE-conjugated anti-c-kit
MoAb for 30 minutes at 4°C. As negative controls, cells were
stained with FITC-, APC-, and PE-conjugated mouse IgG1 (Becton
Dickinson). After two washes,
CD34+CD38+ c-kit+ cells,
CD34+CD38+c-kit- cells,
CD34+CD38-c-kit+ cells, and
CD34+ CD38-c-kit- cells were
individually sorted in 5 mL tubes by a FACStarplus flow
cytometer (Becton Dickinson). The cells in each group were then
resorted into the individual wells of a 96-well U-bottomed tissue
culture plate (#3077; Becton Dickinson) containing 100 µL of the
serum-deprived culture medium supplemented with SCF and/or TPO,
using the FACStarplus flow cytometer equipped with an
automatic cell deposition unit. Ninety-nine percent of the wells
contained a single cell on the first day of culture. The plates were
incubated at 37°C in a humidified atmosphere flushed with a mixture
of 5% CO2, 5% O2, and 90% N2. After 3 weeks, colonies of more than 30 cells were scored in situ on an
inverted microscope, and the constituent cells of colonies were
identified on cytocentrifuged preparations stained with
May-Grünwald-Giemsa.
Flow cytometric analysis and cell sorting.
For the analysis of surface markers on the cultured cells, 1 to 2 × 106 cells were collected in plastic tubes and
incubated with appropriately diluted FITC- or PE-MoAb, as described
previously.13,15 The cells were washed twice, after which
their surface markers were analyzed with the FACScan flow cytometer,
using the Lysis 2 software program (Becton Dickinson).
Viable cells were gated according to their forward light-scatter
characteristics (FSC) and side-scatter characteristics (SSC). The
proportion of positive cells was determined by comparison to cells
stained with FITC- or PE-conjugated mouse isotype-matched Ig.
For the identification of the maturation stage of neutrophilic cells
grown by SCF + TPO, the cultured cells at 3 weeks (1 × 106) were incubated with 5 µL APC-conjugated anti-CD34
MoAb, 20 µL PE-conjugated anti-c-kit MoAb, and 20 µL
FITC-conjugated anti-CD15 MoAb for 30 minutes at 4°C. As negative
controls, cells were stained with APC-, PE-, and FITC-conjugated mouse
isotype-matched Ig (Becton Dickinson and Immunotech S.A.). After two
washes,
CD34 c-kit/lowCD15+
cells were sorted by the FACStarplus flow cytometer.
Cytochemical staining.
Cultured cells were spread on glass slides using a Cytospin II (Shandon
Southern, Sewickly, PA), and stained with May-Grünwald-Giemsa, Sudan black B, Biebrich scarlet, or toluidine blue. Cytochemical reactions with peroxidase (POX), -naphthyl butyrate esterase, acid
phosphatase, and alkaline phosphatase (ALP) were performed by the
conventional methods.20
Immunocytochemical staining.
Reactions with mouse MoAbs against MPO, elastase, lactoferrin, CD2,
CD11b, CD14, CD15, CD19, CD35, GPA, eosinophil POX, and tryptase were
detected using the ALP-anti-ALP method (Dako APAAP Kit System; Dako
Corp, Carpinteria, CA), as described previously.21 The
isotype mouse MoAb was used as a control. Briefly, cytocentrifuged samples were fixed with Carnoy's fluid, washed with PBS, and
preincubated with normal rabbit serum to saturate the Fc receptors on
the cell surface. After being washed with PBS three times, the samples were reacted with mouse MoAb for 30 minutes at room temperature in a
humidified chamber. After three more washes with PBS, the samples were
reacted with rabbit antimouse IgG antibody, washed three times, and
successively reacted with the calf intestinal ALP-mouse monoclonal
anti-ALP complex. Finally, ALP activity was detected with naphthol
AS-MX phosphate, Fast Red TR, and levamisole to inhibit nonspecific ALP
activity. The specimens were counterstained with hematoxylin. Three
hundred cells were examined.
Statistical analysis.
All experiments were performed at least three times and were shown to
be reproducible. Values are expressed as mean ± SD. One-way analysis of variance, followed by post hoc contrasts with Bonferroni limitation, was employed for more than four independent groups.
| |
RESULTS |
Production of neutrophilic cells by combination of SCF and TPO from
CD34+ cord blood cells in long-term serum-deprived liquid
cultures.
To examine the effects of SCF and TPO, alone or in combination, on the
cell production by hematopoietic progenitors, we initiated serum-deprived liquid cultures with 2 × 104
CD34+ cord blood cells per well, and maintained the
cultures with the repeated addition of growth factor(s). G-CSF and
GM-CSF were used as controls. The results are presented in
Fig 1. In the presence of G-CSF, the total
viable cell number increased to a maximum of twice the input quantity
after 2 weeks. A large part of the progenies were neutrophils, and they
did not survive beyond 4 weeks. Significant cell production was
observed in a well containing GM-CSF (16-fold the input cell number
after 4 weeks), and most of the cultured cells were eosinophils. In the
case of TPO, the total number of viable cells increased until 2 weeks,
with a peak of 18-fold the input quantity. More than 95% of the
cultured cells were positive for CD41b, and the cells with other
lineage-specific markers (CD2, CD19, CD11b, CD14, CD15, GPA) were at a
negligible level, as reported previously.16 The number of
viable cells decreased after 2 weeks. SCF exerted a modest stimulatory
effect on the cell growth until 8 weeks. Subsequently, the apparent
cell proliferation was observed. After 4 weeks, most of the progenies reacted with antitryptase MoAb, indicating their mast cell property. In
addition, less than 10% of POX+ cells were generated from
4 weeks to 7 weeks, as shown in Fig 2. The
combination of SCF + G-CSF or SCF + GM-CSF caused a synergistic increase in the numbers of total viable cells and POX+
cells, in particular at 4 weeks. These results are consistent with
previous results.22 However, there were no viable cells under stimulation with SCF + G-CSF or SCF + GM-CSF after 8 weeks. The
combination of SCF and TPO exerted a significant interaction on the
cell production from 4 weeks to 10 weeks, compared with each factor
alone. Neutrophil-like cells positive for POX
(Fig 3) first appeared after 2 weeks of the
culture with SCF + TPO, and accounted for approximately 60% to 80% of
the cells generated between 4 weeks and 7 weeks. The production of
POX+ cells supported by SCF + TPO at 7 weeks was at a
twofold greater level, compared with the results obtained by the other
two-factor combinations at 4 weeks. There was a decrease in the
POX+ cell generation accompanied by an increase in mast
cell growth after 10 weeks.
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| Fig 1.
Combination of SCF and TPO stimulates neutrophilic cell
production by CD34+ cord blood cells in serum-deprived
liquid culture. (A) CD34+ cord blood cells (2 × 104) were plated per well containing serum-deprived liquid
culture medium supplemented with SCF, TPO, G-CSF, or GM-CSF, alone or
in combination. Half of the cells and culture medium were replaced
weekly with fresh medium containing growth factor(s). Numbers of viable
cells were serially counted. (B) The cultured cells were processed for
staining with POX. The results shown are from one representative
experiment of three. Similar results were obtained in the other two
experiments. SCF ( ), TPO ( ), G-CSF ( ), GM-CSF ( ), SCF + TPO ( ), SCF + G-CSF ( ), SCF + GM-CSF ( ).
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| Fig 2.
Time course of relative frequency of POX+
cells and tryptase+ cells grown by SCF, or SCF + TPO.
The cells were identified on cytocentrifuged preparations stained with
POX or with a MoAb against tryptase. POX+ cells (black
bars), tryptase+ cells (gray bars), others (white
bars).
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| Fig 3.
Cytological characteristics of neutrophilic cells grown
by SCF + TPO from CD34+ cord blood cells. Staining of
cultured cells grown by SCF + TPO at 8 weeks with
May-Grünwald-Giemsa (A); with POX (C); with a MoAb for MPO (D),
and with ALP (E). The cells generated by SCF + TPO at 8 weeks were
harvested and recultured with SCF + TPO + G-CSF. After 1 week, the cells were processed for staining with
May-Grünwald-Giemsa (B), and with ALP (F). Original
magnification: ×1,000 (A-D), ×400 (E,F).
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There are four possibilities for POX+-cell lineages:
neutrophilic, eosinophilic, basophilic, and monocytic series. The
POX+ cells grown by SCF + TPO were negative for Biebrich
scarlet, eosinophil POX, toluidine-blue and -naphthyl butyrate
esterase staining. On the other hand, they reacted with anti-MPO MoAb, acid phosphatase and Sudan black B, but not ALP, as presented in Fig 3.
The flow cytometric analysis showed that under stimulation with SCF,
approximately 70% to 80% of the cells grown at 3 to 4 weeks of the
culture were positive for c-kit, and most of the remaining cells
reacted with anti-CD15 MoAb, as shown in
Fig 4. After 9 weeks, the frequency of
c-kit+ cells was higher than 95%. The percentages of
CD34+, GPA+, CD2+, or
CD19+ cells were less than 1% at 3 to 4, 10, 13, and 18 weeks. On the other hand, the relative number of CD15+
cells grown by SCF + TPO was significantly higher between 3 weeks and
10 weeks in comparison with the value obtained by SCF alone, whereas
the percentages of CD14+ and CD41b+ cells were
at a negligible or low level. Most of the cultured cells were positive
for c-kit after 13 weeks. The frequency of CD34+,
GPA+, CD2+, or CD19+ cells was less
than 1% at various time points up to 18 weeks except that 2% to 3%
of the cultured cells expressed CD34 at 3 to 4 weeks of the culture. In
the presence of SCF + G-CSF or SCF + GM-CSF, a large portion of the
cultured cells was positive for CD15, and c-kit+ cells were
virtually negative at 3 to 4 weeks. These results indicate the
significant neutrophilic production by the combination of SCF and TPO
from CD34+ cord blood cells.
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| Fig 4.
Time course of surface marker expression on cultured
cells grown by SCF, SCF + TPO, SCF + G-CSF, or SCF + GM-CSF from
CD34+ cord blood cells. Surface marker expression was
determined by flow cytometry. SCF (), SCF + TPO (), SCF + G-CSF (), SCF + GM-CSF ().
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It is possible that the endogenous secretion of G-CSF or GM-CSF
contributes to the SCF + TPO-dependent neutrophil production, because
of the cytokine liberation by mast cells.23 However, neither G-CSF nor GM-CSF was detected in the supernatants of the 8 weeks- and 12 weeks-cultured cells grown by SCF alone or SCF + TPO.
Moreover, the addition of a 1:10,000 dilution of anti-G-CSF and 2 µg/mL of anti-GM-CSF neutralizing antibodies to the cultures containing SCF + TPO did not influence the neutrophilic cell production until 4 weeks (data not shown).
Maturation stage of neutrophilic cells grown by SCF + TPO.
For the examination of neutrophil-associated proteins in the
neutrophilic cells grown by SCF + TPO, the cultured cells generated by
SCF + TPO at 3 weeks were harvested and recultured with SCF + TPO, SCF + G-CSF, or SCF + GM-CSF for another week. As presented in
Table 1 (Experiment 1), the cultured
neutrophilic cells in the presence of SCF + G-CSF contained
MPO, elastase, lactoferrin, CD11b, CD35, and ALP. In the case of SCF + GM-CSF, all of the neutrophil-associated proteins except ALP were
detectable. In the cultures with SCF + TPO, in contrast, the
neutrophilic cells contained MPO, elastase, lactoferrin, and CD11b, but
not CD35 and ALP. To examine the possibility that SCF + TPO exerted
inhibitory effects on the expressions of CD35 and ALP, neutrophilic
cells grown by SCF + TPO were harvested at 8 weeks and recultured with SCF + TPO, SCF + TPO + G-CSF, or SCF + TPO + GM-CSF for 1 week. As
presented in Table 1 (Experiment 2), similar results were obtained.
Thus, the neutrophilic cells generated by SCF + TPO appeared to be
defective in CD35 and ALP.
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Table 1.
Neutrophil-Associated Proteins in Neutrophilic Cells
Grown by Various Combinations of Growth Factors From
CD34+ Cord Blood Cells in Serum-Deprived Liquid
Culture
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Next, we examined the direct effects of the two-factor combinations on
neutrophilic maturation. For this purpose, we sorted CD34c-kit/lowCD15+
cells grown by SCF + TPO at 3 weeks using the FACStarplus
flow cytometer as shown in Fig 5, and
replated them into cultures containing SCF + TPO, SCF + G-CSF, or SCF + GM-CSF. The results are presented in Table
2. More than 95% of the sorted cells were viable and positive for POX.
They were composed of myeloblasts, promyelocytes, myelocytes,
metamyelocytes, and band cells, but not segmented cells. An obvious
transition from promyelocytes/myelocytes to metamyelocytes/band cells
was observed 24 hours after the culture containing SCF + TPO. However,
segmented cells barely appeared even 72 hours after the culture. The
cultured cells were virtually negative for CD35 and ALP throughout the
culture. In the case of SCF + G-CSF, terminal maturation into band
cells and segmented cells became apparent after 48 hours. The frequency
of cells positive for CD35 and ALP corresponded to that of segmented
cells. In the presence of SCF + GM-CSF, there was an intermediate
differentiation into segmented cells, as compared with the other
two-factor combinations. In accordance with the results shown in Table
1, the cells in the culture containing SCF + GM-CSF reacted with
anti-CD35 MoAb, but not ALP.
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| Fig 5.
Sorting of CD34c-kit/low
CD15+ cells grown by SCF + TPO from CD34+
cord blood cells. The cultured cells grown by SCF + TPO at 3 weeks
were stained with APC-conjugated anti-CD34 MoAb, PE-conjugated
anti-c-kit MoAb and FITC-conjugated anti-CD15 MoAb. As negative
controls, APC-, PE-, and FITC-conjugated mouse isotype-matched Ig were
used. (A) The viable cell region (R1) was gated on the basis of FSC and
SSC. (B) CD34 and CD15 expressions of the cells in the R1 region. (C)
The gate (R2) was set on CD34 cells. (D) The expressions
of c-kit and CD15 on these cells were then examined. The cells in the
R3 region were sorted as
CD34c-kit/low CD15+
cells.
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Production of neutrophilic cells by both
CD34+CD38+c-kit+ cells and
CD34+CD38-c-kit+ cells under
stimulation with SCF plus TPO.
To determine which subpopulations of CD34+ cells were
responsible for the SCF + TPO-dependent neutrophilic cell production, we divided CD34+ cells into 4 subsets on the basis of CD38
and c-kit expression: CD38+c-kit+,
CD38+ c-kit-,
CD38-c-kit+, and
CD38-c-kit- cells, as presented in
Fig 6. Single-cell sorting was performed in
all the subsets by the FACStarplus flow cytometer, and each
of the sorted cells was incubated with SCF or SCF + TPO for 3 weeks.
The results are presented in Table 3.
One-fourth of the CD34+CD38+c-kit+
cells formed neutrophilic colonies or neutrophilic cell/mast cell
colonies in response to SCF + TPO. In addition,
CD34+CD38-c-kit+ cells also
responded to the two-factor combination to generate progenies, 25% of
which were of the neutrophilic or neutrophilic/mast cell lineage.
However, the proliferative response of
CD34+CD38+c-kit- cells and
CD34+CD38-c-kit- cells was at a
negligible level. Under stimulation with SCF alone, a small number of
neutrophilic cell-containing colonies were grown only in the culture
with CD34+CD38+c-kit+cells.
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| Fig 6.
Single-cell sorting of four subpopulations of
CD34+ cord blood cells. Cord blood MNCs were stained with
FITC-conjugated anti-CD34 MoAb, APC-conjugated anti-CD38 MoAb, and
PE-conjugated anti-c-kit MoAb. As negative controls, FITC-, APC-, and
PE-conjugated mouse IgG1 were used. (A) The lymphoblastic region (R1)
was gated on the basis of FSC and SSC. (B) The gate (R2) was set on
CD34+ cells. (C) The expressions of CD38 and c-kit on
CD34+cells were examined. The single-cells in the R3, R4,
R5, or R6 region were sorted as
CD34+CD38+c-kit+,
CD34+CD38+c-kit-,
CD34+CD38c-kit+, or
CD34+CD38 c-kit- cells,
respectively, using an automatic cell deposition unit equipped with the
FACStarplus flow cytometer, as described in Materials and
Methods.
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Table 3.
Production of Neutrophilic Cells by Both
CD34+CD38+c-kit+ Cells and
CD34+CD38c-kit+ Cells Under
Stimulation With SCF Plus TPO
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Kinetics of hematopoietic progenitors in the culture containing SCF + TPO.
Finally, we examined the increase of the POX+ cell count
and of colony-forming cells under stimulation with SCF + TPO. As
presented in Fig 7, the significant
POX+ cell generation was observed up to 10 weeks. The
number of GM progenitors increased continuously for 8 weeks. Erythroid
progenitors and multipotential progenitors were also generated, but the
extent of expansion was inferior to that of GM progenitors. At 13 weeks of culture, the number of POX+ cells was rapidly decreased,
and hematopoietic progenitors could not be detected.
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| Fig 7.
Expansion of hematopoietic progenitors under stimulation
with SCF + TPO. After numbers of viable cells were serially counted,
the cultured cells were incubated in methylcellulose culture
supplemented with IL-3, GM-CSF, G-CSF, SCF, and EPO. After 14 days, GM
colonies, erythroid bursts, and mixed erythroid colonies were scored.
The data are the mean ± SD of the number of colonies per well in
triplicate suspension cultures. The results shown are from one
representative experiment of three. Similar results were obtained in
the other two experiments. GM colonies (), erythroid bursts (),
mixed erythroid colonies (), and total colonies ().
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| |
DISCUSSION |
In the present study, SCF alone induced a modest production of
neutrophilic cells and a remarkable generation of mast cells by cord
blood CD34+ cells after 4 weeks of culture. The
SCF-dependent neutrophilic cell production is consistent with results
reported previously.24,25 More than 95% of the cells grown
by TPO were positive for CD41b, and cells of the neutrophilic lineage
were at a negligible level as reported previously.16 In
contrast, the combination of SCF and TPO significantly stimulated the
generation of neutrophilic cells, identified by immunocytochemical
staining and flow cytometric analysis, which continued up to 2 to 3 months of the culture. It is interesting that the effect of SCF + TPO
on the neutrophilic cell generation was greater than that of SCF + G-CSF or SCF + GM-CSF. The possibility was ruled out that the SCF + TPO-dependent neutrophilic cell production resulted from the secretion
of G-CSF and GM-CSF by concomitantly grown mast cells. In addition, no IL-1 , IL-3, or IL-6 was detected in the supernatants of the 8-week and 12-week cells grown by SCF alone or SCF + TPO (data not shown). The
single-cell culture study showed that neutrophilic cells were generated
by not only CD34+CD38+c-kit+ cells
but also CD34+CD38-c-kit+ cells
under stimulation with SCF + TPO, with no influence of nonhematopoietic
cells or mediators other than the two factors. In the clonal-cell
cultures, GM progenitors as well as erythroid progenitors and
multipotential progenitors expanded in the culture supplemented with
SCF and TPO. Taken together with the previous evidence described by
Kimura et al26 that a combination of IL-3, SCF, and
IL-6/soluble IL-6 receptor supports neutrophilic maturation without the
presence of late-acting lineage-specific factors, our results indicate
the existence of a G-CSF/GM-CSF-independent system of neutrophilic
cell production. It is likely that the neutrophilic generation induced
by SCF + TPO is conducted via the production of committed GM
progenitors by primitive progenitors.
The neutrophilic cells grown by SCF + TPO were at myeloblast to band
cell stages. However, there were scarcely any leukocytes at the
polymorphonuclear stage. Although the cells generated by SCF + TPO were
stained with MoAbs against MPO, elastase, lactoferrin, or CD11b, the
specific contents of secretory vesicles such as ALP and CD35 were at
negligible levels. It is well known that ALP is expressed on the
external aspect of the plasma membrane of the fully differentiated
neutrophils. Moreover, Kumar et al27 indicated that
CD35-containing vesicles in polymorphonuclear leukocytes arise by the
endocytic retrieval of proteins that are on the plasma membrane. The
replating of CD34c-kit/low
CD15+ cells into the culture containing SCF + G-CSF
permitted both the terminal maturation into segmented cells and the
appearance of ALP and CD35. This is in agreement with the previous
finding that G-CSF induces ALP in granulocytes from normal
individuals.28 These results suggest that the maturation
arrest at the band cell stage in the neutrophilic cells grown by SCF + TPO is not due to an intrinsic defect, but is the result of an
insufficient potential of the two-factor combination for supporting the
terminal neutrophilic maturation.
The number of mast cells was significantly higher in the cultures
containing SCF + TPO than in the cultures containing SCF alone between
6 weeks and 10 weeks of the culture (Fig 1A and Fig 2). In this regard,
the addition of TPO resulted in an increase in the colony formation
supported by SCF from
CD34+CD38+c-kit+ cells, or
CD34+CD38c-kit+ cells. In
contrast, the mast cell growth was not influenced when TPO was added to
the cultures containing SCF at the 15-week culture (data not shown).
Thus, the increased level of mast cell generation in the presence of
SCF + TPO may result from the two-factor combination-mediated expansion
at the hematopoietic progenitor level, but not at the mast cell level.
The mast cell population kept expanding up to at least 4 months in the
presence of SCF + TPO. This long-term generation of mast cells is
consistent with the results described previously.29,30 On
the other hand, the differentiation toward the neutrophilic lineage was
apparently time limited. Based on the kinetic study of hematopoietic
progenitors in the culture containing SCF + TPO, it may result from an
exhaustion of hematopoietic progenitors belonging to the myelopoietic
lineages.
Because the activation or suppression of one or more lineage- and
developmental-specific transcription factors in part determines the
developmental expression pattern of neutrophil-specific
genes,31 the neutrophilic cells grown by SCF + TPO may be a
useful model for studying the G-CSF- or GM-CSF-mediated regulation of
genes expressed at late stages of neutrophilic maturation.
| |
FOOTNOTES |
Submitted May 18, 1998;
accepted September 14, 1998.
This work was supported by Grants-in-Aid No. 09670796 and 09770537 from
the Ministry of Education of Japan.
The publication costs of this
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.
Address correspondence to Kenichi Koike, MD, Department of Pediatrics,
Shinshu University School of Medicine, 3-1-1, Asahi, Matsumoto,
390-8621, Japan.
| |
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Abstract
Introduction