Leukemia (1998) 12, 907–911
 1998 Stockton Press All rights reserved 0887-6924/98 $12.00
Recombinant human megakaryocyte growth and development factor (MGDF)
increases the numbers of megakaryocyte progenitor cells to normal values in long-
term bone marrow cultures of patients with AML in first remission
C Kasper1, A Schwarzer1, EA De Wynter1, J Chang1, TM Dexter1, D Ryder2 and NG Testa1
1
Cancer Research Campaign Department of Experimental Haematology, Paterson Institute for Cancer Research, and 2Department of Medical
Statistics, Christie Hospital, Manchester, UK
The megakaryopoietic potential in the bone marrow (BM) of
patients in first remission after treatment for acute myelogen-
ous leukaemia (AML) was investigated using long-term bone
marrow cultures (LTC) stimulated with megakaryocyte growth
and development factor (MGDF). The baseline number of
megakaryocyte colony-forming cells (Meg-CFC) was very low.
However, there was a 10 to 100-fold increase of Meg-CFC in
cultures treated with 10 ng/ml MGDF with mean numbers within
the normal range for the first 4 weeks of culture with a 24-fold
increase in their cumulative numbers. Similarly, a 12-fold
increase in the numbers of megakaryocytes (MKs) was found
by CD61 immunostaining. These effects were lost at the dose
of 100 ng/ml. In contrast, the cumulative mean numbers of Meg-
CFC in the control cultures from normal bone marrow (NBM)
were not significantly different from those in cultures treated
with 10 or 100 ng/ml MGDF. These results demonstrate that
MGDF stimulates megakaryocytopoiesis in patients with AML
in first remission, restoring the Meg-CFC compartment to
normal values, a result with potential clinical implications for
their treatment with autologous transplantation.
Keywords: megakaryocyte growth and development factor
(MGDF); thrombopoietin; colony assay; long-term bone marrow
culture (LTC); AML
Introduction
Megakaryocyte growth and development factor (MGDF) or
thrombopoietin, the ligand of the c-mpl receptor, has been
purified and cloned by several groups independently.1–6
MGDF has potent effects on the generation, proliferation and
differentiation of megakaryocyte (MK) progenitor cells in vitro
and in vivo.7–10
Patients treated for acute myelogenous leukemia (AML) who
achieved first remission show severe and persistent haemato-
poietic damage manifested by substantially decreased num-
bers of clonogenic progenitor cells and of stem cells.11 AML
patients in first or subsequent remissions may be treated with
autologous transplantation following high-dose chemo-
therapy.12,13 Severe and prolonged thrombocytopenia is usu-
ally observed after this treatment.
The effect of MGDF on long-term cultures (LTC) derived
from patients with AML in first complete remission was inves-
tigated in order to assess their megakaryopoietic potential and
response to stimulation. The results indicate that MGDF has
the ability to increase the numbers of MK progenitors (Meg-
CFC) and CD61+ cells from a very low baseline to within
normal values.
Correspondence: C Kasper, Department of Internal Medicine (Cancer
Research), West German Tumor Center, University of Essen, Hufe-
landstr. 55, 45122 Essen, Germany; Fax: 0201 723 5924
Received 3 October 1997; accepted 30 January 1998
http://www.stockton-press.co.uk/leu
Materials and methods
Patients
BM samples were collected with informed consent from 13
patients with AML in first complete remission. Patients’
characteristics are shown in Table 1.
As induction and consolidation treatment patients received
two to three cycles of DAT chemotherapy, containing dauno-
rubicin (60 mg/m2 i.v. days 1–3), cytosine arabinoside
(200 mg/m2 continuous infusion, days 1–7) and thioguanin
(200 mg/m2, days 1–7), followed by up to two cycles of inter-
mediate dose cytosine arabinoside (1.5 g/m2 at 12 h intervals,
days 1–3). Two patients also received high-dose busulfan
(16 mg/kg body weight) with peripheral blood progenitor cell
rescue prior to the collection of the bone marrow sample, and
one patient an allogenic BM transplantation.
Long-term cultures (LTC)
BM samples were obtained after informed consent from heal-
thy donors (NBM) for BM transplantation or AML patients
(AMLBM). Cells were separated from the red cells by gravity
sedimentation in 0.1% methylcellulose for 45–60 min at room
temperature, and washed twice in Iscove’s modified Dulbec-
co’s medium (IMDM, Gibco BRL) plus 2% fetal calf serum
(FCS, Gibco BRL, Life Technologies, Paisley, UK). 1.5–2 × 106
buffy coat cells/ml diluted in 10 ml LTC medium that con-
tained 80% IMDM, 10% FCS, 10% horse serum (HS, Sera Lab-
oratories, Crawley Down, UK), and 5 × 10−7 M hydrocortisone
were inoculated into tissue culture flasks (Falcon, Becton
Dickinson, Franklin Lakes, NJ, USA), gassed with 5% CO2 in
air and incubated at 33°C.14 Rh MGDF (a gift from Amgen
Corporation, Thousand Oaks, CA, USA) was added to the LTC
at two concentrations, 10 or 100 ng/ml. The cultures were
maintained by weekly feeding replacing half of the growth
medium with fresh medium and MGDF; control cultures with-
out MGDF were performed concurrently. The non-adherent
cells removed at feeding were used for the assays described
below.
Clonogenic assays
Cells (105) were plated in a mixture containing 30% FCS, 1%
deionized bovine serum albumin (BSA), 10% conditioned
medium from the bladder carcinoma cell line 5637 (as a
source of growth factors), 2 U erythropoietin (Boehringer
Mannheim, West Lothian, UK) and 1.35% methylcellulose
(Sigma, St Louis, MO, USA). Cultures were plated in triplicate
and incubated at 37°C for 14 days in a fully humidified atmo-
sphere of 5% CO2, 5% O2 in nitrogen. Colonies consisting of
 MGDF stimulates megakaryocytopoiesis in first remission AML
C Kasper et al

908 Table 1 Patients’ characteristics

No. Age Sex FAB Cytogenetics No. of chemotherapy Time between CR

prior to BM sample and BM sample
DAT ID Ara-C (month)

1 53 M M2 t(8;21) 3 2 6
2 42 M M5b ND 2 1 2
3 54 M M1 ND 2 1 6
4 36 F M2 normal 3 2 8
5 48 M M4 del (9) 1 0 0
6 62 M M2 −7 2 0 5
7 63 M M2 normal 2 0 0
8 58 F M0 normal 3 2 8
9a 26 M M2 normal 2 2 8
10 32 M M4 t(8;21), t(4;10) 3 0 3
11 20 M M2 t(3;5), del (9) 3 0 3
12b 28 M M2 ND 2 0 106
13a 23 M M2 ND 2 1 56

a
Patients studied two (No. 9) and 50 months (No. 13) after autologous transplantation with mobilized peripheral blood progenitor cells.
b
This patient was studied 101 month after allogeneic bone marrow transplantation.
FAB, French–American–British classification; ID Ara-C, intermediate-dose cytosine arabinoside; CR, complete remission; BM, bone marrow;
ND, not documented.

50 translucent cells or more were counted as GM-CFC- Results

derived colonies using an inverted microscope.
To assay Meg-CFC 1–2 × 105 cells were plated in a mixture In NBM cultures, Meg-CFC were detected for 7 weeks. How-
of 30% pretested human aplastic anaemia serum, 5 × 10−5 M ever, they were detected only until week 5 in the cultures
2-mercaptoethanol, 5% conditioned medium from phyto- from patients with AML (Figure 1). In those cultures only very
haemagglutinin-stimulated leukocytes (PHA-LCM) and 1.35%
methylcellulose. Cultures were plated and incubated as
described above. We used the established criteria for defining
MK colonies: colonies of at least five conspicuously large cells
with translucent ‘crystal’ clear cytoplasm and a well-delin-
eated cell border of high refractility were scored as Meg-
CFC.14 Their megakaryocytic nature was confirmed by picking
some colonies for CD61 immunostaining. All results were cal-
culated as total colony-forming cells in the supernatant (SN)
per culture; the data in Figures 1–3 are expressed as mean
± s.e.m.

Cell morphology

Cytospins were prepared with 5 × 104 cells for Pappenheim

stain and CD61 immunostaining (DAKO, Glostrup, Denmark)
as previously described,15 to determine the number of
megakaryocytic cells.

Statistics

Formal statistical analysis was confined to the MK profiles. It

was felt that the cumulative output in weeks 1–4 captured
the aspect of primary interest as it encloses the time of acute
regeneration after chemotherapy and also covers the time pre-
viously used for autologous transplants with cultured AMLBM
cells.16 On occasion, however, when data were missing for
some of the weeks, the observed cumulative output continued
to be right censored for such individuals (ie it is assumed that
the actual output is known only to be larger than that
observed). Right censored data may be analysed with ‘sur- Figure 1 The upper panel shows the numbers of Meg-CFCs per
culture in the supernatant of AML for untreated control cultures (쐽,
vival’ methods. The statistical analysis was done with a Cox
n = 13), or treated with 10 ng/ml (쎲, n = 8) or 100 ng/ml MGDF (왖,
regression model with appropriate adjustment for the corre- n = 7). The lower panel shows data from NBM cultures for untreated
lation of repeated observations for the same individual.17 All (n = 13), or treated with 10 ng/ml (n = 6) or 100 ng/ml MGDF (n =
tests performed in this framework were Wald type tests. 9). Data are expressed as mean numbers of Meg-CFC ± s.e.m.

low levels of Meg-CFC were seen during the first 4 weeks of
culture. The output of Meg-CFC in untreated AML cultures
was at least one log lower than in NBM cultures (P = 0.001).
CD61 immunostaining indicated the presence of MK until
week 3 for both NBM and AMLBM. However, treatment of
AML cultures with MGDF resulted in a two log increment in
week 1 and a one log increase in week 2, reaching similar
Meg-CFC numbers to those observed in NBM cultures. In
these and following experiments, patient 12, who received an
allogenic bone marrow transplantation, showed similar results
as the rest of the patients, so the results were analysed
together. There were no MK colonies detected in clonogenic
assays performed from the adherent cells from NBM (n = 4)
and AMLBM treated with 10 ng/ml TPO (n = 4) after week 5
of culture.
Analysis of these results showed that the cumulative output
of Meg-CFC in weeks 1–4 (Figure 2) was significantly lower
in cultures derived from AMLBM than from NBM (P = 0.01).
However, treatment of AML cultures with 10 ng/ml MGDF
resulted in a 24.3-fold increase in the cumulative mean num-
ber of Meg-CFC at week 4 reaching similar numbers of Meg-
CFC as those seen in the NBM cultures (P = 0.21). This
enhancement was statistically significant (P ⬍ 0.001). The sig-
Figure 2 Percentage of Meg-CFC above an absolute number of
Meg-CFC (x). Data are expressed as cumulative numbers of Meg-CFCs
from weeks 1 to 4 in AML cultures (upper panel) and NBM (lower
panel) for all experiments, discriminated between control (—),
10 ng/ml MGDF (---), 100 ng/ml MGDF (– – –). Censored values are
marked with ticks. The percentage of Meg-CFC in AMLBM using
10 ng/ml MGDF were significantly different from the values of control
cultures and cultures using 100 ng/ml MGDF but not significantly dif-
ferent from cultures derived from NBM.
MGDF stimulates megakaryocytopoiesis in first remission AML
C Kasper et al
909
nificance was lost in the cultures treated with 100 ng/ml
MGDF (P = 0.19). Treatment of NBM with MGDF had no sti-
mulating effect on the numbers of Meg-CFC (P = 0.21),
although there appeared to be a trend towards an increased
yield (Figure 1).
Although the number of experiments using immunostaining
with CD61 was smaller similar results were found. In the NBM
(n = 8) there was a cumulative mean number of 353 positively
stained cells by week 4; this was not different in both treat-
ment groups (290 and 370 for 10 and 100 ng/ml MGDF,
respectively). However, in AML cultures (n = 3) the corre-
sponding number was 109 for the control with an increase
to 1252 in two experiments treated with 10 ng/ml MGDF. In
experiments with NBM (Figure 3a), or with AMLBM (Figure
3b), MGDF had no effect on the number of non-adherent
cells, nor on the numbers of GM-CFC up to week 4 of culture.
However, in agreement with our previously published data11
from week 5 onwards the numbers of GM-CFC declined in
the AML cultures compared to those derived from NBM. This
was not changed by administration of MGDF. Treatment with
10 or 100 ng/ml MGDF had no effect on the erythroid colony
formation (data not shown).
Discussion
Patients treated for AML develop severe neutropenia and
thrombocytopenia which might cause treatment delay and
could lead to fatal infections or bleeding. There is hope that
MGDF may act in thrombocytopenia similarly to G-CSF in
neutropenia, which decreases the median duration of neutro-
penia in elderly patients after treatment for AML.18
However, it is known that the haematopoietic status of AML
patients in remission shows a marked and persistent
deficiency in primitive haematopoietic cells, both at the level
of committed progenitors, and of stem cells.11 It is therefore
important to establish that there are enough residual primitive
cells able to respond to megakaryopoietic stimulation in
response to the appropriate cytokine. This is also relevant for
patients who receive an allogenic bone marrow transplan-
tation (like one of the patients in this series), since it is known
that these patients have a long-term deficiency in the numbers
of CFC in their BM.19
In these experiments LTC were used to investigate the effect
of MGDF on BM from patients in first complete remission.
The results demonstrate a several-fold enhancement of Meg-
CFC to numbers within the normal range, as well as a similar
increase in CD61-positive cells in LTC treated with MGDF at
a dose of 10 ng/ml. However, there was no stimulation of
Meg-CFC in NBM above normal ranges. Thrombopoietin is
synthesized by BM stromal cells.20 Although the MGDF levels
were not measured in these experiments it is known that the
concentrations of MGDF used here were well above the levels
measured in LTC20 and human serum.21 It appears to be likely
that there is a deficiency in the microenvironment in AMLBM
while the megakaryopoiesis in cultures derived from NBM
may be already maximally stimulated by stromal cells.
MGDF effects appear not to be restricted to megakaryocyto-
poiesis. Recently, it has been shown that MGDF possesses the
ability to enhance erythroid proliferation,22 and in combi-
nation with other cytokines, stimulates the proliferation of
granulocytes, macrophages23 and primitive haematopoietic
cells;24–26 these effects were not observed using MNC from
umbilical cord blood (A Schwarzer, personal
communication). Furthermore, in those experiments MGDF
 MGDF stimulates megakaryocytopoiesis in first remission AML
C Kasper et al
910
Figure 3 The left panel shows the mean numbers (± s.e.m.) of nucleated cells (top) and of GM-CFCs (bottom) per culture in the supernatant
of untreated control cultures of AML patients (쐽, n = 13), or treated with 10 ng/ml (쎲, n = 8) or 100 ng/ml MGDF (왖, n = 7). The right panel
shows the corresponding data for cultures of NBM for untreated control (n = 13), or treated with 10 ng/m (n = 6) or 100 ng/ml MGDF (n = 9).
had no influence on the number of CFC or of granulopoietic
or monocytic cells in the cultures. A similar effect has been
noted previously using G-CSF which has only a small and
transient stimulatory effect on granulopoiesis in LTC of human
BM,27 perhaps because granulopoietic cell production is
already adequately stimulated in this system. No attempts
were made in these experiments to optimize the system for
erythroid growth. These results are in contrast with the
increase in granulopoiesis in LTC of murine BM23 seen
together with stimulation of megakaryopoiesis after treatment
with thrombopoietin. The lack of influence of MGDF after 5
weeks of culture to generate Meg-CFC in LTC is likely to be
due to the poor status and short life of BM cultures of AML
in remission, reported previously11 and confirmed here.
It is known that AML cells may express the c-mpl proto-
oncogene28 and that recombinant human thrombopoietin may
induce proliferation of AML cells in vitro.29,30 A similar in vitro
stimulating effect of G-CSF (and also of other cytokines) on in
vitro proliferation of AML cells has been reported.31 In spite
of this, randomised clinical trials in patients with AML have
not detected an effect of G-CSF administration on leukaemic
cell growth.18 Thus, it remains to be determined whether the
presence of the c-mpl receptor in AML cells will have any
clinical relevance for the administration of MGDF to AML
patients.
In summary, the present results indicate that the BM of AML
patients have enough potential reserve capacity to be able to
respond to stimulation of megakaryopoiesis by MGDF, not
only by the expected stimulation of platelet production, but
also by correcting the serious defect in the numbers of MK
progenitors in BM.
Acknowledgements
CK is supported by the ‘Aktion Kampf dem Krebs’, Germany.
This work was supported by CRC, UK.
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