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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 830-843
Effects of Cytokines on Platelet Production From Blood and Marrow
CD34+ Cells
By
Françoise Norol,
Natacha Vitrat,
Elisabeth Cramer,
Josette Guichard,
Samuel A. Burstein,
William Vainchenker, and
Najet Debili
From INSERM U 91, Hôpital Henri Mondor, Créteil, France.
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ABSTRACT |
The late stages of megakaryocytopoiesis, consisting of the terminal
processes of cytoplasmic maturation and platelet shedding, remain
poorly understood. A simple liquid culture system using CD34+ cells in serum-free medium has been developed to
study the regulation of platelet production in vitro. Platelets
produced in vitro were enumerated by flow cytometry. A truncated form
of human Mpl-Ligand conjugated to polyethylene glycol (PEG-rHuMGDF)
played a crucial role in both proplatelet formation and platelet
production. A combination of stem cell factor (SCF), interleukin-3
(IL-3), and IL-6 was as potent as PEG-rHuMGDF for the growth of
megakaryocytes (MKs). However, the number of proplatelet-displaying MKs
and platelets was increased 10-fold when PEG-rHuMGDF was used.
Peripheral blood mobilized CD34+ cells gave rise to a
threefold augmentation of platelets compared with marrow
CD34+ cells. This finding was related to the higher
proliferative capacity of the former population because the proportion
of proplatelet-displaying MKs was similar for both types of
CD34+ cells. The production of platelets per MK from
CD34+ cells was low, perhaps because of the low ploidy of
the cultured MKs. This defect in polyploidization correlated with the
degree of proliferation of MK progenitors induced by cytokines. In
contrast, ploidy development closer to that observed in marrow MKs was
observed in MKs derived from the low proliferative CD34+
CD41+ progenitors and was associated with a twofold to
threefold increment in platelet production per MK. As shown using this
CD34+ CD41+ cell population, PEG-rHuMGDF
was required throughout the culture period to potently promote platelet
production, but was not involved directly in the process of platelet
shedding. IL-3, SCF, and IL-6 alone had a very weak effect on
proplatelet formation and platelet shedding. Surprisingly, when used in
combination, these cytokines elicited a degree of platelet production
which was decreased only 2.4-fold in comparison with PEG-rHuMGDF. This
suggests that proplatelet formation may be inhibited by non-MK cells
which contaminate the cultures when the entire CD34+ cell
population is used. Cultured platelets derived from PEG-rHuMGDF- or
cytokine combination-stimulated cultures had similar ultrastructural features and a nearly similar response to activation by thrombin. The
data show that this culture system may be useful to study the effects
of cytokines and the role of polyploidization on platelet production
and function.
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INTRODUCTION |
IN CONTRAST TO the early stages of
megakaryocyte (MK) differentiation, the ultimate step of
megakaryocytopoiesis, that of platelet shedding, is incompletely
understood. Most investigators consider that platelet shedding, both in
vitro and in vivo, requires the formation of long cytoplasmic
extensions termed proplatelets.1-5 Another hypothesis
concerning the mechanism of platelet release proposes that MK
demarcation membranes delineate platelet territories and that future
platelets are released subsequent to membrane fragmentation.6,7 The late stages of megakaryocytopoiesis have remained poorly understood because of the difficulty of acquiring large populations of MKs capable of shedding platelets in vitro. Culture systems permitting all stages of megakaryocytopoiesis might be
particularly valuable for understanding platelet formation and its
regulation by cytokines. Several culture systems have been designed to
study the late stages of thrombocytopoiesis. In mice, guinea pigs and
humans, MKs have been purified and cultured on a short-term basis to
study proplatelet formation.8-10 In the rat, MK progenitors
have been purified based on the expression of glycoprotein (GP)
IIb/IIIa and proplatelet formation was analyzed after 2 to 3 days of
culture in the presence of cytokines.8,10-12 Recently, Choi
et al13,14 described an in vitro system in which platelet-sized fragments that are morphologically and functionally similar to blood-derived platelets were generated from human MKs derived from CD34+ cells cultured in liquid medium.
The role of cytokines in proplatelet formation and platelet shedding is
also poorly understood. It has been suggested that MPl-Ligand (Mpl-L),
also called thrombopoietin or megakaryocyte growth and development
factor (MGDF, a nonglycosylated truncated form of
Mpl-L),15-17 which plays a crucial role in MK maturation, is not directly involved in the process of platelet shedding and at
high concentrations might even inhibit proplatelet
formation.18,19 Among the other cytokines, it has been
shown in rodents that IL-6, IL-11, and erythropoietin can induce
proplatelet formation.8,20,21 The effect of IL-6 on
proplatelet formation in humans is controversial.18,19 Unlike Mpl-L, the other cytokines such as IL-6 are regulators of
platelet production in stress conditions such as inflammation.
In this report we describe the effects of culture conditions and
several cytokines on MK proplatelet formation, together with the
ultrastructural and functional characteristics of the platelet-like particles produced in vitro.
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MATERIALS AND METHODS |
Blood and Bone Marrow (BM) Cells
Blood CD34+ cells were isolated from leukapheresis samples
performed on patients undergoing autologous peripheral blood stem cell
transplantation. BM cells were obtained from normal adult donors
undergoing hip surgery. Informed consent was obtained from all donors.
BM cells were collected by vigorous shaking of bone fragments in
Iscove's modified Dulbecco medium (IMDM; GIBCO, Paisley, UK)
supplemented with 100 ng/mL of DNAse (Sigma, St Louis, MO). Blood cells
were diluted to a concentration of ~50 × 103/µL in a
phosphate-buffered saline (PBS; GIBCO) solution containing 5 ng/mL EDTA
(Sigma). Mononuclear cells were separated on a Ficoll gradient
(Lymphoprep; Nycomed Pharma, Oslo, Norway). Low-density cells (<1.077 g/cm3) were recovered, washed, and then
used for isolation of CD34+ cells.
Antibodies
Directly conjugated monoclonal antibodies (MoAbs)
R-phycoerythrin-(PE)-HPCA2 (anti-CD34) and fluorescein isothiocyanate
(FITC) anti-CD41a (Becton Dickinson, Mountain View, CA; and Pharmingen, San Diego, CA; respectively) were used for cell sorting. FITC-TAB (anti-CD41b; TAB provided by Dr R. McEver, Oklahoma Medical Research Foundation), R-PE anti-CD41a MoAb (Pharmingen), and an R-PE anti-CD62 MoAb (anti-P-selectin; Becton Dickinson) were used for analysis of MKs
and platelets by flow cytometry. FITC- and R-PE-conjugated immunoglobulin (Ig) G1 MoAb controls were obtained from
Becton Dickinson.
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
CD34+ cells recovered was determined by flow cytometry
using PE-HPCA2 and was greater than 80%.
Cell Sorting
BM CD34+ cells were treated with neuraminidase to prevent
binding of platelets.22 Cells (1 × 106)
were incubated in 100 µL PBS and 0.2% bovine serum albumin (BSA) with 0.2 U/mL neuraminidase for 1 hour at 37°. Cells were washed and
incubated with R-PE-HPCA2 (20 µL) and FITC anti-CD41a (20 µL) in
100 µL volume at 4°C for 45 minutes. After one wash, cells were
suspended in IMDM at a concentration of 5 × 105
cells/mL and separated by cell sorting. Cells were sorted on a FACS
Vantage flow cytometer (Becton Dickinson). A morphologic gate including
80% of the events and all the CD34+ cells was determined
on two-parameter histograms (side scatter [SSC] versus forward
scatter [FSC]). Compensation for two-color labeled samples was set up
with singly stained samples. Positivity or negativity for the CD41
antigen among the CD34+ cells was determined using control
cells labeled with PE-HPCA2 and an irrelevant IgG1 MoAb. Cells were
sorted into a CD34+ CD41 (~96% of the
CD34+ cells) and a CD34+ CD41+ cell
fraction (~2% of the CD34+ cells).
Human Cytokines and Cytokine 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
polyethylene glycol (PEG)-rHuMGDF (gifts of Amgen Corp, Thousand Oaks,
CA) were usually used at a final concentration of 50 ng/mL and 10 ng/mL, respectively. CD34+ CD41+ cells were
cultured with increasing concentrations of PEG-rHuMGDF ranging from
0.01 to 500 ng/mL. In some experiments, a murine soluble Mpl receptor
(gifts of ZymoGenetics, Seattle, WA) and full length rHuMpl-L (TPO;
Genzyme, Cambridge, MA) were added at a concentration of 5 to 10 µg/mL and 10 ng/mL, respectively.
Cell Cultures
CD34+ cells were cultured in IMDM with
penicillin/streptomycin/glutamine and 11.5 µmol/L  -thioglycerol
(Sigma). Cultures were usually performed in serum-free conditions in
which IMDM was supplemented with 1.5% BSA (Cohn's fraction V; Sigma),
sonicated lipids, and iron-saturated human
transferrin.23,24 Two types of serum-containing cultures
were used as controls. In the first, IMDM was supplemented with 1% BSA
and 10% normal human AB serum, and in the second with 1% BSA and 10%
normal human serum derived from platelet-poor plasma (PPP).24 In some experiments, heparin (3 U/mL, Fragmine;
Pharmacia, Orsay, France) or an anti-TGF- antibody (R&D
Systems, Minneapolis, MN) were added to the medium.
CD34+ cells were cultured in 6- or 24-well tissue culture
plates, in 3 or 1 mL volumes, respectively. CD34+
CD41+ cells were grown in 96-well plates in a 100 µL
volume. Cultures were incubated at 37°C in a fully-humidified
atmosphere containing 5% CO2 in air. Two O2
concentrations (21% or 7%) were compared. The cultures were examined
with an inverted microscope at 40× and 100× magnification.
Determination of MKs and Platelet Numbers Produced in Culture
The total numbers of MKs were determined by flow cytometry or cytology.
Platelet numbers were determined by flow cytometry. Flow cytometry
quantification was performed after FITC-TAB (anti-CD41) or R-PE
anti-CD41a MoAb labeling. After collecting and rinsing with PBS/EDTA,
cultured cells were centrifuged at 350g for 15 minutes,
incubated with the MoAb for 30 minutes, and fixed with 0.5%
paraformaldehyde (Serva, Heidelberg, Germany) for 20 minutes. Cells
from each culture condition were distributed in the same volume (400 µL). For each sample, the acquisition rate was 1 µL/second for 100 seconds. For quantitation of MKs, a linear scale was used for FCS and
SSC. MKs were defined as brightly positive CD41 cells with scatter
properties of nucleated cells. For quantitation of platelets, events
were collected without gating using a log scale for FSC and SSC. An
analytical gate was determined based on scatter properties of normal
blood platelets treated similarly. This gate excluded large
contaminating cells (MKs) and small debris or microparticles. Culture-derived platelets were enumerated as CD41+ events
with the same scatter properties as blood platelets. Samples were
analyzed with a FACSort flow cytometer (Becton Dickinson).
Proplatelet-displaying MKs were defined as cells exhibiting one or more
cytoplasmic processes with areas of constriction. The percentage of MKs
with such processes was quantitated with a hemocytometer at various
times during the culture period depending on the starting cell
population (blood or marrow CD34+ cells or marrow
CD34+ CD41+ cells).
Determination of MK Ploidy
MK ploidy was measured by a double-staining technique and flow
cytometry.25 Cultured cells were counted, fixed with 0.5% paraformaldehyde for 20 minutes at 4°C, and then washed in calcium and magnesium-free PBS. Cells were kept at 4°C until analysis (<2
days). MKs were identified after labeling with FITC-TAB, while DNA
staining was performed by incubating the cells in a solution of
propidium iodide (50 µg/mL in isotonic NaCl containing 100 µg/mL
RNAse [Merck, Darmstadt, Germany] and 0.1% Tween 20 [Sigma]) for 1 hour. Control cells were stained with an irrelevant FITC-IgG1 MoAb and
propidium iodide. The flow rate used was 500 to 1,000 cells/s. MKs were
identified by the expression of CD41b. The ploidy distribution was
determined by setting markers at the nadirs between peaks, whereas the
frequency of MKs in different ploidy classes was evaluated on 5,000 cells.
Detection of the Activation-Dependent Antigen P-Selectin (CD62) on
Platelets
Cultured cells were stimulated for 10 minutes at 37°C with 2 U/mL
thrombin (Stago) added directly to the culture well. Activated and
nonactivated platelets were incubated for 30 minutes with both
R-PE-anti-CD62 (2 µg/mL) and FITC-TAB. The cells were then fixed for
1 hour with an equal volume of 0.5% paraformaldehyde. Control cells
were fixed in the same manner without prior activation. Cells were
subsequently resuspended in PBS.
Activation of normal plasma-derived platelets was performed in
parallel; blood platelets were purified by gel filtration on Sepharose
2B in a buffer (containing NaCl, 129 mmol/L; Na3 citrate, 13.6 mmol/L; glucose, 11.1 mmol/L; KH2PO4, 1.6 mmol/L; and NaH2PO4, 8.6 mmol/L, pH 7.3) and
then treated as above. Activated and nonactivated blood and
culture-derived platelets were analyzed by flow cytometry.
Ultrastructural Studies
Cultured cells were examined by electron microscopy. The cells were
fixed with 1.25% glutaraldehyde in 0.1 mmol/L phosphate buffer for 1 hour at 22°C washed, postfixed with osmium tetroxide, dehydrated, and
embedded in Epon (TAAB, Aldermaston, Berkshire, UK). Thin
sections were examined with a Philips CM 10 electron microscope
(Philips, Eindoven, The Netherlands) after lead citrate staining.
Statistical Analysis
The numbers of MKs, of MKs bearing proplatelets, and of platelets were
expressed as the mean ± 1 SD. In each experiment, cultures were
performed in parallel in different conditions (medium or cytokines).
Results of these different conditions were compared with those obtained
in serum-free medium or with PEG-rHuMGDF-stimulated cultures,
respectively. Statistical analysis was thus performed using a paired
t-test.
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RESULTS |
Optimization of Culture Conditions
Various approaches to improve proplatelet formation and platelet
production were evaluated. Reproducible proplatelet formation required
a relatively pure population of CD34+ cells. This was
attainable using an immunobead selection technique with which a purity
of greater than 85% CD34+ cells was routinely observed. As
free oxygen radicals produce toxicity on maturing cells in many culture
systems,26-28 the effects of low O2
concentration (7%) on platelet production were compared with ambient
O2 concentrations (21%) in six experiments. The average number of proplatelet-bearing MKs and platelets observed was not significantly different at these two O2 concentrations but
was always higher at 7%. In these experiments, we did not remove the antioxidant (-thioglycerol) which may have minimized
differences.28 Finally, initial densities of 5 × 104 cells/mL ensured satisfactory MK proliferation, with
only one refeeding performed during the 14- to 16-day culture period.
Based on these preliminary data, all subsequent cultures were
established using purified populations of CD34+ cells at an
initial density of 5 × 104 cells/mL cultured at 7%
O2.
Platelets produced in culture were enumerated by flow cytometry as
particles having the same scatter properties as blood platelets and
expressing a high level of CD41 (Fig 1A, B, and
C). This definition seems the more
appropriate to define and quantitate platelets produced in vitro. In
addition, as previously shown,29 there are numerous other
CD41+ elements with much lower forward scatter which may
correspond to platelet microparticules or cell fragments (Fig 1B).
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| Fig 1.
Flow cytometric characteristics of platelets produced in
culture. Culture-produced platelets were defined as CD41+
elements with the same scatter properties as blood platelets. (A)
Scatter properties of blood platelets after FITC-TAB (anti-CD41b) labeling. (B) Scatter properties of culture platelets labeled in the
same conditions and the gate chosen for the analysis. Black points are
CD41 elements, grey points CD41+ elements.
(C) CD41 expression of platelets produced in culture before (---) and
after ( ) thrombin activation. The isotype control (....) is shown.
(D) Forward scatter height of platelets produced in culture before
(---) and after () thrombin activation. (E) Side scatter height of
platelets produced in culture before (---) and after () thrombin
activation.
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Kinetics of MK growth, proplatelet formation, and platelet production
(Fig 2) were first determined. A plateau in
MK growth was observed between day 9 and day 14 for marrow
CD34+ cells. This plateau was delayed for MK derived from
blood CD34+ cells and was reached at day 11 to day 12 (Fig
2A). Proplatelet formations were observed from day 6 to day 14 for
marrow-derived MKs whereas they were detected from day 12 to day 14 for
blood-derived MKs (Fig 2B). Platelet production paralleled proplatelet
formation and was maximum at day 14 for blood CD34+
cell-derived cultures (Fig 2C). The vast majority of cells began to
lyze after day 14. Therefore, proplatelet formation and platelet production were studied between day 6 and day 12 for marrow
CD34+ cell-derived cultures and at day 13 or day 14 for
those derived from blood CD34+ cells.
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| Fig 2.
Kinetics of MK growth (A), proplatelet formation (B), and
platelet production (C) from marrow or blood
CD34+ cells. CD34+ cells from marrow ( )
and blood CD34+ cells ( ) were grown in serum-free
conditions in the presence of PEG-rHuMGDF (10 ng/mL). MKs were
enumerated as CD41+ cells by flow cytometry at different
days of culture. In this and all subsequent figures, the number of MKs
bearing proplatelets was determined using an inverted microscope and
hemocytometer as MKs showing one or more cytoplasmic expansions with
constriction areas. In this and all subsequent figures, culture
platelets were enumerated by flow cytometry as CD41+
events with the same scatter properties as blood platelets shown in Fig
1. For the kinetics of MK growth and proplatelet formation, results are
the average of three experiments. For determination of platelet
production, the entire kinetics was only performed in one experiment
from a blood CD34+ cell-derived culture.
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The effects of the serum on platelet production were evaluated. Six
experiments comparing serum-free medium and medium supplemented with
10% serum or PPP were performed. Blood- and marrow-derived CD34+ cells (n = 3 for each) were cultured with 10 ng/mL
PEG-rHuMGDF. MK growth was twofold to threefold higher in serum-free
medium compared with serum and PPP (Fig
3A). The percentage of MKs displaying proplatelet was similar in the different conditions, ranging from 42%
to 51%. Therefore, the differences in the number of
proplatelet-displaying MKs paralleled those of MK growth
(4.6 × 103/103 CD34+ cells in
cultures without serum compared with
2 × 103/103 CD34+ cells or
1.1 × 103/103 CD34+ cells;
P = .04, in cultures containing serum or PPP; Fig 3B). In
these experiments, platelet number was 50-fold the number of proplatelet-bearing MKs (Fig 3C).
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| Fig 3.
Effects of culture conditions on MK growth (A),
proplatelet formation (B), and platelet production (C).
CD34+ cells from marrow (n = 3,  ) or blood
(n = 3,  ) were grown during 14 days in the presence of
PEG-rHuMGDF (10 ng/mL) in serum-free conditions, in serum derived from
PPP, or normal serum in the absence or presence of heparin (H) or an
anti-TGF- antibody. MKs and platelet bearing MKs were enumerated
using an inverted microscope and hemocytometer. The results represent
the mean ± SD of three independent experiments, and are expressed per
1 × 103 plated CD34+ cells. Asterisks
denote a significative difference (P < .05) in comparison
with culture in serum-free conditions.
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We next tested if addition of heparin or an anti-TGF- antibody
might improve these serum or PPP-containing cultures. Addition of
heparin (3 U/mL) to PPP-containing medium increased the number of MKs
and proplatelet-bearing MKs by twofold to threefold, as well as
increasing the number of platelets produced. In contrast, addition of
heparin to serum-containing medium or of an anti-TGF- antibody in
media containing serum or PPP increased the number of MKs and platelets
produced, but not significantly. Nevertheless, the numbers of MKs,
proplatelet-bearing MKs, and platelets observed were much lower than in
serum-free medium (Fig 3A, B, and C). The mean ploidy remained nearly
identical in all tested culture conditions, but was low (range, 3.9 to
4.5; data not shown).
In these various conditions, the percentage (about 50%) of
proplatelet-bearing MKs was similar using either blood- or
marrow-derived CD34+ cells, but as shown in Fig 3, their
absolute numbers per CD34+ cell and the number of platelets
produced were on average threefold higher for peripheral blood
CD34+ cells (P < .01). The mean MK ploidy was
similar (in serum-free medium, 5.1 and 4.9 for blood- and
marrow-derived cells, respectively; data not shown).
Effects of Cytokines on the Production of Proplatelet-Bearing MKs and
Platelets
The effects of cytokines on the entire CD34+ cell
population, and on CD34+ CD41+ cells, highly
enriched in mature MK progenitors, were assessed in serum-free medium
at 7% O2.
Effects of cytokines on CD34+ cells.
Cytokines were tested on purified populations of blood- and
marrow-derived CD34+ cells. In a first experiment, we
determined that full-length rHu Mpl-L-stimulated cultures
supplemented with the same molarity of PEG as Mpl-L- and
PEG-rHuMGDF-stimulated cultures gave the same platelet production (91, 100, 101 × 103 platelets/103 BM
CD34+ cells, respectively, average from one experiment).
This confirms previous experiments showing that PEG does not modify the
in vitro biological activity of a molecule.30 Subsequently,
PEG-rHuMGDF was used alone or in different combinations with IL-3 plus
SCF, or SCF plus IL-6. These effects of PEG-rHuMGDF were compared
with those of different combinations of IL-3, SCF, and IL-6 without PEG-rHuMGDF (Fig 4). PEG-rHuMGDF had a
major effect on the production of MKs, proplatelet-bearing MKs, and
platelets associated with a high proportion (mean, 79%) of MKs in the
cultures. From blood CD34+ cells, PEG-rHuMGDF induced an
average of 4.3 × 103 proplatelet-bearing MKs (54%
of cultured MKs) and 199 × 103 platelets/103
CD34+ cells. Addition of IL-3, SCF, or the combination of
SCF and IL-6 to PEG-rHuMGDF increased the number of MKs by 2.2, 1.6, and 1.5; proplatelet-bearing MKs by 1.9, 1.9, and 1.7; and the number
of platelets by 1.7, 1.5, and 1.2; respectively (Fig 4A, B, and C). Of
interest, addition of IL-6 to the combination of SCF and PEG-rHuMGDF had no effect on the number of proplatelet-bearing MKs. Other than
PEG-rHuMGDF, IL-3 was the only cytokine which induced MK differentiation from CD34+ cells. The number of MKs
obtained from blood CD34+ cells was only 1.8-fold lower
than with rHuMGDF, but MKs represented only 6% of the output. The
number of proplatelet-bearing MKs and platelets was ~14-fold lower
using IL-3 than with PEG-rHuMGDF (P = .01). This difference
with PEG-rHuMGDF-stimulated cultures remained significant despite
addition of SCF or of a combination of SCF plus IL-6 to the IL-3, which
induced the growth of a similar number of MKs than with PEG-rHuMGDF.
Therefore, the more significant effect of PEG-rHuMGDF concerns the
transition from MKs to proplatelet-bearing MKs. The mean ploidy on day
9 in PEG-rHuMGDF-stimulated cultures was 5, and was not significantly
different from that observed with IL-3 alone (4.6). Ploidy was lower on
day 12 (4.4 and 4.3, respectively). A combination of several cytokines
increased MK numbers but led to a significant decrease in ploidy (Fig
5). As previously observed, marrow
CD34+ cells showed a threefold lower capacity to produce
MKs than their blood counterparts.
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| Fig 4.
Effects of PEG-rHuMGDF and of SCF, IL-3, and IL-6 on MK
growth (A), proplatelet formation (B), and platelet production (C). Marrow (n = 3, ) or blood (n = 6,  ) CD34+
cells were grown from 11 to 14 days in serum-free conditions, in the
presence of PEG-rHuMGDF (10 ng/mL), IL-3 (100 U/mL), and different
combinations of these two different cytokines with SCF (50 ng/mL) and
IL-6 (100 U/mL). Asterisks denote a significative difference
(P < .05) in comparison with culture with 1 ng/mL
PEG-rHuMGDF alone.
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| Fig 5.
Effects of PEG-rHuMGDF and of SCF, IL-3, and IL-6 on MK
ploidy. MK ploidy was measured by a double staining technique with an
FITC-anti-CD41b MoAb and propidium iodide solution using flow cytometry. The mean ploidy was calculated. The results are the average
of six experiments performed starting with blood CD34+
cells. Asterisks denote a significative difference
(P < .05) in comparison with culture with 1 ng/mL
PEG-rHuMGDF alone. ( ), MGDF; ( ), IL3; (), MGDF + SCF; (),
MGDF + SCF + IL6; ( ), MGDF + IL3; (), IL3 + SCF; ( ),
IL3 + SCF + IL6.
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In subsequent experiments, cultures were initially stimulated in two
different manners (either with a combination of IL-3 plus IL-6 and SCF,
or with PEG-rHuMGDF) and after 7 days, cultures initially stimulated
with the cytokine combination were switched to PEG-rHuMGDF. The data
indicated that the presence of PEG-rHuMGDF in the second phase of the
culture (corresponding to the terminal phase of MK maturation) did not
induce a high proportion of proplatelet-displaying MKs or the
production of a large quantity of platelets (15% of MKs displaying
proplatelets and 68 × 103 platelets/103
CD34+ cells in the switch conditions v 41% and
220 × 103, for cultures initiated with PEG-rHuMGDF).
Nevertheless, the proportion of MKs displaying proplatelets and the
number of platelets was threefold higher than in cultures stimulated
with IL-3, SCF, and IL-6 throughout the culture period. These data
indicated that in this culture system, the presence of PEG-rHuMGDF at
all phases of megakaryocytopoiesis maximizes platelet production.
The CD34+ cell population is highly heterogenous and
contains all types of hematopoietic progenitors. Depending on the
culture conditions, the percentage of MKs was extremely variable, from 79% in PEG-rHuMGDF stimulated cultures to 4% with a combination of
cytokines. Thus, we investigated the effects of cytokines on proplatelet formation and platelet shedding using a more homogenous population of MK progenitors. In this purpose, the CD34+
CD41+ cells were purified. This cell fraction is highly
enriched in late MK progenitors which synchronously differentiate in 5 to 7 days. However, the majority of the MK progenitors responsible for
platelet production at day 12 are present in the CD34+
CD41 cell population.
Effects of cytokines on CD34+ CD41+ cells.
To reduce the risk of false positivity due to platelet binding to
CD34+ cells, the purification of CD34+
CD41+ cells from BM cells was performed in the presence of
PBS/EDTA and the cells were treated with neuramidinase.
CD34+ CD41+ cells (~2% of CD34+
cells) were cultured in the presence of increasing PEG-rHuMGDF
concentrations (0.01 to 500 ng/mL). Without PEG-rHuMGDF,
CD34+ CD41+ cells disintegrated after 2 days.
MK differentiation and maturation were more rapid at PEG-rHuMGDF
concentrations greater than 1 ng/mL, with development of the first
proplatelet-bearing MKs observed at day 4 using 10 ng/mL, at day 5 at 1 ng/mL, and at day 7 at lower PEG-rHuMGDF concentrations. As shown in
Fig 6, MK proliferation and the percentage
of proplatelet-bearing MKs increased with PEG-rHuMGDF concentrations up
to 1 ng/mL; there was no significant inhibitory effect at higher
PEG-rHuMGDF concentrations up to 500 ng/mL, although some diminution
was observed. The frequency of proplatelet-bearing MKs was 1%, 4%,
44%, 45%, 43%, and 38% at a concentration of 0.01, 0.1, 1, 10, 100, and 500 ng/mL, respectively. Platelet production precisely paralleled
the number of proplatelet-bearing MKs and reached a plateau at 1 ng/mL
PEG-rHuMGDF. The production of platelets per MK was threefold higher in
cultures initiated with CD34+ CD41+ cells than
in those commencing with the total CD34+ population (Fig
4). The ploidy of MK grown from CD34+ CD41+
cell population was much higher (mean ploidy value = 8; Fig
7A) than in cultures derived from
CD34+ cells (mean = 5).
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| Fig 6.
Effects of increasing concentrations of PEG-rHuMGDF on
proplatelet formation (A) and platelet production (B) from
CD34+ CD41+ cells. CD34+
CD41+ cells sorted from marrow were grown in serum-free
conditions in the presence of increasing concentrations of PEG-rHuMGDF
(0.01 to 500 ng/mL). A murine soluble Mpl receptor was added at day 4 (time of differentiation into proplatelet-bearing MKs,  ) or day 6 (time of platelet shedding,  ) to cultures stimulated with 1 ng/mL
PEG-rHuMGDF. PEG-rHuMGDF (500 ng/mL) was also added at day 4 to
cultures initiated with 1 ng/mL of this cytokine ( ). MKs bearing
proplatelets and platelets were enumerated at days 6 and 7, respectively, as in Figs 3 and 4. Asterisks denote a significative
difference (P < .05) in comparison with culture with 1 ng/mL PEG-rHuMGDF alone.
|
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| Fig 7.
Comparison of ploidy distribution of MKs and the
expression of CD41b in different ploidy classes in the MKs obtained in
the presence of PEG-rHuMGDF or the combination of IL-3, SCF, and IL-6 from CD34+ CD41+ cells. Marrow
CD34+ CD41+ cells were grown from 6 days in
serum-free conditions, in the presence of PEG-rHuMGDF (10 ng/mL) or
IL-3 (100 U/mL) plus SCF (50 ng/mL) plus IL-6 (100 U/mL). Ploidy of the
CD41+ cells obtained in the presence of PEG-rHuMGDF (A)
and IL-3 plus SCF plus IL-6 (B). Comparison of the expression of CD41b
in MK cultivated in the presence of PEG-rHuMGDF (solid line) and the combination of IL-3, SCF and IL-6 (broken lines) in the 2N (C), 4N (D),
8N (E), 16N (F), and 32N MKs (G). MKs were considered as
CD41b+ cells and analysis was performed in each ploidy
class.
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The effect of PEG-rHuMGDF on platelet production was investigated
further in three experiments by adding a soluble Mpl receptor or a high
concentration (500 ng/mL) of PEG-rHuMGDF after 4 to 5 days of culture
in the presence of 1 ng/mL PEG-rHuMGDF (these additions were made at
the time of differentiation into proplatelet-bearing MKs). A high dose
of PEG-rHuMGDF had no effect on proplatelet formation and the number of
platelets produced, whereas a soluble Mpl receptor markedly inhibited
their production. In contrast, a soluble Mpl receptor added at day 6 (at the time of platelet shedding) had no effect (Fig 6).
The effects of the three other tested cytokines (IL-3, SCF, and IL-6)
were also determined on this population of mature MK progenitors
(n = 7). The only other cytokine promoting MK proliferation and
differentiation into proplatelet-bearing MKs was IL-3, but its effect
was weak compared with that of PEG-rHuMGDF. In contrast, a combination
of two cytokines gave a higher number of proplatelet-bearing MKs and
platelets with IL-3 + SCF > IL-3 + IL-6 > SCF + IL-6. The
combination of the three cytokines (SCF, IL-3, and IL-6) gave the best
results by inducing the growth of a number of MKs quite similar to that
obtained with PEG-rHuMGDF (1.2 or 0.8 × 103
proplatelet-bearing MKs in presence of PEG-rHuMGDF or combination of
the three cytokines, respectively). Platelet production was decreased
but only 2.4-fold in comparison with PEG-rHuMGDF-stimulated cultures
(46 or 111 platelets per 1 × 103 CD34+
CD41+ cells, respectively). Ploidy distribution (Fig 7A and
B) and expression of CD41 in each ploidy class (Fig 7C through G) were also identical to that noted in PEG-rHuMGDF-stimulated cultures.
As SCF and IL-6 do not permit MK proliferation when used alone, we
studied their effect on MK differentiation and platelet production by
adding them to a suboptimal concentration (1 ng/mL) of PEG-rHuMGDF.
Both cytokines did not significantly modify the number of MK and
platelets produced. In contrast, addition of IL-3 to PEG-rHuMGDF
inhibited proplatelet formation (Fig 8).
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| Fig 8.
Effects of PEG-rHuMGDF associated with other cytokines on
platelet production from CD34+ CD41+ cells.
Marrow-derived CD34+ CD41+ cells were grown
in serum-free conditions in the presence of 1 ng/mL PEG-rHuMGDF alone
or combined with IL-3 (100 U/mL), SCF (50 ng/mL), and IL-6 (100 U/mL).
Platelets were enumerated at day 7 of culture by flow cytometry.
Asterisk denotes a significative difference (P < .05) in
comparison with culture with 1 ng/mL PEG-rHuMGDF alone.
|
|
Structure and Function of Platelets Produced in Culture
We previously described the ultrastructure of platelet formation by
human MKs cultured with PEG-rHuMGDF.29 No substantial differences were observed in the ultrastructure of the
platelet-shedding MKs produced in the presence of either PEG-rHuMGDF or
the combination of IL-3, SCF, and IL-6. In the two conditions, platelet
shedding MKs showed several cytoplasmic expansions which correspond to proplatelets (Figs 9a and
10a); constriction zones already
individualize distinct platelet fields. In the same way, platelets
derived from culture with either PEG-rHuMGDF or the
combination of IL-3, IL-6, and SCF were of similar size and exhibited
- and dense granules. Many of these platelets were adherent to MKs;
however, they did not exhibit ultrastructural evidence of activation
(Figs 9b and 10b). By fluorescent labeling, circumferential microtubule
coils and actin filaments were observed with an antitubulin MoAb and phalloidin in the vast majority of the culture-derived platelets (data
not shown).
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| Fig 9.
Ultrastructure of platelet shedding MKs and platelets
obtained in the presence of PEG-rHuMGDF from CD34+
CD41+ cells. (a) A mature MK presenting signs of platelet
formation. This MK displays dilatation of the demarcation membranes
(dm) located at the periphery. They individualize a zone of cytoplasm (arrows) which will form a future proplatelet (N, nucleus). (b) Platelet-sized-fragments (P) exhibit the usual cytoplasmic organelles: granules (A), smooth connected canalicular system (sccs), and endoplasmic reticulum (er).
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| Fig 10.
Ultrastructure of platelet shedding MKs and platelets
obtained in the presence of the combination of SCF, IL-3, and IL-6 from CD34+ CD41+ cells. (a) A similar mature MK
as in Fig 9a with dilatation of demarcation membranes (dm) at the
periphery (arrows) (N, nucleus). (b) Platelet-sized-fragments (P) are
present with the combination of three cytokines. They are similar to
those obtained with PEG-rHuMGDF (Fig 9b). On a proplatelet (PP), a
constriction zone area is disposed along the cytoplasmic extension
individualizing a distinct platelet field with a vacuole at the level
of the future rupture (arrow) (A, granules; sccs, smooth connected
canalicular system; er, endoplasmic reticulum).
|
|
Expression of CD62 on culture-derived platelets produced in various
culture conditions was subsequently investigated. Before activation,
less than 10% of platelets expressed CD62 on their surfaces (Fig
11A). A majority were capable of being
activated. Activation was associated with slight changes in the scatter
properties of culture-derived platelets (Fig 1D and E). The percentage
of activated platelets in cultures using PEG-rHuMGDF
(92%) was similar to that of cultures using a combination of SCF,
IL-3, and IL-6 (70%) (Fig 11B).
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| Fig 11.
Expression of CD62 on the surface of platelets produced
in vitro from CD34+ cells (A) or CD34+
CD41+ (B). MKs were cultured either from
CD34+ in the presence of PEG-rHuMGDF (A) or
CD34+ CD41+ in the presence of PEG-rHuMGDF
or the combination of IL-3, SCF, and IL-6 (B). At day 13 or 7 for
cultures deriving from CD34+ or CD34+
CD41+, respectively, cells were stimulated for 10 minutes
at 37°C with 2 U/mL thrombin. Activated and nonactivated platelets
were incubated with phycoerythrin (PE) anti-CD62 and FITC-TAB and
analyzed relative to PE-conjugated IgG1MoAb control by flow cytometry
in a morphological gate corresponding to blood platelets. (A)
Serum-free cultures in the presence of PEG-rHuMGDF (10 ng/mL) from
CD34+ cells. Expression of CD62 (solid line) in
thrombin-activated platelets in comparison with nonactivated platelets
(broken line). (B) Serum-free cultures in the presence of PEG-rHuMGDF
(10 ng/mL) or rHuSCF (50 ng/mL), rHuIL-6 (100 U/mL), and rHuIL-3 (100 U/mL) from CD34+ CD41+ cells. Expression of
CD62 in thrombin-activated platelets from PEG-rHuMGDF (thick solid
line) or SCF, IL-3, and IL-6 (thin solid line). Isotype control is
shown (broken line).
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|
In some experiments, it was observed that CD41+ elements
with high scatter properties and which were excluded from the
analytical gate expressed CD62 before activation. The CD62 level was
not increased by thrombin stimulation, suggesting that a proportion of
the platelets quickly activate or become permeable to anti-CD62 during
or following production in vitro.
| |
DISCUSSION |
We have developed a culture system capable of producing large numbers
of MKs which release platelets after terminal differentiation. This
culture system is similar to that described by Choi et
al,13,18,31 and is initiated with CD34+ cells.
However, the cultures reported here were performed in only one step.
The present culture model is quite easy and can be used to study the
terminal stages of megakaryocytopoiesis and platelet production. One
pitfall of these cultures is the definition of platelets produced in
vitro. Circulating platelets have precise cytological and
ultrastructural features. Most workers define platelets by their
discoid form, the presence of circumferentially arranged microtubules
(relative to the plasma membrane), and specific organelles
(-granules and an open canalicular system). In cultures containing a
majority of mature MKs, it is more difficult to define platelets. Such
cultures can contain (1) functional platelets with typical
ultrastructural characteristics, (2) detached proplatelets or giant
platelets that may further fragment to evolve into functional platelets, (3) lytic, nonfunctional fragments of MKs, and (4) platelet
microparticles. Apart from the studies of Choi et al,13 there are no published reports on platelet production in vitro. Those
investigators used a double-centrifugation procedure to isolate cell
fragments produced in culture that have features of circulating
platelets, including size (6 to 10 fL). Using this size criterion, they
enumerated culture-derived platelets with an electrical impedance
device. Employing the same conditions, we were unable to reproduce
their results, because most of the cultured platelets could not be
separated from MKs by centrifugation. At the ultrastructural level, we
observed that a portion of the platelets produced in culture adhere to
MKs during centrifugation, although there was no sign of activation.
Therefore, in this study cultured platelets were defined on the basis
of cytometric criteria: scatter properties and GPIIb expression
identical to blood platelets. This definition has the advantage of not
requiring centrifugation, and platelets produced in varying culture
conditions can be readily quantitated. Moreover, these platelet-like
particles were capable of being activated by thrombin, providing an
additional criterion for a "viable" platelet. According to this
definition, nonviable platelets were also present, and exhibited high
side scatter properties and constitutive CD62 expression.
During optimization of the culture conditions, two main observations
were made. First, substantial numbers of proplatelet-displaying MKs and
platelets were noted only in serum-free conditions. In the guinea pig,
it has been shown that serum prothrombin was responsible for inhibition
of proplatelet formation and its effects could be reversed with
glycosaminoglycans.11,12 Addition of heparin to the
serum-containing cultures improved proplatelet formation and platelet
production. Only a slight improvement was noted when an anti-TGF-
antibody was added. TGF- is involved in the negative regulation of
megakaryocytopoiesis,24,32,33 but does not seem to inhibit
proplatelet formation.12 Therefore, other serum or plasma
molecules which remain to be identified may inhibit platelet production
in vitro. Second, blood CD34+ cells obtained from
cytapheresis after mobilization gave rise to a threefold higher number
of MKs and platelets than their marrow counterparts, but with a delay
in their kinetics of growth. This may be caused by either a higher
percentage or greater proliferative capacities of the CD34+
colony-forming unit-MK contained in the blood in comparison with the
marrow.
Although this culture system was quite efficient in the production of
large numbers of MKs (4 MKs/CD34+ cell) and
proplatelet-displaying MKs (up to 60% of the MKs), the number of
platelets produced per MK is low (<50 per proplatelet-bearing MK).
This is partly due to an underestimation of the number of platelets
produced because cultures are not synchronous and platelets are not
released simultaneously. Consequently, a portion of the produced
platelets are not viable at the time of analysis. However, it is likely
that another important factor explaining this platelet production
defect is the low ploidy of MKs produced in vitro. This is not directly
caused by the cytokines employed because the ploidy was identical using
either PEG-rHuMGDF or IL-3, but appears to be related to the degree of
progenitor cell proliferation. The combinations of cytokines which
increased MK number to the greatest degree markedly diminished
polyploidization. Moreover, we did not observe an increase in ploidy
with time, but rather a marked decrease, in contrast to a previous
report.34 A reasonable hypothesis is that mature MK
progenitors which have low proliferative ability give rise to high
ploidy MKs within several days, whereas more primitive progenitors will
proliferate at the expense of endomitosis during terminal
differentiation. This hypothesis is supported further by the fact that
CD34+ CD41+ cells gave rise to MKs with an 8N
modal ploidy in 5 to 7 days. An inverse relationship between
proliferation and polyploidization has been described previously for
IL-3-stimulated cultures.35-37 These data may have
consequences for in vitro approaches to expand MKs from
CD34+ cells for clinical purposes. Although use of multiple
cytokines augments the total number of CD41+ or
CD61+ cells,38-40 these cytokine combinations
may expand low ploidy MK with limited platelet production capacity.
Choi et al14,31 reported a higher number (>200) of
platelets produced per MK. This difference can be explained by the use
of velocity sedimentation for MK purification. That procedure depletes
the 2, 4, and 8N cell populations and thus enriches 16N
cells.41
PEG-rHuMGDF has a critical effect on the in vitro production of
proplatelet-displaying MKs and of platelets. IL-3 or a combination of
SCF plus IL-6 and IL-3 were capable of giving rise to a similar or
greater number of MKs than PEG-rHuMGDF. However, stimulation with
PEG-rHuMGDF yielded a greater percentage of MKs bearing proplatelets and a higher total number of platelets than did stimulation using other
cytokines. Differences in platelet production between PEG-rHuMGDF and
this combination of three cytokines were much less when
CD34+ CD41+ cells were used instead of the
entire CD34+ cell population. The combination of SCF, IL-3,
and IL-6 stimulated primarily the growth of non-MK cells from
CD34+ cells, whereas a nearly pure population of MKs was
derived from CD34+ CD41+ cells. It is likely
that these contaminating cells inhibit proplatelet formation and
platelet shedding. In favor of this hypothesis, it has been recently
described that accessory cells synthesize proteases which inhibit
growth of hematopoietic progenitors in serum-free
conditions.42 However, this study clearly shows that Mpl-L
is not absolutely required in culture to observe full MK development as
previously suggested,43 since "viable" platelets capable of being activated by thrombin are produced in culture in its
absence. Whether the platelets produced in the presence of Mpl-L or
other cytokines have similar functions remains to be determined. These
in vitro results are comparable to those observed in c-mpl or
Mpl-L knockout mice which have a 90-fold decrease in their number of
platelets.44-46 Also valuable for interpreting the effects
of cytokines on MK differentiation may be p45 NF-E2 knockout mice. Mice
lacking p45 NF-E2 have a lethal thrombocytopenia with an increased
number of MKs. These MKs exhibit abnormal demarcation membranes and a
decreased number of granules.47 Because no binding site for
NF-E2 is found in the promoter region of MK-specific genes,
Shivdasani48 has hypothesized that NF-E2 regulates another set of genes involved in the regulation of MK cytoplasmic maturation. At the level of the MK progenitor, expression of MK-restricted genes
could be regulated by several cytokines including Mpl-L, IL-3, or IL-6,
whereas later in differentiation genes involved in MK cytoplasmic
maturation may be regulated essentially by Mpl-L. However unlike the in
vivo approach, our in vitro system using IL-3 and IL-6 may more mimick
stress conditions of platelet regulation than a physiological
regulation.
By testing the dose-response characteristics of PEG-rHuMGDF on
CD34+ CD41+ cells, it was noted that
PEG-rHuMGDF accelerated MK maturation and increased platelet formation
up to 1 ng/mL. Higher doses did not significantly modify these results
and only a slight decrease (<10%) was observed when 500 ng/mL was
added at the stage of proplatelet formation. Others have reported a
more marked inhibitory effect of PEG-rHuMGDF on proplatelet formation
at high concentrations18,19 and it has been hypothesized
that this paradoxical effect of PEG-rHuMGDF is due to an inhibition of
MK apoptosis.49 As in the present report, Horie et
al50 recently described a crucial effect of PEG-rHuMGDF
vis-à-vis subsequent proplatelet production at the level of the
rat MK progenitor, but with an optimal dose of 0.037 ng/mL. Larger MKs
subsequently forming prominent proplatelets were observed at higher
concentrations. The difference between the present study and that of
Horie et al50 may be related to the species. Addition of
SCF and IL-6 to PEG-rHuMGDF-stimulated cultures did not modify
proplatelet formation. In contrast, in the presence of PEG-rHuMGDF,
addition of IL-3 inhibited this process. A negative effect of IL-3 was
also observed in the rat; however, it was found that IL-6 or IL-11 were
very potent inducers of proplatelet formation.50 This
effect of IL-6 on proplatelet formation seems to be restricted to
rodents and is not observed with human cells.49
Finally, we investigated whether PEG-rHuMGDF is necessary for the final
stages of thrombocytopoiesis. Addition of a soluble Mpl receptor to a
homogenous population of mature proplatelet-displaying MKs derived from
CD34+ CD41+ cells did not inhibit platelet
production 1 or 2 days later. However, when the same experiments were
performed on unseparated CD34+-derived MKs which were at
different stages of maturation, or on CD34+
CD41+ cells prior to proplatelet formation, addition of the
soluble Mpl receptor inhibited proplatelet formation and induced
apoptosis of maturing MKs. This difference may be explained by the fact that Mpl-L is not necessary for platelet shedding, which may be an
intrinsic property of mature MKs,18,49 but is absolutely required for survival and terminal differentiation of immature MKs.
In conclusion, we have developed a simple liquid culture method to
study the late stages of platelet production. This culture technique
may permit testing the effects of cytokines or stromal cells on
platelet formation. It may also facilitate the characterization of the
genes involved in platelet formation by investigating cultures from
patients with congenital thrombocytopenia.
| |
FOOTNOTES |
Submitted May 20, 1997;
accepted September 23, 1997.
Supported by the Institut National de la Santé et de la Recherche
médicale, the Institut Gustave Roussy, and by grants from the
Association de la Recherche contre le Cancer (ARC), la Ligue Nationale
contre le Cancer, and a Fogarty International Fellowship (to S.A.B.).
Address reprint requests to Najet Debili, PhD, INSERM U 362, PR1,
Institut Gustave Roussy, Villejuif, 94800, France.
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 grateful to J.-L. Nichol (Amgen, Thousand Oaks, CA) for
providing the rHuSCF and PEG-rHuMGDF, to D. Foster (ZymoGenetics, Seattle, WA) for the murine soluble Mpl receptor, to D. Cosman (Immunex, Seattle, WA) for rHuIL-3, and to A. Katz and M. Zohar for
cell sorting. We are indebted to J.-M. Massé for photographic assistance.
| |
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Abstract
Introduction
Abstract