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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3703-3712
By
From Department of Pediatrics, Shinshu University School
of Medicine, Matsumoto, Japan; Blood Transfusion Service, Shinshu
University Hospital, Matsumoto, Japan; Pharmaceutical Research
Laboratory, Kirin Brewery Co, Ltd, Takasaki, Japan; and Research & Development, Mitsubishi Kagaku Bio-Clinical Laboratories, Inc, Tokyo,
Japan.
The effects of thrombopoietin (TPO) and/or stem cell factor (SCF) on
the development of human mast cells from CD34+ bone
marrow (BM) cells were investigated using a serum-deprived liquid
culture system. Mast cells were identified by measurement of
intracellular histamine content, immunocytochemical staining, and flow
cytometric analysis. Whereas SCF alone generated only a small number of
tryptase+ cells, the addition of TPO to the culture
containing SCF resulted in an apparent production of mast cells from 3 weeks until at least 15 weeks. Some of the cells reacted with an
antichymase monoclonal antibody as well. Based on the effects of growth
factor(s) on a later phase of the mast cell growth, TPO may stimulate
an early stage of mast cell development in combination with SCF, whereas subsequent growth seems to be supported by SCF alone. Single-cell culture studies indicated that the
CD34+CD38
HUMAN MAST CELLS are distributed over the
whole body in tissues including skin, lungs, gut, and nasal mucosa, and
are classified into two phenotypically distinct subpopulations on the
basis of their protease expression:
tryptase+chymase In the murine system, it has been elucidated that mast cells originate
from hematopoietic stem cells in vivo2 or multipotential hematopoietic progenitors in vitro.3 Mast cell precursors
depart from bone marrow (BM) and migrate into connective or mucous
tissues, where they differentiate into the mature form. The stem cell
factor (SCF)-c-kit receptor signal transduction pathway is essential for the development of murine mast cells, because both W
and steel (SI) mice, which have mutations in the locus of c-kit
receptor and SCF, respectively, are deficient in mast
cells.4 In addition to SCF, interleukin (IL)-3, IL-4, IL-9,
and IL-10 promote the proliferation and differentiation of mast
cells.5-8 In the human system, SCF has been shown to act as
a major growth and differentiation factor for mast cell development
from cord blood (CB) mononuclear cells (MNCs),9 BM
cells,10,11 and fetal liver cells.12 However,
there are several differences between the human and murine mast cell
development systems. First, it was shown that the addition of IL-6 or
IL-11 to cultures containing SCF enhances the growth of mast cells from
CD34+ CB cells.13 Second, neither IL-3 nor IL-4
induces the differentiation of human mast cells.14-16
Although the hypothesis that monocytic precursors or basophils are
candidates for mast cell precursors was previously
proposed,17,18 Agis et al19 clearly showed that
human mast cells are derived from CD34+ cells. However, the
clonal origin of human mast cells from multilineage BM-derived
progenitor cells has not been established because of the
limitation of assays available.
Thrombopoietin (TPO) was cloned by several groups as a potent
stimulator in megakaryocytopoiesis.20-24 Recent studies
showed that TPO can act on the growth of hematopoietic progenitors
(especially multipotential progenitors) in combination with other
cytokines, including SCF, IL-3, and flt3 ligand (FL).25,26
In the present study, we examined, using CD34+ BM cells,
whether TPO can stimulate the SCF-dependent proliferation and
differentiation of human mast cell progenitors/precursors in a
serum-deprived culture system.
Factors and antibodies.
Human recombinant TPO, SCF, IL-3, granulocyte-macrophage
colony-stimulating factor (GM-CSF), and erythropoietin (EPO) were provided by Kirin Brewery Co Ltd (Takasaki, Japan). Human recombinant FL was purchased from PeproTech EC Inc (Rocky Hill, NJ). Human recombinant granulocyte colony-stimulating factor (G-CSF) was a gift
from Chugai Pharmaceutical Co (Tokyo, Japan). Human recombinant IL-6
was kindly provided by Ajinomoto Co (Kawasaki, Japan). Human recombinant IL-11 was purchased from R & D Systems (Minneapolis, MN).
Isolation of CD34+ cells from bone marrow MNCs.
BM cells were aspirated in heparinized plastic syringes from healthy
volunteers after informed consent was obtained. BM MNCs were separated
by density centrifugation over Ficoll-Paque (Pharmacia, Piscataway,
NJ), washed twice, and suspended in Ca2+- and
Mg2+-free phosphate-buffered saline (PBS) containing 1 mmol/L EDTA 2-Na and 2.5% fetal bovine serum (Hyclone, Logan, UT). The
cells (2 × 106) were incubated with 20 µL of
FITC-conjugated anti-CD34 MoAb for 30 minutes at 4°C. As negative
controls, the cells were stained with FITC-conjugated mouse IgG1
(Dako). After two washes, CD34+ cells were sorted by a
FACStarplus flow cytometer (Becton Dickinson), as described
previously.27
Serum-deprived suspension culture.
Serum-deprived liquid cultures were carried out in 24-well culture
plates (#3047; Becton Dickinson), using the technique described previously.28,29 Five to 10 × 103
CD34+ BM cells were cultured in each well containing 2 mL
of Serum-deprived single-cell culture.
Single-cell sorting was performed by two-step sorting, as described
previously.29 BM MNCs (2 × 106) were
incubated with 20 µL of FITC-conjugated anti-CD34 MoAb, 5 µL of
APC-conjugated anti-CD38 MoAb, and 20 µL of PE-conjugated anti-c-kit
MoAb for 30 minutes at 4°C. As negative controls, the 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 Flow cytometric analysis.
For the analysis of surface markers on the cultured cells, the cells
were collected in plastic tubes and incubated with appropriately diluted FITC- or PE-MoAbs, as described previously.29 The
cells were washed twice, and their surface markers were analyzed with a
FACScan flow cytometer (Becton Dickinson) 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 with cells stained with FITC- or PE-conjugated mouse isotype matched Ig. To analyze the surface expression of c-mpl on CD34+ BM cells, cultured mast cells,
and cultured megakaryocytic cells, the cells were incubated with 20 µL anti-c-mpl MoAb for 30 minutes at 4°C. Isotype MoAb was used as
a control. The cells were washed three times and stained with
FITC-conjugated goat antimouse immunoglobulin (GAM; Becton Dickinson)
for 15 minutes. CD34+ BM cells and cultured cells grown by
TPO were washed three times and treated with mouse serum for 15 minutes. Then, the cells were stained with PE-conjugated anti-CD34 MoAb
or biotinized anti-CD41 MoAb, followed by Cy-Chrome-labeled
streptavidin (PharMingen, San Diego, CA). PE-conjugated and biotinized
isotype antibodies were used as controls.
Cytochemical and immunologic stainings.
Cultured cells were spread on glass slides using a Cytospin II (Shandon
Southern, Sewickly, PA) and stained with May-Grünwald-Giemsa. A
cytochemical reaction with peroxidase (POX) was performed by the
conventional method.
Histamine assay.
Cultured cells were lysed with 0.5% Nonidet P-40 (Nakalai
Tesque Inc), and the content of histamine in the cell lysate was measured by a radioimmunoassay (RIA; Immunotech S.A.). All assays were
conducted in triplicate.
Reverse transcription-polymerase chain reaction (RT-PCR).
RT-PCR was performed according to a modification of the procedure
described previously.28 CD34+ BM cells,
15-week-old c-kit+ cultured cells grown by SCF+TPO, and day
10-cultured CD41+ cells grown by TPO were sorted by the
FACStarplus flow cytometer, as described
previously.27 Total RNA was individually isolated from the
cells, using Isogen (Wako Pure Chemical Industries, Osaka, Japan).
Next, 200 ng of RNA was reverse-transcribed in 200 U SuperScript II
(Life Technologies, Gaithersburg, MD), 10 Statistical analysis.
All experiments were carried out at least three times and were shown to
be reproducible. Values are expressed as mean ± standard deviation (SD). One-way analysis of variance, followed by
post hoc contrasts with Bonferroni limitation, was employed for four independent groups.
Combination of SCF and TPO stimulates the growth of mast cells by
CD34+ BM cells in long-term serum-deprived liquid cultures.
CD34+ BM cells were cultured at 10,000 cells per well
containing serum-deprived liquid culture medium supplemented with 10 ng/mL of TPO and/or 10 ng/mL of SCF. The results are shown in Fig
1A. In the absence of the growth factors,
almost all of the cells degenerated within 2 weeks. The addition of SCF
alone supported the growth of approximately 1,000 to 2,000 viable cells
at 2 to 3 weeks. Although portions of the cells were megakaryocytes,
neutrophils, and mast cells, the remaining cells were blastic and
degenerated without terminal differentiation within 3 weeks. Even 100 ng/mL of SCF failed to increase the cell production (data not shown). Under stimulation with TPO, a small number of megakaryocytes
(approximately 200 cells) were generated between day 7 and day 10, but
there were no viable cells beyond 2 weeks. In contrast, a combination of SCF and TPO caused marked cell production. The total viable cell
number increased to approximately 13 times the input quantity after 2 weeks. It decreased after 3 weeks, but remained unchanged from 6 to 15 weeks. Under this culture condition, a low percentage of megakaryocytes
was seen until 2 weeks. After 3 weeks of culture, a large part of the
cultured cells became positive for either POX or tryptase (a mast
cell-specific protease), as shown in Fig 1B. The POX+
cells were of the neutrophilic lineage, in the light of the reaction with anti-MPO MoAb. The frequency of tryptase+ cells was
25% at 3 weeks of culture and reached levels higher than 95% after 12 weeks. Consequently, the absolute numbers of cultured mast cells grown
by SCF + TPO were relatively constant between 3 weeks and 15 weeks.
Some tryptase+ cells showed nuclear lobulation. The
frequency of the chymase+ cells was 5% ± 2%
(mean ± SD) at 3 weeks and 31% ± 3% at 15 weeks of culture in
three independent experiments. In addition, the 9-week-old cultured
cells contained a significant amount of histamine (3 to 8 ng per
5 × 104 cells). The percentage of GPA-, CD2-, or
CD19-positive cells was less than 1% at 2, 3, 6, and 12 weeks of the
culture.
Comparison of the ability of various two-factor combinations to
generate mast cells.
Because Nakahata et al13 described the generation of a
sufficient number of mast cells by CB MNCs in the culture with SCF+IL-6 or SCF+IL-11, but not SCF + G-CSF, we compared effects of these two-factor combinations with those of SCF + TPO on the growth of mast
cells from CD34+ BM cells. SCF, IL-6, IL-11, and G-CSF were
used at 10 ng/mL, 50 ng/mL, 50 ng/mL, and 10 ng/mL, respectively. After
3 weeks, numbers of c-kit+CD15 Effects of FL or GM-CSF on the SCF + TPO-dependent mast cell growth.
Next, we examined the effects of FL on the SCF + TPO-dependent mast
cell production by CD34+ BM cells. Five thousand
CD34+ cells were plated in a well containing 10 ng/mL of
SCF, 10 ng/mL of TPO, or 50 ng/mL of FL, alone or in combination. The
cultured mast cells and myeloid cells were distinguished on the basis
of c-kit and CD15 expressions, ie, the
c-kit+CD15
Production of mast cells by
CD34+CD38+c-kit+ cells and
CD34+CD38
Differentiative potentials of mast cell-producing colony-forming
cells supported by SCF plus TPO.
To assess the differentiative potentials of mast cell-producing
colony-forming cells supported by various types of growth factor
combinations, we performed a two-step culture assay. Each of the sorted
cells was initially incubated with SCF + TPO, SCF + FL, TPO + FL, or
SCF + TPO + FL. When the cell number in each well reached more than 10 cells between day 10 and day 32, the cells were divided into two
aliquots. One half of the sample was replated in a well containing SCF + TPO for the expression of neutrophil and mast cell lineages. The
other half was recultured in a well containing G-CSF, SCF, TPO, FL,
GM-CSF, IL-3, and EPO (GFs) for the expression of neutrophil,
macrophage, megakaryocyte, and erythroid lineages. We defined the sum
total of cell lineages expressed under stimulation with SCF + TPO or
GFs as the full potentials of hematopoietic progenitors. The results
are presented in Table 2. In the
CD34+CD38+c-kit+ subset, all of the
SCF + TPO-responsive progenitors yielded progenies including mast
cells. Half of them had a differentiative capacity restricted to
neutrophil/mast cell lineages, and the remaining half could
differentiate into neutrophil/macrophage/mast cell lineages. In the
CD34+CD38
Activation via c-kit is essential for murine mast cell development,
because SCF- or c-kit-deficient mice lack mast cells. As in mice,
human SCF by itself can induce human mast cell development from
hematopoietic progenitors in serum-containing culture
condition.9,11,12,19 However, in our serum-deprived culture
of CD34+ BM cells, SCF seldom generated mast cells, and TPO
was required for the substantial growth. The identification of mast
cells was performed by measurements of intracellular histamine,
immunocytochemical staining, and flow cytometric analysis. One possible
explanation for the poor mast cell growth obtained from
CD34+ BM cells stimulated by SCF alone may be an inferior
proliferative potential of SCF-responsive mast cell-containing
colony-forming cells under our culture condition. Fetal bovine serum
has been thought to be a potential endogenous source of hematopoietic
factors.33,34 Coupled with the evidence that both adult
peripheral blood and CB contain significant levels of
TPO,35,36 BM mast cell progenitors may be dependent on the
synergistic factor(s) present in the serum. Nevertheless, the
SCF-c-kit signaling system seems to be important for the generation of
mast cells from human BM progenitors, based on the finding that the
progenitors responding to SCF + TPO were exclusively restricted to
those expressing c-kit receptors.
Submitted October 1, 1998; accepted January 22, 1999.
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 reprint requests to Kenichi Koike, MD, Department of
Pediatrics, Shinshu University School of Medicine, 3-1-1, Asahi,
Matsumoto, 390-8621, Japan.
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TOP
ABSTRACT
INTRODUCTION
c-kit+ cells and
CD34+CD38+c-kit+ cells were
responsible for the SCF + TPO-dependent mast cell production.
Two-step culture assays clearly showed that mast cells originated from
multilineage colony-forming cells that had potential to differentiate
into neutrophil/mast cell lineages, neutrophil/macrophage/mast cell
lineages, or neutrophil/macrophage/mast cell/erythroid lineages. These
results suggest that TPO plays an important role in the development of
human mast cells from CD34+ BM cells in concert with SCF,
and provide direct evidence of the differentiation into the mast cell
lineage of human multipotential BM-derived progenitors.
-medium (Flow Laboratories Inc, Rockville, MD) supplemented with
1% deionized bovine serum albumin (Sigma Chemical Co, St Louis, MO),
600 µg/mL fully iron-saturated human transferrin (approximately 98%
pure; Sigma), 16 µg/mL soybean lecithin (Sigma), and 9.6 µg/mL
cholesterol (Nakalai Tesque Inc, Kyoto, Japan) in the presence of 10 ng/mL TPO, 10 ng/mL SCF, 10 ng/mL GM-CSF, 50 ng/mL FL, 50 ng/mL IL-6, 50 ng/mL IL-11, or 10 ng/mL G-CSF, 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 cell-free supernatant was replaced with
fresh medium containing growth factor(s) every 5 to 7 days. The number
of viable cells was determined by a trypan-blue exclusion test using
hemocytometers, and the cells were processed for the cytochemical and
immunologic stainings and flow cytometric analysis.
-actin cDNA, 5'-CTGGACTTCGAGCAAGAGAT-3' (nt
702-721), and 5'-TCGTCATACGCCTGCTTGCT-3' (nt 1132-1113).
), no growth
factors; (
), SCF; (
), TPO; (
), SCF + TPO. (B) Time course of
the relative frequency of tryptase-positive cells and
peroxidase-positive cells grown by SCF + TPO. The cultured cells
grown by SCF + TPO were stained with a MoAb against tryptase or with
POX. (
), POX-positive cells; (
), labeled with PE-conjugated anti-c-kit MoAb or anti-c-mpl
MoAb followed by FITC-conjugated GAM. (---), labeled with PE-conjugated
mouse IgG1 or control mouse IgG1 followed by FITC-conjugated GAM. The
abscissa shows the fluorescence intensity of each surface marker, and
the ordinate shows the number of cells. (b) RT-PCR analysis: lane 1, no
RNA; lane 2, CD34+ BM cells; lane 3, cultured
c-kit+ cells grown by SCF + TPO; lane 4, cultured
CD41+ cells grown by TPO.
