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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3338-3346
Characterization of Mast Cell-Committed Progenitors Present in
Human Umbilical Cord Blood
By
Duraisamy Kempuraj,
Hirohisa Saito,
Azusa Kaneko,
Kazumi Fukagawa,
Masaharu Nakayama,
Hano Toru,
Morimitsu Tomikawa,
Hiroshi Tachimoto,
Motohiro Ebisawa,
Akira Akasawa,
Toko Miyagi,
Hiromitsu Kimura,
Toshiharu Nakajima,
Kohichiro Tsuji, and
Tatsutoshi Nakahata
From the Department of Allergy and the Department of Experimental
Surgery, National Children's Medical Research Center, Tokyo, Japan;
the Department of Clinical Oncology, The Institute of Medical Science,
University of Tokyo, Tokyo, Japan; and the Department of Bioregulatory
Function, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
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ABSTRACT |
Human mast cells are derived from CD34+ hematopoietic
cells present in cord blood, bone marrow, and peripheral blood.
However, little is known about the properties of the
CD34+ cells. We demonstrated here that mast cell
progenitors that have distinct phenotypes from other hematopoietic cell
types are present in cord blood by culturing single, sorted
CD34+ cells in 96-well plates or unsorted cells in
methylcellulose. The CD34+ mast cell-committed
progenitors often expressed CD38 and often lacked HLA-DR, whereas
CD34+ erythroid progenitors often expressed both CD38 and
HLA-DR and CD34+ granulocyte-macrophage progenitors often
had CD33 and sometimes expressed CD38. We then cultured single cord
blood-derived CD34+CD38+ cells under
conditions optimal for mast cells and three types of myeloid cells, ie,
basophils, eosinophils, and macrophages. Of 1,200 CD34+CD38+ cells, we were able to detect 13 pure mast cell colonies and 52 pure colonies consisting of either one
of these three myeloid cell types. We found 17 colonies consisting of
two of the three myeloid cell types, whereas only one colony consisted
of mast cells and another cell type. These results indicate that human mast cells develop from progenitors that have unique phenotypes and
that committed mast cell progenitors develop from multipotent hematopoietic cells through a pathway distinct from myeloid lineages including basophils, which have many similarities to mast cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MAST CELLS AND BASOPHILS are unique cell
types that possess metachromatic granules composed of highly sulfated
proteoglycans and histamine and that release their granular contents on
cross-linking of their high-affinity receptors for IgE.1
However, mast cells are known to originate from bone marrow progenitors
that migrate into various tissues via blood circulation as immature
cells and undergo complete maturation in the tissues,2,3
whereas basophils complete their maturation within the bone marrow
itself.4 Because of the functional similarities between the
two cell types and the demonstration of human leukemic cells that
possess hybrid granules usually specific for either basophils or mast
cells,5 the possibility that mast cells and basophils share
common bipotent progenitors is still accepted by some investigators. On
the other hand, human basophils were found to possess major basic
protein and Charcot-Leyden crystals, a property they share with
eosinophils, but not mast cells.6 Basophils and eosinophils
also share common progenitors as detected using an in vitro colony
assay,7-9 although these studies cannot completely exclude
the possibility that the common progenitors were multipotent
hematopoietic cells cultured under conditions unsuitable for mast cells.
In contrast to mouse interleukin-3 (IL-3),10,11 which can
act as a mast cell growth factor, human IL-3 by itself does not support
the development of mast cells but instead supports the development of
human basophils and eosinophils.12 The development of human
mast cells was therefore detected in cultures that did not contain
IL-3.13-15 The human mast cell growth factor constitutively expressed on the fibroblast membrane was subsequently cloned and given
several different names, including steel factor (SF), stem cell factor
(SCF), and c-kit ligand.16-18 It is now widely accepted that human mast cells originate from CD34+
cells19,20 and undergo optimal development in the presence of SF.21-23
The primitive human hematopoietic progenitor cells express CD34 but not
CD3824,25 or HLA-DR,26 whereas committed
progenitors, especially erythroid and granulocyte progenitors, often
coexpress CD38 with CD34.23 HLA-DR is often coexpressed on
dendritic cell progenitors,27 B-cell
progenitors,28 erythroid progenitors,29 and
progenitors lacking the ability of stromal cells.30 In the present study, using a single-cell culture system of sorted
CD34+cells, we elucidated the CD34+ progenitors
for human mast cells often coexpressing CD38 and often lacking HLA-DR.
Furthermore, by cultivating singly sorted cord blood-derived
CD34+CD38+ cells under a combined culture
condition suitable for human mast cells,31,32 basophils,
eosinophils, and macrophages,33 we demonstrated that human
mast cells develop from committed progenitors distinct from the three
other myeloid cells.
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MATERIALS AND METHODS |
Cell preparation.
Human umbilical cord blood samples were obtained from normal full-term
deliveries according to the hospital's legal guidelines. Cord blood
was collected in heparinized tubes containing 10 U/mL heparin and
diluted with twice the volume of phosphate-buffered saline (PBS).
Nonphagocytic mononuclear cells were separated by density-gradient
centrifugation using Lymphocyte Separation Medium (LSM; Organon Teknika
Corp, Durham, NC) after depletion of phagocytes with silica (Immuno
Biological Laboratories, Fujioka, Japan). The interface containing
mononuclear cells was collected after density-gradient centrifugation.
Cytokines and antibodies.
Recombinant human thrombopoietin (rhTPO), recombinant human IL-6
(rhIL-6), rhIL-3, and recombinant human erythropoietin (rhEPO) were
generously provided by Kirin Brewery Co, Ltd (Tokyo, Japan). rhSF and
recombinant human granulocyte colony-stimulating factor (rhG-CSF) were
kindly provided by Amgen Biologicals (Thousand Oaks, CA) and Chugai
Pharmaceutical Co (Tokyo, Japan), respectively. The concentrations of
these cytokines used in the first experiments were 100 ng/mL SF, 100 ng/mL IL-6, 40 ng/mL IL-3, 10 ng/mL G-CSF, 2 U/mL EPO, and 4 ng/mL TPO.
Those used in the second series of experiments were 100 ng/mL SF, 50 ng/mL IL-6, and 5 ng/mL rhIL-3 (purchased form Intergen Co, Purchase, NY).
For flow cytometry, cells were treated with monoclonal antibodies
(MoAbs) recognizing various CD antigens. Most of them were provided
from the 6th International Workshop and Conference on Human Leukocyte
Differentiation Antigens.34 CD88 (C5aR, W17/1) and CD123
(IL-3R, 6H6) were purchased from Pharmingen (San Diego, CA) and Serotec
(Kidlington, Oxford, UK), respectively. Fluorescein isothiocyanate
(FITC)-conjugated antihuman CD34 (My10), phycoerythrin (PE)-conjugated
antihuman CD38, PE-conjugated antihuman CD33, and PE-conjugated
antihuman HLA-DR were purchased from Becton Dickinson (San Jose, CA).
Clone sorting.
Clone sorting was performed using the FACS-Vantage (Becton Dickinson,
Mountain View, CA) equipped with an automated cell deposition unit
(ACDU; Becton Dickinson) using a modified method, as previously described.35 Briefly, cells stained with FITC-labeled
anti-CD34 and either one of PE-labeled anti-CD38, CD33, or HLA-DR and
cells stained only with anti-CD34 were respectively sorted from cord blood mononuclear cells into 96-well flat-bottomed plates (Falcon; Becton Dickinson). As negative controls, cells were stained with FITC-
or PE-conjugated mouse IgG1 (Becton Dickinson).
Cell culture.
In the first series of experiments, the clone-sorted cells were singly
cultured in 96-well plates. Each well contained 200 µL  -medium
(Flow Laboratories, Rockville, MD) containing 0.9% methylcellulose
(Shin-etsu Chemicals, Tokyo, Japan), 30% fetal bovine serum (FBS;
Hyclone Laboratories Inc, Logan, UT), 1% deionized bovine serum
albumin (BSA; Sigma Chemical Co, St Louis, MO), 0.05 mmol/L
2-mercaptoethanol (2-ME), SF, IL-6, IL-3, G-CSF, and EPO. All cultures
were scored at day 14 according to criteria reported previously.36,37 To assess the accuracy of the in situ
identification of colonies, individual colonies were lifted with an
Eppendorf micropipette under direct microscopic visualization, spread
onto glass slides using a Cytospin II (Shandon Southern Instruments Inc, Sewickley, PA), and stained for morphological examination using
May-Grünwald-Giemsa stain.
For the detection of mast cell development, the sorted single cells
were cultured in 96 wells in -medium containing 20% FBS, 2-ME, SF,
and IL-6 for 8 weeks with replacement of the half volume of the medium
every week. The colonies were then spread on a slide and stained with
antitryptase MoAb (see below).
In the second series of experiments, the 1,200 CD34+CD38+ cells obtained from 7 different cord
blood donors were plated as single cells into 96-well plates. Each
single cell was suspended in 200 µL IBL Media I (containing insulin,
transferrin, 2-ME, HEPES, and NaSeO3; Immuno Biological
Laboratories) supplemented with 5% FBS (Cansera, Rexdale, Ontario,
Canada), 100 ng/mL SF, 50 ng/mL IL-6, and 5 ng/mL IL-3
and plated into 96-well plates. The CD34+CD38+
cells were cultured for 7 days in the presence of SF, IL-6, and IL-3.
After 7 days of culture, colonies were divided into two aliquots. As
shown in Fig 1, half of the colonies was
cultured for 7 more weeks in the presence of 100 ng/mL SF and 50 ng/mL IL-6 in 96-well plates (plate A). The other aliquot of colonies was
cultured for 3 more weeks in the presence of 5 ng/mL IL-3 in 96-well
plates (plate B). When cells were not found in either one of the two
counterpart plates on day 8, they were excluded from the study. For the
differential count of cultured cells, the samples were centrifuged onto
slides using the Cytospin II and stained with the May-Grünwald
and Giemsa staining method or with immunostaining using the
antitryptase MoAb. The cell counts were determined based on a total of
100 cells, except in cases in which there were fewer than 100 cells.
Colonies that consisted of at least 10 cells in smears were finally
evaluated as colonies.
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| Fig 1.
Experimental design for morphological analysis of
CD34+CD38+ cell-derived colonies. The
clone-sorted CD34+CD38+ cells were singly
cultured for 7 days in the presence of SF, IL-6, and IL-3 in 96-well
plates. After 7 days of culture, colonies were divided into two
aliquots. An aliquot of the colonies was cultured for 7 more weeks in
the presence of SF at 100 ng/mL and IL-6 at 50 ng/mL in 96-well plates
(plate A). The other aliquot of colonies was cultured for 3 more weeks
in the presence of IL-3 at 5 ng/mL in 96-well plates (plate B).
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In some experiments, CD34+ cells were separated using a
magnetic separation column according to the manufacturer's
instructions (Macs II, 441-01; Miltenyl Biotec, Bergisch Gladbach,
Germany). Magnetic microbeads were removed with the CD34-isolation kit
(Miltenyl Biotec). The CD34+ cells were further separated
with either anti-CD38 antibody or anti-HLA-DR antibody and followed by
antimouse IgG1 magnetic microbeads (Miltenyl Biotec). The
300 cells of each fraction were seeded in 1 mL methylcellulose (0.9%)
supplemented with 100 ng/mL SF, 50 ng/mL IL-6, and 5 ng/mL IL-3 (with
or without 10 ng/mL IL-4; purchased from Genzyme Co, Cambridge, MA) in
35-mm Falcon's Petri dish (Becton Dickinson). On day 14 in culture, 1 mL methylcellulose (0.6%) supplemented with SF and IL-6 (without
cells) was further layered over the methylcellulose culture. They were
cultured for total of 28 days. A single colony was lifted with a
Pasteur pipette under an inverted microscope, divided into two parts,
and stained with May-Giemsa or antitryptase MoAb.
For the phenotypic assay of the cultured mast cells and basophils, the
CD34+ cells were cultured in Media I supplemented with 10%
FBS, 100 ng/mL SF, and 50 ng/mL IL-6 in 75-cm2 flasks
(Iwaki Glass, Tokyo, Japan) at 37°C in 5% CO2, for
greater than 10 weeks. For basophil development, a proportion of the
cells on day 7 was subsequently cultured in Media I supplemented with 5 ng/mL IL-3.
Immunostaining for tryptase.
Immunostaining for tryptase was performed using a modified method
described by Craig et al.38 The cytospin smears were first air-dried for a few hours at room temperature and then fixed with Carnoy's solution (60% ethanol, 30% chloroform, and 10% glacial acetic acid) for 1 minute. After fixation, the smears were stained for
granular tryptase by the alkaline phosphatase antialkaline phosphatase
(APAAP) method using the Dako APAAP Kit (Dako Corp, Carpinteria, CA)
according to the manufacturer's instructions. Briefly, the smears were
incubated overnight at 4°C with the mouse antihuman tryptase MoAb
(Chemicon, Temecula, CA; diluted to a final concentration of 1 µg/mL
in Tris-HCl-PBS, pH 7.6, + 10% FBS). The smears were then brought to
room temperature and incubated with the Ig fraction of rabbit antiserum
to mouse Igs for 30 minutes. The smears were then incubated with the
alkaline phosphatase mouse antialkaline phosphatase immune complex for
30 minutes. Between each incubation, the smears were rinsed in
Tris-buffered saline (TBS; pH 7.6) for 10 minutes. Finally, the
reaction was developed with the alkaline phosphatase substrate solution
(containing naphthol AS-MX phosphate, Fast Red, and Levamisole) for 20 minutes and then rinsed briefly in a water bath. Negative controls were
performed either by the omission of the primary antibody or by using an isotype-matched mouse IgG1 antibody instead of the primary antibody.
Immunofluorescence staining.
Expression of cell surface antigens on cultured mast cells or basophils
was analyzed by flow cytometry. Briefly, the cells were incubated with
saturating concentrations of the relevant MoAbs for 30 minutes
(4°C), washed, incubated with the FITC-conjugated goat antimouse
IgG+IgM+IgA antibody (30 minutes; Pharmingen), and analyzed by flow
cytometry using a FACScan (Becton Dickinson, San Jose, CA). In each
experiment, negative controls were performed by using isotype-matched
irrelevant control MoAbs.
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RESULTS |
Phenotypic analysis of mast cell progenitors.
First, we examined the phenotypes of other lineage-committed
hematopoietic progenitors by culturing clone-sorted cells in methylcellulose to compare them with the mast cell-committed
progenitors. As shown in Table 1, most of
the eosinophil progenitors and erythroid progenitors expressed CD34 and
CD38, whereas cord blood cells that gave rise to immature blast cells
in 14 days were mostly CD34+CD38
(94/96). All of the 10 blast cell colonies randomly chosen could give
rise to 19 to 719 secondary colonies (data not shown).
CD34+HLA-DR+ cells gave rise to pure colonies
of either granulocytes, macrophages, eosinophils, or erythroid cells,
respectively, at a frequency of 4 of 13, 30 of 77, 4 of 11, or 22 of
30, respectively. The majority of granulocyte/macrophage colonies were
CD34+CD33+ cells, whereas mixed colonies were
generated predominantly from CD34+CD33
cells. These observations were mostly in agreement with previous observations.24-30
Next, we examined the phenotype of the mast cell progenitors by
culturing the clone-sorted cells in liquid suspension culture suitable
for mast cell development. As shown in
Table 2, 768 CD34+CD38+ cells gave rise to 34 pure mast cell
colonies, whereas 576 of 576 CD34+CD38
cells failed to give rise to pure mast cell colonies. We confirmed the
presence of pure mast cell colonies by examining all the colonies containing the moderate sized cells (Fig
2A) with antitryptase immunostaining. We judged the type of colonies by
in situ appearance as mixed cell type colonies and pure macrophage
colonies (Fig 2B). In contrast to the observation that coexpression of
HLA-DR was preferentially expressed on erythroid progenitors or was not biased on the other cell lineage-committed progenitors (Table 1), the mast cell progenitors rarely coexpressed HLA-DR. In
addition, the pure mast cell colonies were found in both
CD33+ and CD33 cell-derived colonies
(Table 2).
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| Fig 2.
Morphology of single-cell-derived colonies. A typical
mast cell colony grown in the presence of SF + IL-6 (A). A typical
macrophage colony (B) and a typical basophil colony (C) grown in the
presence of IL-3. The colony (A)-derived mast cells stained with
May-Giemsa (D) or antitryptase immunostaining (E). May-Giemsa staining
of a basophil (top) and an eosinophil (bottom) found in a mixed
basophil-eosinophil colony (F). Original magnification: ×183 (A, B,
and C), ×1100 (D and F), and ×733 (E).
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We also examined the phenotypic features of mast cell committed
progenitors by using multifactors, ie, SCF, IL-6, and IL-3 (in the
presence or absence of IL-4), on CD34+/HLA-DR+
versus CD34+/HLA-DR cells and on
CD34+/CD38+ versus
CD34+/CD38 cells. From 1,200 CD34+ cells, 29 pure mast cell colonies are found in the
HLA-DR fractions, whereas 11 pure mast cell colonies
are found in the HLA-DR+ fractions
(Table 3). Similar results were obtained in
the experiments shown in the Tables 2 and 3 for CD38 expression,
although 2 pure mast cell colonies were detected in the experiment
shown in the Table 3.
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Table 3.
Pure Mast Cell Colony Formation Derived From
CD34+ Cells in the Presence of SF, IL-6, and IL-3 (With
or Without IL-4)
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Morphological analysis of
CD34+CD38+ cell-derived colonies.
In the first series of experiments, we thus demonstrated that mast cell
progenitors preferentially expressed unique phenotypes by culturing
single hematopoietic cells under a condition suitable for mast cells
but not for myeloid cells. In the second part of this study, therefore,
we used a combined culture method that optimally supported at least the
development of mast cells, basophils, eosinophils, and macrophages. We
first cultured the clone-sorted CD34+CD38+
cells in the presence of IL-3, IL-6, and SF for 7 days. The
proliferated cells in each well were then split into 2 parts on day 7 of culture and thereafter cultured either in the presence of SF and
IL-6 for 7 more weeks or in the presence of IL-3 only for 3 more weeks (Fig 1).
In preliminary experiments, the addition of IL-3 for 1 week did not
inhibit the development of mast cells, although the continuous addition
of IL-3 for 8 weeks inhibited the mast cell development as previously
reported.32 The transient addition of SF and IL-6 at the
beginning of the culture slightly enhanced IL-3-dependent basophil
growth (data not shown).
The colonies that consisted of basophilic cells by
May-Grünwald-Giemsa staining and of tryptase-positive cells by
immunostaining were defined as mast cell colonies. Of 1,200 single
CD34+CD38+ cells, we were able to detect 13 pure mast cell colonies (Fig 2a, d, and e), 12 pure basophil colonies
(Fig 2c), and 4 pure eosinophil colonies, as shown in
Table 4. In 3 of the 13 mast cell colonies,
tryptase-positive mast cells were also detected in the
IL-3-supplemented wells. We detected 17 double cell type colonies
consisting of eosinophils, basophils (Fig 2g), or macrophages and only
detected 1 colony consisting of mast cells and another cell type. We
also detected 7 triple cell type colonies consisting of basophils,
eosinophils, and macrophages and only 1 colony was found to consist of
mast cells and the two other cell types.
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Table 4.
The Types and the Number of Colonies That Could Be
Morphologically Identified at 4 to 8 Weeks in Culture (See Fig 1)
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As previously reported, mast cells31,32 and
basophils33,39 grown in the present culture conditions
released histamine by IgE-dependent stimuli (data not shown). The
surface antigens expressed on these cultured cells were almost
identical to those expressed on the primary cells, as previously
reported by Valent et al.40,41 The CD34+
cell-derived cultured basophils, but not cultured mast cells, expressed
CD11b, CDw17, CD18, CD31, CD54, CD88, and CDw123 (IL-3R), as did the
primary cells. The cultured mast cells, but not basophils, expressed
CD51, CD61, and CD117 (c-kit). Less than 10% of the cultured mast
cells expressed CD38 after 8 weeks in culture, whereas 10% to 40% of
the cells were judged to be CD38+ until 8 weeks in culture.
Effect of IL-4 on the development of mast cells from
CD34+ or CD34 cells at 3 weeks in
culture.
We have previously reported that IL-4 induces the differentiation of
chymase-positive mast cells when it is added after 10 weeks of culture
in the presence of SF and IL-6.42 One may raise the
question as to whether a particular type of mast cell progenitors such
as HLA-DR+ cells gives rise to chymase-positive mast cells
only in the presence of IL-4. However, IL-4 has been reported to
inhibit the mast cell development, probably via downregulation of c-kit
when it is added at the beginning of culture.43,44 Thus, to
answer the question, CD34+ cells and
CD34 cells were separated with MACS from cord
blood-derived cells cultured in the presence of SF and IL-6 for 3 weeks
and were further cultured in the presence of SF and IL-6 with or
without IL-4 for 3 more weeks. Before the addition of IL-4, the
CD34 cells consisted of 67.5% (53% to 83%)
tryptase-positive cells, whereas less than 5% of tryptase-positive
cells were contaminated in the CD34+ fraction. The
CD34+ fraction formed 26 ± 8 colonies per 1,000 cells
after 3 weeks in the absence of IL-4, whereas the
CD34 cells failed to form cell clusters of more than
10 cells under the same condition. As shown in
Table 5, IL-4 significantly inhibited the
colony formation of the CD34+ fraction. On the other hand,
IL-4 slightly enhanced the SF/IL-6-dependent increase in the number of
tryptase-positive cells from the CD34 fraction
(407% v 243%).
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DISCUSSION |
The present study was designed with the central premise of elucidating
the ontogeny of human mast cells. The CD34+ mast
cell-committed progenitors frequently coexpressed CD38, whereas
primitive CD34+ hematopoietic cells did not express CD38.
The CD38 molecule is expressed on mature granulocytes, some
CD34+ myeloid cells, and lymphocytes at a certain
maturational stage. Mature human mast cells have been
reported to lack CD38 cells.40,41 These results and reports
collectively indicate that the committed progenitors transiently
express this molecule during the development of human mast cells from
multipotent hematopoietic cells. Human CD38 has been recently reported
to be a molecule involved in the regulation of leukocyte adhesion to
endothelial cells,45 indicating that
CD34+CD38+ cells preferably migrate into the
tissues where the appropriate chemoattractants are present, as compared
with CD34+CD38 cells.
The CD34+ mast cell progenitors often lacked HLA-DR. HLA-DR
is not expressed on the primitive hematopoietic cells capable of producing myeloid, lymphoid, and stromal cells,30 although
it has been reported to often be coexpressed on CD34+
erythroid progenitors,29 as shown in the present study. The results shown in Tables 1, 2, and 3 are summarized in
Fig 3. The phenotype of the mast
cell-committed progenitors was mainly CD34+CD38+HLA-DR (Fig 3),
although CD34+CD38+HLA-DR
cells also gave rise to pure neutrophil or eosinophil colonies under a
certain culture condition.
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| Fig 3.
Phenotypic properties of various lineage-committed
progenitors. The results shown in Tables 1 and 2 are summarized. The
phenotype of cells that gave rise to pure mast cell colonies was mainly
CD34+CD38+HLA-DR and that of
erythroid progenitors was
CD34+CD38+HLA-DR+. Cells that
gave rise to pure macrophages, neutrophils, or eosinophils were found
in various phenotypes of CD34+ cells.
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In the first series of studies, we thus demonstrated that mast cell
progenitors have unique phenotypes by culturing single hematopoietic
cells under conditions suitable for mast cell growth but not optimal
for the growth of myeloid cells. In the second series of studies,
therefore, we used a combined culture method that optimally supports
the development of at least mast cells, basophils, eosinophils, and
macrophages. Thus, by culturing 1,200 single
CD34+CD38+ cells in the presence of SF, IL-6,
and IL-3 for 7 days and then culturing them for 3 more weeks in the
presence of IL-3 or for 7 more weeks in the presence of SF and IL-6, we
were able to obtain 13 pure mast cell colonies, 12 pure basophil
colonies, 26 pure macrophage colonies, and 4 pure eosinophil colonies.
Of 14 pure mast cell colonies grown in the presence of SF and IL-6, 1 colony was found to consist of mast cells and another cell type in the presence of IL-3. The remaining 13 mast cell progenitors did not give
rise to any other cell types even in the presence of IL-3, ie, the
culture condition suitable for basophils, eosinophils, and macrophages.
These results suggest that these cell lineages were independent at the
level of CD34+ committed progenitors.
In 5 of the 13 mast cell colonies, tryptase-positive mast cells were
also detected in the IL-3-supplemented wells, indicating that IL-3
also can support the development of a proportion of human mast cells,
as has been previously reported.19 These results suggest
that some populations of mast cell progenitors express receptors for
IL-3.
We detected 17 double cell type colonies consisting of two cell types
among eosinophils, basophils, and macrophages, whereas we could only
detect 1 double colony consisting of mast cells and another cell type.
We also detected 7 triple cell type colonies consisting of basophils,
eosinophils, and macrophages, whereas another colony was found to
consist of mast cells with two other cell types. These results suggest
that human multipotent hematopoietic cells for inflammatory cells tend
to loose their potency for the mast cell lineage, although the pathway
of differentiation is identical to the stochastic model, ie, the
differentiation of multipotent hematopoietic cells is through
progressive and stochastic restriction in cell
lineages.46,47 However, it is necessary to determine
whether the mast cell lineage is closer to other as yet unexamined lineages.
Basophils and eosinophils have been reported to share some common cell
surface antigens, including cytokine receptors, and some common granule
proteins such as major basic protein and are also known to share common
progenitors resulting in mixed eosinophil-basophil colonies.7-9 However, basophils also share several common
features with mast cells and even express low levels of
tryptase.48 Yet, based on these reports, it is rather
difficult to precisely define whether basophils share common precursors
with mast cells or eosinophils. The present conclusion that mast cells
may originate from progenitors different from myeloid progenitors,
including the basophil lineage, can be compared with the observations
made by Agis et al,41 who proposed different progenitors
for mast cells and basophils. These researchers concluded that human
mast cells originate differently from basophils or monocytes, because
they express distinct cell surface antigens from those of basophils or
monocytes, especially cytokine receptors and adhesion receptors.
However, we have recently reported that human cultured mast cells can
express high levels of some integrins such as CD11b when cultured in
the presence of IL-4, which otherwise is expressed by granulocytes,
including basophils,41 but not mast cells.49
Therefore, it is quite difficult to define the ontogeny of mast cells
based only on the differential expression of cell surface antigens. In
this context, our present results provide the first direct evidence
that human mast cells originate from progenitors distinct from those
for myeloid cells, including basophils.
We have previously reported that IL-4 induces the differentiation
chymase-positive mast cells after 10 weeks in culture.42 However, IL-4 inhibited the mast cell development probably via downregulation of c-kit when it is added at the beginning of culture, as previously reported by other investigators.43,44 To
exclude the possibility that a particular type of mast cell progenitors gives rise to chymase-positive mast cells in the presence of IL-4, CD34+ cells and CD34 cells at 3 weeks in
culture were separated and were further cultured in the presence or
absence of IL-4 for 3 more weeks. When the CD34
fraction containing 67.5% tryptase-positive cells was cultured, these
cells proliferated by forming many small cell clusters in methylcellulose. IL-4 slightly enhanced the proliferation. However, colony formation was found only in the CD34+ fraction, and
IL-4 significantly inhibited the formation. These results suggest that
IL-4 is acting only on CD34 and tryptase-positive
cells and that may not be applicable to the ontogeny assay, because it
inhibits the development of CD34+ cells.
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ACKNOWLEDGMENT |
The authors thank Dr Ryuichi Kaku and the staff of the Kaku Obstetric
Hospital at Hino-shi; Dr Kiyoshi Kawashima; Dr Shigenobu Shoda; and the
staff of the Department of Obstetrics, Gyoda Chuo Hospital for their
continuous support by generously providing the umbilical cord blood. We
also thank Dr Bruce Bochner, Dr Ruby Pawankar, Dr Hidetoshi Kawahara,
Dr Ichiro Nomura, Tomohide Hasegawa, and Hisashi Tomita for their
reviewing of the manuscript, assistance, and advice.
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FOOTNOTES |
Submitted July 27, 1998; accepted January 11, 1999.
Supported in part by grants to H.S. from the Japanese Ministry of
Health and Welfare (Pediatric Research Grant No. 9-04) and the Japan
Health Sciences Foundation (Grant No. 5114, 1997) and by a grant from
the Japanese Ministry of Education and Culture to T.N.
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 Hirohisa Saito, MD, PhD, The Department of
Allergy, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509, Japan; e-mail:
hsaito{at}nch.go.jp.
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REFERENCES |
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ABSTRACT
INTRODUCTION