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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4065-4073
Thrombopoietin Enhances the Production of Myeloid Cells, but not
Megakaryocytes, in Juvenile Chronic Myelogenous Leukemia
By
Nobukuni Sawai,
Kenichi Koike,
Tsukasa Higuchi,
Kinya Ogami, and
Megumi Oda
From the Department of Pediatrics, Shinshu University School of
Medicine, Matsumoto; Pharmaceutical Research Laboratory, Kirin Brewery
Co Ltd, Takasaki; and the Department of Pediatrics, Okayama University,
Medical School, Okayama, Japan.
 |
ABSTRACT |
We previously reported the aberrant growth of granulocyte-macrophage
(GM) progenitors induced by a combination of stem cell factor (SCF) and
granulocyte-macrophage colony-stimulating factor (GM-CSF) in juvenile
chronic myelogenous leukemia (JCML). We examined here the effects of
thrombopoietin (TPO) on the proliferation and differentiation of
hematopoietic progenitors in JCML. In serum-deprived single-cell
cultures of normal bone marrow (BM)
CD34+CD38high cells, the addition of TPO to
the culture containing SCF + GM-CSF resulted in an increase in the
number and size of GM colonies. In the JCML cultures, in contrast, the
number of SCF + GM-CSF-dependent GM colonies was not increased by
the addition of TPO. However, the TPO addition caused an enlargement of
GM colonies in cultures from the JCML patients to a significantly
greater extent compared with the normal controls. There was no
difference in the type of the constituent cells of GM colonies with or
without TPO grown by JCML BM cells. A flow cytometric analysis showed
that the c-Mpl expression was found on CD13+ myeloid
cells generated by CD34+CD38high BM cells
from JCML patients, but was at an undetectable level in normal
controls. The addition of TPO to the culture containing SCF or SCF + GM-CSF caused a significant increase in the production of GM
colony-forming cells by JCML CD34+CD38neg/low
population, indicating the stimulatory effects of TPO on JCML primitive
hematopoietic progenitors. Normal BM cells yielded a significant number
of megakaryocytes as well as myeloid cells in response to a combination
of SCF, GM-CSF, and/or TPO. In contrast, megakaryocytic cells
were barely produced by the JCML progenitors. Our results may provide a
fundamental insight that the administration of TPO enhances the
aberrant growth of GM progenitors rather than the recovery of
megakaryocytopoiesis.
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INTRODUCTION |
JUVENILE CHRONIC myelogenous leukemia
(JCML) is a rare myeloproliferative disorder that occurs in infancy and
early childhood.1 The International Juvenile Myelomonocytic
Leukemia Working Group has proposed the term of juvenile myelomonocytic
leukemia.2 In general, the prognosis of patients with JCML
is poor. One reason is that intensive chemotherapy is not superior to
nonintensive chemotherapy or even to no chemotherapy.2
Supportive care such as control for thrombocytopenia is important in
most cases.
Thrombopoietin (TPO), c-Mpl ligand, has been cloned by several
independent groups3-7 as a growth factor specific for the megakaryocyte-platelet lineage. Like the administration of
granulocyte-colony-stimulating factor (G-CSF) for neutropenia, the
clinical application of TPO may be a potent strategy for
thrombocytopenia in patients with JCML. Methia et al8
showed that c-mpl transcript is detected in human CD34+
cells as well as megakaryocytes and platelets. Kobayashi et
al9 reported that TPO synergizes with stem cell factor
(SCF) and/or interleukin-3 (IL-3) in support of the formation
of multilineage colonies, granulocyte-macrophage (GM) colonies, and
erythroid colonies. In addition, TPO has been indicated to stimulate
the proliferation of acute myeloblastic leukemia cells.10
Thus, it is necessary to elucidate the effects of TPO on the growth of
hematopoietic progenitors in JCML.
Emanuel et al11 proposed that the primary mechanism for the
myeloproliferation in JCML is hypersensitivity of the GM progenitors to
GM-CSF. In our previous study, JCML GM progenitors showed the favorable
response to GM-CSF plus SCF.12 We examined here whether TPO
exerted the effects on the proliferation and differentiation of
hematopoietic progenitors in JCML using a serum-deprived culture system.
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MATERIALS AND METHODS |
Patients.
Bone marrow (BM) cells were obtained from five patients who showed the
clinical and laboratory characteristics of JCML at the time of
diagnosis, ie, hepatosplenomegaly and/or skin manifestations, leukocytosis with monocytosis, erythroblastosis with or without anemia
and thrombocytopenia. The clinical data of four of the five patients
(cases 1 to 4) were presented in our previous report.12 The
laboratory findings of case 5 (a 5-month-old girl) were as follows:
white blood cell (WBC) count, 68,600/µL with 12% immature myeloid
cells and 13% monocytes; hemoglobin (Hb) level, 10.2 g/dL; erythroblast count, 2/100 WBC; platelet count, 151,000/µL; and fetal
hemoglobin (HbF) level, 7.8%. A BM examination showed myeloid hyperplasia and a decreased number of megakaryocytes. No chromosomal abnormalities were observed. The patient's BM specimens yielded high
numbers of GM colonies in the absence of hematopoietic growth factors.
When the BM cells were obtained, two of the five patients had been
treated with 6-mercaptopurine (6-MP) and the remaining two with either
etoposide or a combination of 6-MP and cytosine arabinoside. No
treatment had been given to case 5. Four patients treated with the
antileukemic drug(s) had hepatosplenomegaly when the samples were
obtained. The relevant hematological findings were as follows: WBC
counts 4,900 to 14,070/µL; Hb levels 11.0 to 12.2 g/dL; and platelet
counts 27,000 to 321,000/µL. The myeloid hyperplasia, elevated HbF
level, and spontaneous GM colony formation by BM cells were still
observed.
Factors and antibodies.
Human recombinant TPO, SCF, IL-3, and GM-CSF were generously provided
by Kirin Brewery Co Ltd (Takasaki, Japan). Human recombinant G-CSF was
provided by Chugai Pharmaceutical Co (Tokyo, Japan).
For the flow cytometric analysis, monoclonal antibodies (MoAbs) for
CD34 (HPCA-2, fluorescein isothiocyanate, FITC) and CD38 (Leu17,
phycoerythrin, PE) were purchased from Becton Dickinson Immunocytometry
Systems (Mountain View, CA); MoAbs for CD34 (581, phycoerythrin-cyanin
5, PE-Cy 5), CD13 (SJ1D1, PE), and CD 41b (SZ.22) were from Immunotech
S.A. (Marseilles, France). An MoAb against human c-Mpl domain 1 (M113) was obtained from Genzyme Co (Cambridge, MA). In our
preliminary experiments, the Western blot analysis showed that M1 MoAb
recognized the c-Mpl with the molecular weight of 82 kD in
a platelet lysate.
Cell sorting.
BM cells were aspirated in heparinized plastic syringes from the five
patients with JCML and from the three healthy volunteers after
provision of informed consent. BM mononuclear cells (MNCs) were
separated by density centrifugation over Ficoll-Paque (Pharmacia, Piscataway, NJ), washed twice, and suspended in  -medium (Flow Laboratories Inc, Rockville, MD). One milliliter of cell suspension containing 5 × 106 cells in -medium with a final
volume of 10% dimethyl sulfoxide (Sigma Chemical Co, St Louis, MO) and
10% fetal bovine serum (FBS; Hyclone, Logan, UT) was kept in a
freezing tube (Sarstedt, Rommelsdorf, Germany). The samples were frozen
with liquid nitrogen until the study. The MNCs were rapidly thawed, and
then passed through a 200-µm monofilament nylon filter and suspended
in -medium consisting of Ca2+- and Mg2+-free
cold phosphate-buffered saline (PBS), 1 mmol/L EDTA 2 Na, and 2.5%
FBS. Cells (2 × 106) were incubated with both 20 µL
FITC-conjugated anti-CD34 MoAb and 20 µL PE-conjugated anti-CD38 MoAb
for 30 minutes at 4°C. As negative controls, cells were stained with
FITC- and PE-conjugated mouse IgG1 (DAKO, Glostrup,
Denmark). After two washes, CD34+CD38high cells
or CD34+CD38neg/low cells were sorted by a
FACStar plus flow cytometer (Becton Dickinson), as described
previously.12
Serum-deprived single-cell culture.
Single-cell sorting was performed by two-step sorting. BM
CD34+CD38high cells were collected in 5-mL
tubes, and were resorted into the individual wells of a 96-well
U-bottomed tissue culture plate (#3077; Becton Dickinson) containing
100 µL of -medium supplemented with 1% crystallized deionized
bovine serum albumin (BSA; Sigma), 600 µg/mL of fully iron-saturated
human transferrin (approximately 98% pure; Sigma), 16 µg/mL of
soybean lecithin (Sigma), 9.6 µg/mL of cholesterol (Nakalai Tesque
Inc, Kyoto, Japan), and 10 ng/mL of TPO, 10 ng/mL of SCF, 10 ng/mL of
GM-CSF, 100 U/mL of IL-3, 10 ng/mL of G-CSF, alone or in combination,
using the FACStar plus flow cytometer equipped with an
automatic cell deposition unit (Becton Dickinson), as described
previously.14 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. On day
14, GM colonies consisting of more than 50 cells were scored in situ on
an inverted microscope according to the criteria described previously.15,16
Serum-deprived suspension culture.
To examine the effects of TPO on JCML primitive hematopoietic
progenitors, serum-deprived liquid culture was performed in a 96-well
U-bottomed tissue-culture plate according to the procedure described by
Kobayashi et al.9 One hundred
CD34+CD38neg/low cells or 2,000 CD34+CD38high cells were cultured in a well
containing 100 µL of -medium supplemented with 1% BSA, 600 µg/mL of fully iron-saturated transferrin, 16 µg/mL of soybean
lecithin, 9.6 µg/mL of cholesterol, and 10 ng/mL of TPO, 10 ng/mL of
SCF, 10 ng/mL of GM-CSF, alone or in combination. After 7 days, the
cultured cells were washed twice and incubated in methylcellulose
culture supplemented with 30% FBS, 1% BSA, 10 ng/mL of SCF, 10 ng/mL
of GM-CSF, 100 U/mL of IL-3, and 10 ng/mL of G-CSF for 14 days. GM
colonies consisting of more than 50 cells were scored.
Determination of colony size, cytochemical staining, and
immunocytochemical staining.
The size of small GM colonies (<500 cells) was determined by direct
cell counting in situ under an inverted microscope at a magnification
of 150×. Colonies consisting of more than 500 cells were individually
lifted with an Eppendorf micropipet and prepared as single-cell
suspensions. The colony size was estimated by using a counting chamber.
After determination of the colony size, 20 GM colonies were obtained
from each of the patients or normal controls with an Eppendorf
micropipet under an inverted microscope and pooled. After one washing
with PBS, the cells were spread on glass slides using a Cytospin II
(Shandon Southern, Sewickly, PA), and stained with
May-Grünwald-Giemsa, -naphthyl butylate esterase (ANB), or
naphthol AS-D chloroacetate esterase (NASDCA), as described previously.17 Differential counts were done on more than
300 cells in all experiments.
To examine megakaryocyte generation in the cultures of
CD34+CD38+ BM cells and SCF + GM-CSF,
immunocytochemical staining was performed on the Cytospin preparations
using a DAKO LSAB Kit (DAKO) as described previously.18
Briefly, the samples were fixed with buffered paraformaldehyde-acetone
for 30 seconds. After treatment with blocking reagents, the specimens
were incubated with anti-CD41b MoAb for 60 minutes at room temperature.
Next, they were incubated with biotinylated goat anti-mouse antibody
for 30 minutes, followed by alkaline phosphatase-conjugated
streptavidin for 10 minutes and substrate-chromogen solution for 15 minutes. The specimens were counterstained with hematoxylin. More than
300 cells were examined.
Fluorescence-activated cell sorter (FACS) analysis.
To analyze surface markers on the cultured cells, 1 × 105
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 anti-mouse Ig (GAM; Becton
Dickinson) for 15 minutes. After the three washings and treatment with
mouse serum for 15 minutes, the cells were stained with PE-conjugated
anti-CD13 MoAb and/or PE-Cy 5-conjugated CD34 MoAb for 30 minutes. PE- and PE-Cy 5-labeled isotype control antibodies were used
as control. After two washings, cells were analyzed with a FACScan flow
cytometer using the Lysis 2 software program.
Statistical analysis.
All experiments were performed at least twice and were shown to by
reproducible. Colony numbers are expressed as the mean ± SD of
triplicate 96-well culture plates containing a single cell per well.
The probability of significant differences was determined according to
Student's t-test. The statistical analysis of the GM colony
size was performed on logarithms of cell numbers of individual
colonies.
| |
RESULTS |
Effects of TPO on the growth of GM progenitors from JCML
CD34+CD38high cells in serum-deprived
single-cell culture.
To examine the effects of TPO on the growth of hematopoietic
progenitors from the patients with JCML, we used BM
CD34+CD38high and
CD34+CD38neg/low cells as the target cells. As
shown in Fig 1, the percentage of
CD34+CD38high cells among the total BM MNCs was
1.5% ± 0.4% (mean ± SD) in the five JCML patients, being
similar to the value of the three normal controls (1.4% ± 0.6%).
The percentage of CD34+CD38neg/low cells was
0.037% ± 0.02% in JCML patients and 0.042% ± 0.01% in the
normal controls. First, we examined whether TPO enhanced the GM colony
growth by stimulation with SCF alone, GM-CSF alone, SCF plus GM-CSF,
and a combination of SCF, GM-CSF, IL-3, and G-CSF (growth factors, GFs)
using BM CD34+CD38high cells, since our
previous study showed that GM progenitors in JCML have an intrinsic
abnormality in response to hematopoietic factors, in particular, to SCF
plus GM-CSF.12 Serum-deprived single-cell cultures were
used to reduce the influence of cytokines secreted by the paracrine
mechanism. The results are presented in Table
1. In the normal control cultures, the
addition of TPO to the culture containing SCF + GM-CSF significantly
increased the number of GM colonies. However, TPO failed to augment the number of GM colonies under stimulation with SCF alone, GM-CSF alone,
or GFs. In the JCML cultures, in contrast, there was no difference in
the number of GM colonies in the presence or absence of TPO, when
CD34+CD38high cells were plated in SCF alone,
GM-CSF alone, SCF + GM-CSF, or GFs.
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| Fig 1.
Sorting of CD34+CD38+ cells
from JCML and normal BM MNCs. BM MNCs were stained with FITC-conjugated
anti-CD34 MoAb and PE-conjugated anti-CD38 MoAb. As negative controls,
FITC- and PE-conjugated mouse IgG1 were used. The lymphocyte/blast
region (R1) were gated according to their FSC and SSC. The cells in the
R2 region were sorted as CD34+CD38high
cells.
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We then compared the size of GM colonies grown by SCF + GM-CSF
with that of GM colonies grown by SCF + GM-CSF + TPO in JCML patients
and normal controls. As shown in Figs 2 and
3, upon
stimulation with SCF + GM-CSF, the JCML
CD34+CD38high cells generated significantly
larger GM colonies compared with the normal
CD34+CD38high cells (P < .001),
which is consistent with our previous result.12 The
addition of TPO resulted in the enlargement of GM colonies by 33.1 ± 13.0 (20.8 to 50.8)-fold in the JCML cultures, and by only 3.5 ± 0.4 (3.1 to 3.8)-fold in the normal control cultures. The difference in the
degree of enlargement was significant (P < .001).
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| Fig 2.
In situ appearance and the constituent cells of SCF + GM-CSF-dependent or SCF + GM-CSF + TPO-dependent colonies. (A)
and (C) show the colonies with the maximal size grown by stimulation
with SCF + GM-CSF or with SCF + GM-CSF + TPO from JCML
CD34+CD38high cells, respectively. (B) and
(D) show the staining of the constituent cells of 20 pooled colonies
grown by SCF + GM-CSF or with SCF + GM-CSF + TPO from JCML
CD34+CD38high cells with both ANB (brown) and
NASDCA (blue), respectively. (E) and (F) show the staining of the
constituent cells of 20 pooled colonies grown by SCF + GM-CSF + TPO
from normal or JCML BM CD34+CD38high cells
with an MoAb for CD41b using the LSAB Kit, respectively.
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| Fig 3.
Effects of TPO on the size of SCF + GM-CSF-dependent
GM colonies. Mean values and SD for 20 GM colonies grown by SCF + GM-CSF ( ) or SCF + GM-CSF + TPO ( ) are indicated.
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Twenty GM colonies from each of the patients and controls were obtained
and pooled. Cell types were determined by differential counting of the
Cytospin preparations stained with May-Grünwald-Giemsa, ANB, and
NASDCA. The results are presented in Figs 2 and
4. In the normal control group, a large
part of the constituent cells of the GM colonies grown by SCF + GM-CSF were positive for NASDCA. The addition of TPO resulted in
no change in the percentage of NASDCA+ cells. In the JCML
cultures, however, approximately 80% to 90% of the constituent cells
of the SCF + GM-CSF-dependent GM colonies were positive for ANB with
or without TPO. It is possible that the generation of megakaryocytes
contributes to the TPO-mediated increase in the SCF + GM-CSF-dependent
GM colony size, based on the presence of undifferentiated cells. As
presented in Fig 2E and F, the immunocytochemical studies showed that
the mean percentages of CD41b+ cells generated by normal BM
CD34+CD38high cells on day 12 were 1.3% in the
presence of SCF + GM-CSF and 6.3% in the presence of SCF + GM-CSF + TPO. The addition of TPO to the culture containing SCF + GM-CSF caused
an approximately sevenfold increase in the absolute number of
CD41b+ cells. In contrast, no CD41b+ cells were
observed in the cultures containing JCML BM cells and SCF + GM-CSF with
or without TPO.
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| Fig 4.
Differential counts of pooled GM colonies generated by
SCF + GM-CSF or SCF + GM-CSF + TPO. Twenty GM colonies from each
JCML patient and normal control were obtained and pooled.
ANB+ cells ( ), NASDCA+ cells ( ),
undifferentiated cells ().
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To elucidate the mechanism underlying the TPO-mediated enlargement of
JCML GM colonies supported by SCF + GM-CSF, we examined the surface
expression of CD34, CD13, and c-Mpl on the cultured cells, using a
three-color FACScan analysis. CD13 was used as a marker for the myeloid
differentiation. The results are presented in Fig
5. At the beginning of the culture, 6.3% ± 3.2% (n = 3) of the JCML BM
CD34+CD38high cells expressed c-Mpl, whereas
3.1% ± 2.9% (n = 3) of the normal CD34+CD38high cells reacted with anti-c-Mpl
MoAb. In the cultures with SCF + GM-CSF, most of the progenies
generated by the JCML BM cells expressed CD13 antigen, and a small
percentage of CD13+ cells were positive for CD34 until day
8, as shown in Fig 5. CD34+ cells were not detectable on
day 12. Most intriguing was the c-Mpl expression on one third of the
JCML CD13+ cells on day 5, and the percentage of
c-Mpl+ cells in the CD13+ cells increased with
culture (approximately 80% on day 12). In contrast, the c-Mpl
expression was not detected on CD13+ cells from normal
CD34+CD38high cells on day 12, as shown in Fig
6.
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| Fig 5.
Time course of c-Mpl and CD34 expression on
CD13+ cells grown by SCF + GM-CSF from JCML
CD34+CD38high cells. The cells generated by
the JCML CD34+CD38high cells in the presence
of SCF + GM-CSF were labeled with anti-c-Mpl MoAb, and then stained
with FITC-conjugated GAM. After treatment with mouse serum, the cells
were stained with PE-conjugated CD13 MoAb and PE-Cy 5-conjugated CD34
MoAb. Surface marker expression was analyzed on days 5, 8, and 12. The
quadrants were determined by the negative controls using
isotype-matched Ig.
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| Fig 6.
Comparison of c-Mpl expression on CD13+
cells generated by JCML or normal
CD34+CD38high cells in the presence of SCF + GM-CSF. The cells generated by JCML or normal
CD34+CD38high cells on day 12 were stained
with anti-c-Mpl MoAb ( ) or mouse IgG1, (---), and then stained with
FITC-conjugated GAM. After treatment with mouse serum, the cells were
stained with PE-conjugated anti-CD13 MoAb. Surface-marker expression on
the cells in R1 region was analyzed.
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Effects of TPO on the production of GM colony-forming cells by JCML
CD34+CD38neg/low cells.
Next we examined the effects of TPO on JCML primitive hematopoietic
progenitors using a serum-deprived suspension culture, as described by
Kobayashi et al.9 The
CD34+CD38neg/low cells sorted by the FACStar
plus flow cytometer were incubated with TPO, SCF, GM-CSF, alone or in
combination for 7 days. GM colony-forming cells were assayed in clonal
cultures containing SCF, GM-CSF, IL-3, and G-CSF. As shown in Table
2, production of GM colony-forming cells by
CD34+CD38neg/low population was significantly
increased by the addition of TPO to the culture containing SCF + GM-CSF
in two JCML patients and by the addition of TPO to the culture
containing SCF in the remaining patient. The combination of SCF + TPO
or SCF + GM-CSF + TPO generated 11 to 39 GM colony-forming cells from
100 JCML CD34+CD38neg/low cells. On the other
hand, only 2 to 15 GM colony-forming cells were produced by 2,000 JCML
CD34+CD38high cells under stimulation with the
two-factor or three-factor combination. The stimulatory effects of TPO
on the SCF-dependent or SCF + GM-CSF-dependent production of GM
colony-forming cells were either absent or present at variable levels.
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Table 2.
Effects of TPO on the Production of GM Colony-Forming
Cells by JCML CD34+CD38neg/low Cells and
CD34+CD38high Cells in Suspension
Culture
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DISCUSSION |
The apparent GM colony growth in the absence of hematopoietic
factors is a hallmark of JCML. The endogenous production of IL-1,
GM-CSF, or tumor necrosis factor- by monocytes has been shown to
account for the spontaneous GM colony growth in JCML.19-21 In addition, FBS is a potential endogenous source of hematopoietic factors, as described previously.22 Therefore, we used a
serum-deprived single-cell culture technique to clarify the direct
effects of TPO on the growth of hematopoietic progenitors in JCML
CD34+CD38high cells. The present study showed
that SCF or GM-CSF alone yielded lower numbers of GM colonies, as
compared with our previous results.12 This may be due to a
difference in the target cells and/or the culture system.
We have previously reported the favorable response of GM progenitors to
SCF + GM-CSF in JCML.12 The present results showed that, in
normal BM cells, the addition of TPO to cultures containing SCF + GM-CSF resulted in increases in both the number and size of GM
colonies. On the other hand, in the case of JCML, TPO failed to
increase the number of GM colonies supported by SCF + GM-CSF, but
enlarged GM colonies to a significantly greater extent, as compared
with the increase in the size of GM colonies grown by normal
CD34+CD38high cells. There was no difference in
the type of constituent cells of GM colonies with or without TPO grown
by JCML BM cells. Although the c-Mpl expression on JCML BM
CD34+CD38high cells was at a low level in JCML,
as in normal BM CD34+CD38high cells, most of
the JCML myeloid progenies in the cultures expressed c-Mpl at a higher
level compared with the normal myeloid progenies. Thus, the combined
effect of SCF and GM-CSF appears to be optimal for the initial stage of
the proliferation of JCML GM progenitors. Alternatively, TPO may not
stimulate the entry of SCF + GM-CSF-dependent GM progenitors to the
proliferative process, but it can enhance their subsequent growth, with
no influence on the differentiation into the myeloid lineage.
In addition to a high level of the c-Mpl expression on the JCML
CD13+ cultured cells generated by SCF + GM-CSF, one third
of the circulating CD13+ cells of the JCML patients reacted
with anti-c-Mpl MoAb. However, c-Mpl expression was not detected on
normal circulating CD13+ cells (unpublished data, January
1998). Thus, a higher level of the c-Mpl expression
implies an essential abnormality in JCML myeloid cells.
It is generally held that the CD34+CD38
immunophenotype defines a primitive subpopulation of progenitors in
fetal liver, fetal BM, cord blood, and adult BM.23-29
Lapidot et al30 indicated that JCML stem cells are also
present in a CD34+CD38 fraction, based on
the results of the transplantation into severe combined immune
deficient mice. In the present study, the addition of TPO to the
culture containing SCF or SCF + GM-CSF caused a significant
increase in the production of GM colony-forming cells by JCML
CD34+CD38neg/low population. These results
suggest that primitive hematopoietic progenitors in JCML can be
responsive to TPO.
In the presence of TPO alone, pure megakaryocyte colony growth was not
found in the cultures containing normal or JCML BM CD34+CD38high cells. Our serum-deprived liquid
culture allowed the generation of a large number of megakaryocytes from
cord blood CD34+ cells.18 However, in the
serum-deprived single-cell culture of normal BM CD34+
cells, only 4% of the wells contained two to four
megakaryocytes by stimulation with TPO alone, whereas half of wells
contained various combinations of progenies including megakaryocytes in the presence of SCF, GM-CSF, G-CSF, IL-3, TPO, and erythropoietin (unpublished data, January 1998). Therefore, the present
results may be explained by a low frequency of TPO-responsive
megakaryocytic progenitors in BM CD34+CD38high
cells rather than the inappropriate culture condition. The normal BM
cells generated a significant number of megakaryocytes as well as
myeloid cells in response to the combination of SCF, GM-CSF, and/or TPO on day 12. In contrast, megakaryocytic cells were
barely produced by the JCML progenitors. These findings suggest that the thrombocytopenia in JCML, in part, results from a defective megakaryocyte production.
In conclusion, our results may provide a fundamental insight for the
clinical application of TPO in JCML, suggesting that the administration
of TPO augments the aberrant growth of GM progenitors rather than the
recovery of megakaryocytopoiesis.
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FOOTNOTES |
Submitted October 27, 1997;
accepted January 15, 1998.
Supported by Grants-in-Aid No. 09670796 and 09770537 from the Ministry
of Education of Japan.
Address reprint requests to Kenichi Koike, MD, Department of
Pediatrics, Shinshu University School of Medicine, 3-1-1, Asahi, Matsumoto, 390, 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.
| |
ACKNOWLEDGMENT |
We are deeply indebted to Prof A. Komiyama (Department of Pediatrics,
Shinshu University School of Medicine) for helpful comments. We also
thank Dr S. Ito (Blood Transfusion Service, Shinshu University Hospital) for his excellent technical assistance. We are grateful to
Drs H. Miyazaki and T. Kato of Kirin Brewery Co, Ltd, for supplying recombinant TPO and other growth factors.
| |
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Abstract
Introduction
Abstract