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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 125-139
By
From INSERM U 362, PR1, Institut Gustave Roussy, Villejuif;
Laboratoire d'hématologie, Hôpital Raymond Poincaré,
Garches; and INSERM U363, ICGM, Hôpital Cochin, Paris, France.
Essential thrombocythemia (ET) and idiopathic myelofibrosis (PMF)
are two myeloproliferative diseases characterized by a marked megakaryocytic (MK) involvement. The pathogenesis of these two diseases
is unknown. Recently it has been shown that overexpression of
Mpl-ligand (Mpl-L) in mice induces thrombocytosis and myelofibrosis. In
this study, we investigated whether Mpl-L was responsible for the
pathogenesis of ET and PMF. Using in vitro cultures of blood or marrow
CD34+ cells, we investigated whether MK growth was
abnormal in these two diseases. Spontaneous MK growth involving only a
fraction (20%) of the MK progenitors, as compared with growth in the
presence of pegylated recombinant human megakaryocyte
growth and development factor (PEG-rhuMGDF), was found in both diseases
(21ET and 14PMF) using serum-free semisolid and liquid cultures,
including cultures at one cell per well. We first searched for a
c-mpl mutation/deletion by sequencing the entire coding region
of the gene by polymerase chain reaction (PCR) in nine ET patients and
five PMF patients, but no mutation was found. We subsequently
investigated whether an autocrine stimulation by Mpl-L could explain
the autonomous MK growth. Addition of different preparations of soluble
Mpl receptor (sMpl) containing a Fc domain of IgG1 (sMpl-Fc) markedly
inhibited MK spontaneous growth in both ET and PMF patients. This
effect was specific for sMpl because a control soluble receptor
(s4-1BB-Fc) had no inhibitory effect and an sMpl devoid of the Fc
fragment had the same inhibitory efficacy as the sMpl-Fc. This
inhibition was reversed by addition of PEG-rhuMGDF or a combination of
cytokines. The sMpl-Fc markedly altered the entry into cell cycle of
the CD34+ cells and increased the apoptosis that occurs
in most patient CD34+ cells in the absence of exogenous
cytokine, suggesting an autocrine stimulation. In contrast, a
neutralizing antibody against Mpl-L did not alter the spontaneous MK
growth, whereas it totally abolished the effects of 10 ng/mL
PEG-rhuMGDF on patient or normal CD34+ cells. Mpl-L
transcripts were detected at a very low level in the patient
CD34+cells and MK and only when a highly sensitive
fluorescent PCR technique was used. By quantitative
reverse-transcription (RT)-PCR, the number of Mpl-L transcripts per
actin transcripts was lower than detected in human Mpl-L-dependent
cell lines, suggesting that this synthesis of Mpl-L was not
biologically significant. In favor of this hypothesis, the Mpl-L
protein was not detected in culture supernatants using either an
enzyme-linked immunosorbent assay (ELISA) or a biological (Ba/F3hu
c-mpl) assay, except in one PMF patient. Investigation of Mpl-L
signaling showed an absence of constitutive activation of STATs in
spontaneously growing patient MKs. Addition of PEG-rhuMGDF to these MKs
activated STATs 3 and 5. This result further suggests that spontaneous
growth is neither related to a stimulation by Mpl-L nor to a
c-mpl mutation. In conclusion, our results show that Mpl-L or
Mpl are not directly implicated in the abnormal proliferation of MK
cells from ET and PMF. The mechanisms by which the sMpl mediates a
growth inhibition will require further experiments.
ABNORMAL MEGAKARYOCYTE (MK) proliferation
is often associated with myeloproliferative diseases, which include
chronic myelogenous leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (PMF). In these two
last diseases, alterations of megakaryocytopoiesis are the predominant
features. ET is defined by a megakaryocytic (MK) marrow hyperplasia and
persistent thrombocytosis, whereas PMF is a more typical
myeloproliferative disease characterized by a myelofibrosis initially
associated with a marked myeloid proliferation. In PMF, there is an
important hyperplasia of morphologically abnormal MK that may be
responsible for the marrow myelofibrosis by a local release of
fibroblast growth mediators.1-5
With the exception of CML, the molecular mechanisms
responsible for the clonal proliferation in these disorders are
presently unknown. In PV, the biological hallmark of the disease is an
erythropoietin (Epo)-independent colony growth that shows that the
control of erythropoiesis is abnormal.6-8 Such endogenous
erythroid colony formation has been also described in a minority of
patients with ET and PMF.9 In these two diseases, the
growth characteristics of MK progenitors have been extensively
studied.10-19 A spontaneous MK growth (in the absence of
exogenous cytokines) was observed in a majority of
patients.10,14-18,20,21-23 However, cultures were mainly
performed in the presence of serum or with unpurified cell populations,
and it cannot be excluded that the abnormal MK growth could be related
to a paracrine stimulation by cytokines secreted in culture or present
in the serum,22 although spontaneous MK growth could not be
blocked by antibodies against interleukin-3 (IL-3),
granulocyte-macrophage colony-stimulating factor (GM-CSF), and
IL-6.24 More recently, it has been reported that antisense oligonucleotides against c-mpl, the receptor for thrombopoietin (TPO), markedly inhibited spontaneous MK growth in ET,25
suggesting that the Mpl-Ligand (Mpl-L)/Mpl loop was involved in the
pathogenesis of this disease.
Mpl-L, also called TPO or megakaryocyte growth and development factor
(MGDF), is a cytokine that regulates platelet
production.26-30 Mpl-L has similarities with Epo. First, at
the structural level, Mpl-L has a 153-amino acid domain that shares
50% similarity with Epo.26,27,29 Second, Mpl-L and Epo are
hormonal factors mainly synthesized in adults by the kidney and the
liver.26,27,29 However, the two cytokines differ markedly
by three properties: (1) Mpl-L has a much broader biological activity
than Epo; it acts not only on the early and late stages of
megakaryocytopoiesis,28,31,32 but also on primitive
hematopoietic cells33-37; (2) Mpl-L is synthesized by
many cell types including marrow stromal cells28,38;
and (3) Mpl-L plasma levels are directly dependent on the platelet/MK mass and Mpl-L synthesis does not appear to be transcriptionally regulated.39,40 It has been shown that platelets
specifically bind Mpl-L with high affinity, internalize, and degrade
the protein.41-43
Overexpression in mice of Mpl-L using retroviral or adenoviral gene
transfer leads to a MK hyperplasia and a thrombocytosis associated with
myelofibrosis.44-48 The disease induced by marked Mpl-L
overexpression has characteristics similar to human PMF.46 Moreover, a truncated form of c-mpl is an oncogene that leads to an acute myeloproliferative disease associated with autonomous growth of all hematopoietic progenitors.49 In this study,
we have analyzed the growth characteristics of MK progenitors from a
large series of 21 patients with ET and 14 patients with PMF. Our data
show that truly autonomous MK development occurs in both diseases,
which do not result from a mutation/deletion in the c-mpl gene.
Nevertheless, we observed that different preparations of the soluble
murine Mpl receptor could efficiently and significantly interfere with
spontaneous MK growth, although we could not detect an autocrine
stimulation by Mpl-L.
Patients
Blood and Bone Marrow Cells
Reagents for Cytometry
Human Cytokines and Cytokine Soluble Receptors Recombinant human (rhu) IL-3 (a gift from Immunex, Seattle, WA) and rhuIL-6 were both used at a final concentration of 100 U/mL (3 and 5 ng/mL, respectively). Recombinant human stem cell factor (rhuSCF) and PEG-rhuMGDF (gifts from Amgen, Thousand Oaks, CA) were usually used at a final concentration of 50 ng/mL and 10 ng/mL, respectively. In some experiments, different preparations of murine soluble Mpl receptor (sMpl) were added at concentrations from 1 to 50 µg/mL. Two preparations of sMpl were chimeric fusion proteins comprising the extracellular domain of the murine Mpl and the Fc fragment of a human IgG1 (generous gifts from ZymoGenetics and Immunex, Seattle, WA). A third preparation consisted of the extracellular domain of a murine Mpl variant (missing part of the fourth exon due to alternate splicing), which was associated with a Tag (a generous gift from Dr S. Lok, ZymoGenetics).Cell Lines Ba/F3 cells (parental) and transfected with human c-mpl cDNA (Ba/F3hu c-mpl) were maintained in alpha-minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS) and 2.5% WEHI-3 conditioned medium (CM) as a source of murine IL-3. For coculture experiments, Ba/F3 cells were twice washed and deprived of WEHI-3 CM for 4 hours before being plated at a concentration of 1 × 103 cells/mL in the upper layer of a transwell (Costar, Cambridge, MA). In the lower chamber, CD34+ cells were cultured. These cultures were performed in serum-free medium supplemented with 1% FCS. Cell growth was examined with an inverted microscope.Isolation of CD34+ Cells Mononuclear cells were separated using a magnetic cell sorting system (mini MACS; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in accordance with the manufacturer's recommendations. The purity of recovered CD34+ cells was determined by flow cytometry using PE-HPCA2 and was routinely over 80%.Cell Sorting Mononuclear marrow cells (1 × 106) were washed and incubated with R-PE-HPCA2 (20 µL) in 100 µL at 4°C for 45 minutes. After one wash, cells were suspended in Iscove's modified Dulbecco's medium (IMDM) at a concentration of 5 × 105cells /mL and separated by cell sorting. Cells were sorted on a FACS Vantage flow cytometer (Becton Dickinson) equipped with an automatic cloning unit device (ACDU). A morphologic gate including 80% of the events and all of the CD34+ cells was determined on two-parameter histograms (side scatter [SSC] versus forward scatter [FSC]). Positivity for the CD34 antigen was determined using control cells labeled with a R-PE-irrelevant IgG1 MoAb. The CD34high cells were sorted.Cell Cultures Liquid culture.
CD34+ cells (from 1 × 104 to 1 × 105 cells per mL) were cultured in IMDM with
penicillin/streptomycin/glutamine and 11.5 µmol/L Semisolid cultures.
Cultures were performed in serum-free fibrin clot in the presence or
absence of cytokines. Ingredients for serum-free cultures were similar
to those of liquid cultures in which bovine plasma fibrinogen (1 mg/mL;
Sigma), 0.01 mol/L Limiting dilution experiments. For limiting dilution experiments, individual CD34+ were sorted with the ACDU of the cell sorter into 96-well plates.32 Serum-free cultures were performed in the absence or presence of 10 ng/mL PEG-rhuMGDF. Plates were examined at days 7 and 12 after incubation at 37°C in an air atmosphere supplemented with 5% CO2. Determination of MK Numbers Produced in Culture Total numbers of MKs were determined by flow cytometry after labeling with FITC-Tab (anti-CD41) or an R-PE anti-CD41 antibody (Pharmingen). After collection and rinsing with phosphate-buffered saline (PBS)/EDTA (3 mmol/L), cultured cells were centrifuged at 350g for 15 minutes and incubated with the antibody for 30 minutes. Cells from each culture condition were distributed in the same volume (400 µL) and rapidly analyzed by flow cytometry. For each sample, the acquisition rate was 2 µL/s for 100 seconds. MKs were defined as brightly positive CD41 cells with scatter properties of nucleated cells. Samples were analyzed with a FACSort flow cytometer (Becton Dickinson).Quantitative Titration of mRNA by PCR The quantitative titration of the different mRNAs was performed as previously described by PCR run to saturation.51RNA and cDNA preparation. Total RNA was isolated using RNA PLUS (Bioprobe Systems, Montrevil, France), a modification of the acid-guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi.52 RNA were reverse transcribed with random hexamers (GIBCO-BRL/Life Technologies, Cergy Pontoise, France) using AMV-reverse transcriptase (Promega Laboratories, Madison, WI). Construction of internal standards.
A Mpl-L plasmid was obtained by cloning the
EcoR1-Xba 1 fragment of the Mpl-L cDNA
into the EcoR1-Xba 1 site of Bluescript pBS SK+
plasmid. The internal standard for Mpl-L was generated by PCR using the
mutational primer A, which created a 5-bp deletion in the sequence of
the Mpl-L cDNA. The PCR product was digested with
EcoR1-BamH1 and directly cloned into the Mpl-L cDNA
Bluescript pBS SK+ plasmid previously cleaved by
EcoR1-BamH1. Quantitative PCR and run-off reaction.
Two coamplifications were performed for quantitation of Mpl-L (primer
C) and Oligonucleotides.
Primer A:
5 DNA Sequencing Genomic DNA was isolated from peripheral blood cells (leukocytes and mononuclear cells) or bone marrow cells using the proteinase K/sodium dodecyl sulfate (SDS) treatment and phenol/chloroform extraction.53 Each of the 12 exons of the c-mpl gene was amplified and sequenced using the PCR technique with the oligonucleotide primers described in Table 2.
PCR with a fluorescent dye.
RT-PCR for Mpl-L transcript detection was performed in a 25-µL
mixture containing 2.5 µL reverse transcribed mRNA, 0.3 U Taq polymerase (ATGC), 200 µmol/L dNTP, and 2 µmol/L of each of the two
primers, in ATGC buffer. The two primers used were
5 STAT Activation CD34+ cells were maintained in culture for 10 days in the presence or absence of the indicated cytokine combination (PEG-rhuMGDF and SCF or IL-3, IL-6 and SCF) and CD41+ cells were purified,washed in PBS, and incubated for 15 minutes in the presence or absence of PEG-rhuMGDF (30 to 100 ng/mL) or interferon- (IFN-;
1,000 U/mL; Boerhinger Mannheim) at 37°C. Incubation was stopped by
a brief centrifugation and washes in PBS medium. Nuclear extracts were
prepared and incubated (1 to 3 µg proteins) with 10 to 15 fmol of a
32P-labeled oligonucleotide as previously
described.54 Protein concentration was evaluated using the
BCA protein assay kit (Pierce, Rockford, IL). The oligonucleotide
probes (m67 SIE, 5 -CATTTCCCGTAAATC-3; IRF1-GAS,
5-GATCCATTTCCCCGAAATGA-3;  -casein-GAS,
5AGATTCTAGGAATTCAAATC-3) were end-labeled using T4
polynucleotide kinase to a specific activity of 3,000 cpm/fmol.54
Mpl-L Enzyme-Linked Immunosorbent Assay (ELISA) The ELISA for the detection of human Mpl-L was the Human TPO Quantikine kit (R&D system, Minneapolis, MN), which was used according to the manufacturer's instructions. The ELISA sensitivity limit was 25 pg/mL of Mpl-L.Cell Cycle and Apoptosis Analysis by Flow Cytometry Cells were washed and labeled overnight in citrate buffer containing 25 µg/mL PI, 50 µg/mL RNase (Merck, Darmstadt, Germany) and 0.1% Nonidet P40 (Sigma) in the dark at 4°C. For determination of apoptosis, cells were stained with anti-CD34 and anti-CD41 MoAbs conjugated to APC and R-PE, respectively, then washed and stained with annexin V-FITC and 7 AAD according to the manufacturer's instruction. Cell cycle and labeling were analyzed by flow cytometry using a FACSort (Becton Dickinson) equipped with laser and a photodiode tuned at 488 nm and 560 nm, respectively.
CD34+ Cells From Patients Twenty-one patients with a typical ET were studied. Most samples (20 out of 21) were obtained by means of BM aspiration. After separation on ficoll gradient, the number of mononuclear cells varied from 0.8 × 106 to 30 × 106 per volume of 1 to 3 mL of BM aspiration. CD34+ cells were purified either by the immunomagnetic bead technique or by flow cytometry. On the average, 1% of the ET BM cells were CD34+ cells (0.01% to 1.6%). Three blood samples from ET patients contained an average of 0.6% CD34+ cells (0.2% to 1.3%) in the mononuclear cell fraction, and CD34+ cells were purified. However, because insufficient numbers of CD34+ cells were collected, studies were only performed with BM samples. In contrast, in patients with PMF, the entire study was performed with blood samples. In 14 blood samples (20 to 50 mL), the average CD34+ cell recovery was 2.3% (0.2% to 8%) of the initial number of mononuclear cells, and more than 3 × 106 CD34+cells per 10 mL of blood were obtained in most samples. A 15th sample was obtained from a cytapheresis sample and contained 0.12% CD34+ cells. This patient (m6) developed a blastic transformation.Presence of Autonomous MK Growth in ET and PMF Patients In a first set of experiments, low-density (d = 1.077) bone marrow cells (5 × 104/mL) from 20 ET patients were cultured in serum-free fibrin clot. All samples exhibited spontaneous MK growth, which represented 34.5% of the MK colony number obtained after PEG-rhuMGDF stimulation (Table 3). In the majority of cases, spontaneous colonies were composed of a low MK number (from 3 to 6). Spontaneous erythroid colony formation was also observed in only 8 of 20 ET samples tested.
Absence of Mutation in the Coding Region of the c-mpl Gene in
ET and PMF Patients
Search for Autocrine Stimulation by Mpl-L in ET and PMF
Blocking of spontaneous MK growth in ET and PMF by sMpl. Experiments were performed in semisolid and liquid media cultures. In semisolid medium, cultures were performed with CD34+ cells from 3 ET and 1 PMF patients at a concentration of 103 cells per mL (Table 4). A partial and significant inhibition (from 66% to 98%, P < .01) of the spontaneous growth was elicited by the chimeric sMpl (sMpl-Fc, 5 µg/mL) in all these cases. Addition of 10 ng/mL PEG-rhuMGDF did not induce a complete reversion of the inhibitory effect mediated by the sMpl-Fc (P < .05). A larger number of experiments (9 ET and 3 PMF patients) were performed in liquid cultures with CD34+ cells (10 to 50 × 103 per mL; Table 5). Quantitation of MKs was performed using either an inverted microscope when a low number of CD34+cells was available (Table 5A) or by flow cytometry after labeling with an R-PE or FITC anti-CD41 antibody (Table 5B). MK spontaneous growth was significantly inhibited by the sMpl-Fc (40% to 100% inhibition, P < .02). A dose-response curve showed that 5 µg/mL of sMpl-Fc had the maximum inhibitory effect (data not shown). This effect was nearly completely reversed by 10 ng/mL PEG-rhuMGDF (no significant difference with PEG-rhuMGDF cultures, P > .05). In contrast, sMpl-Fc had no effect on ET or PMF CD34+ cells stimulated by a combination of IL-3, IL-6, and SCF (Fig 2). When tested on normal CD34+ cells, 5 µg/mL of sMpl-Fc inhibited the effect of 1 ng/mL of PEG-rhuMGDF (77% inhibition). This effect was nearly totaly reversed by concentration of PEG-rhuMGDF exceeding 20 ng/mL (on the average, a 15% residual inhibition that was not statistically significant). A similar and nonsignificant inhibition was also found by 5 µg/mL of sMpl-Fc on the MK growth stimulated by a combination of SCF, IL-3, and IL-6. To confirm the specificity of the effect of the sMpl-Fc, two other preparations of sMpl (sMpl-Fc and sMpl-Tag) were tested. Both inhibited spontaneous MK growth from CD34+ cells in the two cases of PMF tested (data not shown). In contrast, a control irrelevant soluble receptor (s4-1BB-Fc; 5 µg/mL) had no inhibitory effect (Fig 3).
Neutralizing anti-Mpl-L antibody does not inhibit spontaneous MK
growth in ET and PMF.
To show a possible autocrine stimulation by Mpl-L in spontaneous MK
growth, the effect of a neutralizing anti-Mpl-L antibody was studied
in two cases of ET and PMF. The antibody used at 10 µg/mL had no
inhibitory activity on the spontaneous growth in liquid cultures
performed with 2 to 10 × 103 CD34+cells.
In contrast, the antibody totally inhibited the stimulatory effect of
10 ng/mL PEG-rhuMGDF but had no inhibitory effects on MK cultures
stimulated by three cytokines (SCF, IL-3, IL-6;
Fig 6). This antibody at 10 µg/mL
abolished the stimulatory effects of 10 ng/mL PEG-rhuMGDF on the growth
of MK progenitors from normal CD34+ bone marrow cells (data
not shown).
Studies of Mpl-L Transcripts by RT-PCR in CD34+
Cells or CD34+-Derived MK from ET and PMF Patients
Search for Mpl-L in Patient Serum and the Supernatant of
CD34+-Derived MK From ET and PMF
The Autonomous Growth of Patient MKs Does Not Correlate With a
Detectable Activation of STAT Factors
ET and PMF are two chronic myeloproliferative diseases characterized by
predominant MK involvement, although both diseases are clonal malignant
disorders originating from a pluripotent stem cell.3,56,57
Numerous studies have shown that spontaneous MK colony formation is
frequently observed in ET.14,15,17,20,22 However, a recent
study indicates that this growth defect was not observed with purified
CD34+cells,58 suggesting that the spontaneous
growth could be related to a paracrine cytokine stimulation. In
contrast to patients with ET, only a few patients with PMF have been
previously studied, and it was reported that their MK progenitors also
grew spontaneously.10,24,25 In the present study, we have
investigated a large series of 21 patients with ET and 14 patients with
PMF. We confirm that the spontaneous growth of MKs is one of the
biological hallmarks of these two diseases and is really due to
autonomous MK differentiation because it is still observed at limiting
dilution and in serum-free conditions. However, in both diseases MK
progenitor cell growth remains partially dependent on growth factors
because the exogenous addition of PEG-rhuMGDF increases the cloning
efficiency by threefold to fourfold, confirming previous
observations.15,18 These results are quite similar to those
obtained for erythroid progenitors in PV, where spontaneous erythroid
colony formation is constantly observed.6-8,11 Furthermore,
Epo addition also increases the cloning efficiency of erythroid
progenitors in PV. This augmentation is not exclusively due to the
recruitment of erythroid progenitors belonging to normal
clones,59 therefore showing that the abnormal erythroid
progenitors have different Epo sensitivity.60 Similarly in
ET and PMF patients, we observed not only autonomous MK growth, but
also hypersensitivity to SCF and PEG-rhuMGDF in cell culture studies
(data not shown), suggesting that the majority of MK progenitors belong
to the malignant clone. More surprisingly, when CD34+cells
from PMF patients were cultured in liquid medium, more than 90% of the
cells that grew in the absence of cytokines belonged to the MK lineage.
In contrast, a combination of IL-3, IL-6, and SCF not only induced a
megakaryocytic growth but also a marked myeloid proliferation. This
result further suggests that the abnormal MK proliferation plays a
crucial role in the pathogenesis of PMF.1,4,61
We are grateful to J.-L. Nichol (Amgen, Thousand Oaks, CA) and C. Caillot (Amgen, Neuilly, France) for providing the rhu-SCF, PEG-rhuMGDF, and the poyclonal antibody against Mpl-L; to D. Foster and
Z. Lok (ZymoGenetics, Seattle, WA) for soluble murine Mpl receptors
(sMpl-Fc and sMpl-Tag); and to D. Cosman (Immunex, Seattle, WA) for
rhu-IL-3, murine Mpl (sMpl-Fc), and 4-1BB-Fc soluble-receptors. We are
grateful to P. Bourquelot and O. Hermine (Hôpital Necker, Paris,
France) for providing patient blood. We thank B. Forget for improving
the English manuscript.
Submitted February 11, 1998;
accepted September 2, 1998.
Address reprint requests to Najet Debili, PhD, INSERM U
362, PR1, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805, Villejuif, France; e-mail: denali{at}igr.fr.
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Top
Abstract
Introduction
-thioglycerol
(Sigma). In cultures performed in serum-free conditions, IMDM was
supplemented with 1.5% bovine serum albumin (BSA; Cohn's fraction V;
Sigma), sonicated lipids, and iron-saturated human transferrin.
amino caproic acid, and horse thrombin (6 mU/mL;
Stago, Asnières, France) were added. Target cells were either
low-density marrow cells (5 × 104 to 1 × 105 cells/mL) or CD34+ cells (1 × 103 cells/mL). Cultures were incubated at 37°C in a
fully humidified atmosphere containing 5% CO2 in air and
scored after 10 to 12 days. Colonies were quantitated by an indirect
immuno-alkaline phosphatase labeling technique using an anti-GPIIIa
MoAb (CD61, Y2-51) as previously described.
20°C
until ready for sequencing on a DNA Sequencer (Model 373 A; Applied Biosystems). All computed sequence analyses were performed using the
SeqEd software (Applied Biosystems).
8N MK: 43.7% v 22.4%,
respectively). In controls (bone marrow or cytapheresis), no
spontaneous MK growth from CD34+ cells was observed in
semisolid or liquid cultures.
) or with
PEG-rhuMGDF (10 ng/mL) (
). In 8 ET patients (t) and 1 PMF patient
(m). Results are derived from 288 wells.
), with a combination of rhuIL-3, rhuIL-6, and rhuSCF
(IL-3+IL-6+SCF) (
). CD34+
cells were seeded at 2 × 104 cells/mL in 100 µL in
triplicate, and MKs were quantitated by morphological analysis.
cells was observed without
growth factor (Fig 
), or with three cytokines (IL-3, IL-6, and SCF) associated with
the anti-Mpl-L antibody (IL-3, IL-6, and SCF+Ab ) (
).
CD34+ cells were seeded at concentration ranging from
from 2 × 104 cells/mL to 1 × 105 cells/mL
in 100 µL in triplicate. MKs were quantitated by morphological
analysis and results are expressed as the number of MKs per 1 × 103 seeded CD34+ cells.
