Bone Marrow Transplantation (2001) 27, 1075–1080
 2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00
www.nature.com/bmt
Stem cell expansion
Platelet-derived growth factor enhances ex vivo expansion of
megakaryocytic progenitors from human cord blood
RJ Su1, K Li1, M Yang1, XB Zhang1, KS Tsang2, TF Fok1, CK Li1 and PMP Yuen1
1
Department of Paediatrics, 2Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University
of Hong Kong, Hong Kong
Summary:
Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is a strategy for shortening the duration of
thrombocytopenia after haematopoietic stem cell transplantation. The cell dose after expansion has emerged
as a critical factor for achieving the desired clinical outcomes. This study aimed to establish efficient conditions
for the expansion of the MK lineage from enriched
CD34+ cells of umbilical cord blood and to investigate
the effect of platelet-derived growth factor (PDGF) in
this system. Our results demonstrated that thrombopoietin (TPO) alone produced a high proportion of
CD61+CD41+ cells but a low total cell count and high
cell death, resulting in an inferior expansion. The
addition of interleukin-1␤ (IL-1␤), Flt-3 ligand (Flt-3L)
and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines
produced efficient expansions of CFU-MK up to 400fold with the highest yield observed in the presence of
TPO, IL-1␤, IL-3, IL-6 and Flt-3L. CD34+ cells were
expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61+CD41+ cells, CFU-MK
and CD34+ cells increased by 101%, 134% and 70%,
respectively. On day 14, the CD61+ population consisted
of diploid (86.5%), tetraploid (11.8%) and polyploid
(8N–32N; 1.69%) cells. Their levels were not affected
by PDGF. TPO, IL-1␤, IL-3, IL-6, Flt-3L and PDGF
represented an effective cytokine combination for
expanding MK progenitors while maintaining a moderate increase of CD34+ cells. This study showed, for the
first time, that PDGF enhanced the ex vivo expansion
of the MK lineage, without promoting their in vitro
maturation. PDGF might be a suitable growth factor to
improve the ex vivo expansion of MK progenitors for
clinical applications. Bone Marrow Transplantation
(2001) 27, 1075–1080.
Keywords: platelet-derived growth factor; ex vivo expansion; megakaryocytic progenitors; ploidy
Correspondence: Dr K Li, Department of Paediatrics, The Chinese University of Hong Kong, 6th fl, The Prince of Wales Hospital, Shatin, NT,
Hong Kong
Received 12 January 2001; accepted 11 March 2001
Thrombocytopenia remains a serious problem in patients
treated with intensive high-dose chemotherapy and haematopoietic stem cell transplantation. This condition is
especially apparent after umbilical cord blood transplant
when the platelet recovery time is frequently delayed compared with transplants using bone marrow or mobilised
peripheral blood stem cells.1,2 The infusion of ex vivo
expanded megakaryocytic (MK) progenitors to transplant
patients has been proposed as a strategy for accelerating
platelet recovery. Several clinical trials using expanded peripheral blood stem cells3,4 and bone marrow5 have demonstrated some success in alleviating thrombocytopenia after
high-dose chemotherapy.
A number of cytokines are known to regulate
megakaryocytopoiesis in vivo and enhance the expansion
of MK cells in vitro. TPO has been identified as the most
effective cytokine for megakaryocytopoiesis as it acts on
different developmental stages.6,7 IL-1 enhances the proliferation of MK progenitors.8,9 IL-3 increases the absolute
number of CD34+CD41+ cells in liquid culture6,7 and
expands the number of MK colonies.10,11 IL-6 or IL-11
enhance effects of other growth factors, such as IL-3 and
IL-1, on the induction of MK differentiation, and they
stimulate late stages of MK development.12,13 Flt-3L and
SCF promote the growth of MK colonies, possibly via their
direct stimulation of CD34+ cells.14,15
Platelet-derived growth factor (PDGF) is a member of
the connective tissue growth factor family which includes
vascular endothelial growth factor, basic fibroblast growth
factor and tumor necrosis factor-␤. Initially it was isolated
from human platelets as a protein with a molecular mass of
28–31 kDa.16,17 PDGF consists of two polypeptide chains,
designated A and B, linked together by disulphide bonds.18
PDGF exists in three isoforms (AA, AB and BB). PDGFBB binds to both PDGF ␣- and ␤-receptors and possesses
stronger mitogenic activity than the other two isoforms.19
PDGF does not only stimulate the growth and differentiation of connective tissue cells, such as fibroblasts, endothelial cells and smooth muscle cells,20 but also affects
haematopoiesis. PDGF-B and ␤-receptor knock-out mice
develop haemorrhage and edema,21,22 but the mechanism
remains unclear. PDGF also promotes in vitro multipotent
haematopoietic progenitors and erythropoiesis.23,24 In previous studies, we demonstrated the presence of functional
PDGF receptors on human MK and MK cell lines25 and
PDGF enhances ex vivo expansion
RJ Su et al
1076
showed that PDGF promoted MK colony formation.26 In
this study, we aimed to establish a cytokine combination
for the efficient ex vivo expansion of the MK lineage from
umbilical cord blood CD34+ cells, and to investigate the
effect of PDGF in this system. Our read-out criteria
included CD34+ stem and progenitor cells, colony-forming
progenitor cells of the MK lineage (CFU-MK),
CD61+CD41+ cells and their ploidy/maturation status.
Materials and methods
Human cord blood collection
Umbilical cord blood samples were collected into preservative free heparin (100 IU/ml, David Bull Laboratories,
Victoria, Australia) following normal, full-term vaginal
delivery. These samples were kept at room temperature and
processed within 24 h. Informed parental consent was
obtained for all blood collections and this study was
approved by the Ethics Committee for Clinical Research of
The Chinese University of Hong Kong.
CD34+ cell enrichment and ex vivo culture
Whole cord blood specimens (mean volume 38 ⫾ 1.68 ml,
range 30–45 ml) were diluted with Ca++ and Mg++-free
phosphate-buffered saline (PBS)/0.2% bovine serum albumin (BSA) supplemented with 0.6% ACD. Mononuclear
cells (MNC) were obtained by Ficoll density gradient
(1.077 g/ml; Pharmacia Biotech, Uppsala, Sweden). CD34+
cells were enriched by immunomagnetic cell selection
(VarioMACS system; Miltenyi Biotec, Gladbach,
Germany) according to the manufacturer’s instructions.
Enriched CD34+ cells at 4 × 104/ml were cultured in
Iscove’s modified Dulbecco’s medium (IMDM; Gibco,
Grand Island, NY, USA) supplemented with 10% fetal calf
serum (FCS; Gibco) in a 24-well plate (Costar, Cambridge,
MA, USA) at 37°C in a fully humidified atmosphere containing 5% CO2. Cultures contained the indicated combinations of growth factors. On days 7 and 10, 2 and . volume
of the media were changed, respectively. On days 7 and 14,
CFU-MK and cell surface expression of CD41 and CD61
antigens were analysed. On day 14, the ploidy status of MK
cells was measured by flow cytometry. Viable cell counts
were determined by trypan blue exclusion and nucleated
cell were counted using a haemocytometer.
Cytokines used in the experiments were: recombinanthuman PDGF-BB (50 ng/ml), TPO (50 ng/ml), IL-1␤ (20
ng/ml), IL-3 (20 ng/ml), IL-6 (20 ng/ml), Flt-3L (20 ng/ml).
All cytokines, unless otherwise specified, were products of
Pepro Tech (Rocky Hill, NJ, USA). The optimal concentrations of these cytokines were determined in previous
studies.9,14,25–27
Flow cytometry analysis of cell surface markers and
ploidy
+
Enriched CD34 cells or cultured cells were incubated with
fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)labeled monoclonal antibodies or their respective isotypic
Bone Marrow Transplantation
controls, for 30 min at room temperature. These cells were
washed with PBS/0.2% BSA and analysed by the FACScan
instrument (Becton Dickinson (BD), San Jose, CA, USA).
Ten thousand and 30 000 events were acquired for each
sample before and after expansion, respectively, with dead
cells and debris being gated out by the forward and side
scatter profiles during data analysis. The following monoclonal antibodies were used: IgG1-FITC (BD); IgG1-PE
(BD); CD61-FITC (Dako, Copenhagen, Denmark); CD41PE (Dako); CD34-FITC (BD).
MK ploidy was measured by a two-colour, flow cytometric technique described by Debili et al7 with slight
modifications. On day 14, cells were harvested and washed
with PBS/0.2% BSA. They were then labeled with 20 ␮l
of CD61-FITC/1 × 106 cells at room temperature in the
dark for 30 min. After washing, these cells were resuspended in 0.3 ml Saponin (0.1%, Sigma, Saint Louis, MO,
USA) and incubated at room temperature for 10 min before
the addition of 0.1 ml of RNase (2.5 mg/ml; Bio-Rad, Hertfordshire, UK) for another 10 min. Finally, 0.2 ml of propidium iodide (250 ␮g/ml; Sigma) was added and incubated
for 10 min before analysis by flow cytometer. Seventy
thousand events were acquired for ploidy analysis. The
doublet discrimination function of the CellFIT software
(BD) was used to gate in single cells only.
CFU-MK assay
On days 0, 7 and 14, 3000 cells /ml were seeded into 24well plates in triplicate with 0.5 ml in each well. The culture contained IMDM supplemented with 30% FCS, 1%
BSA, 1.3% methylcellulose, 50 ng/ml TPO and 20 ng/ml
IL-3. After 14 days of incubation at 37°C, 5% CO2, CFUMK were identified microscopically and scored as a group
of more than three MK cells in a cluster.8
Morphological analysis
Cultured cells (day 14) were cytospun on to glass slides
and stained with May–Grunwald-Giemsa.27 Cell
morphology was examined by light microscopy.
Statistical analysis
Statistical analysis was performed using the SigmaStat
software (Jandel Scientific Software, San Rafael, USA).
Comparisons between treatment groups were performed by
the analysis of variance and the paired t-test or Wilcoxon
sign rank test, where appropriate. A P value of ⭐0.05 was
considered as statistically significant. All values were
expressed as mean ⫾ standard error of mean (s.e.m.).
Results
Expansion of nucleated cells and CD34+ cells
The purity of the enriched CD34+ cells was 95.1 ⫾ 0.71%
(range 93.3–98.3%; n = 7). The absolute numbers of
expanded cells were significantly higher on day 14 when
compared with those from day 7 in all treatment groups (P
PDGF enhances ex vivo expansion
RJ Su et al
⬍ 0.05, Table 1). On day 14, total cell counts were significantly lower in cultures treated with TPO or TPO + IL-1␤
when compared with those treated with three or more
growth factors (P ⬍ 0.05). The significant enhancing
effects of PDGF were observed in most cytokine groups
on days 7 and 14 (Table 1). Cell viability as tested by trypan blue exclusion assay was high on day 7 (mean value
over 95% for all groups). On day 14, cell viability was well
maintained in groups treated with three or more cytokines
(mean 86.3–92.2%) but was significantly lower in the TPO
or TPO + IL-1␤ groups with or without PDGF (mean
67.2–73.5%).
CD34+ cells were not expanded in the presence of TPO
or TPO + IL-1␤ on day 7 (mean 0.70–1.05-fold) or day
14 (mean 0.27–1.04-fold). Additional cytokines appeared
to enhance the CD34+ cell yield. Significant increases in
CD34+ cells were observed in cultures with TPO + IL-6 +
Flt-3L when compared with those containing one or two
cytokines (P ⬍ 0.05) with or without PDGF. Flt-3L significantly increased the numbers of CD34+ cells on day 7
(P ⬍ 0.006) and day 14 (P ⬍ 0.05) when compared with
the respective controls but IL-1␤ or IL-3 did not have such
effects. The addition of PDGF significantly increased the
yield of CD34+ cells in most treatment groups (Table 1) to
up to 31.4-fold on day 14.
Expansion of CD41+CD61+ cells
The proportions of CD41+CD61+ cells were high with mean
values over 75% in cultures expanded with TPO alone
(Table 1). However, due to relatively low cell counts, the
Table 1
number of CD41+CD61+ cells was lowest. On day 7, the
addition of IL-1␤ maintained the proportion of these cells
and significantly increased total cell number when the
respective groups were compared (P ⬍ 0.05). Similar
trends were observed on day 14 but the differences were
not significant. While the addition of Flt-3L did not have
such effect, IL-3 increased CD41+CD61+ cells on day 7 (P
⬍ 0.016) but not on day 14. The addition of PDGF to these
cytokine combinations enhanced the yield of total
CD41+CD61+ cells but had little effect on their proportions
in the culture (Table 1).
1077
Expansion of megakaryocytic progenitor cells (CFU-MK)
On days 7 and 14, the trends in CFU-MK expansion were
in general similar to those of CD41+CD61+ cells as indicated in Table 1 and Figure 1. The expansion outcomes
were inferior with TPO alone or with TPO + IL-1␤ (P ⬍
0.05). At day 7, either IL-1␤ or Flt-3L significantly
increased CFU-MK when compared with respective groups
without the cytokines (P ⬍ 0.05). A similar effect was
observed on day 14 at which time a significant enhancement of CFU-MK by IL-1␤ was observed in the TPO
groups with or without PDGF (P ⬍ 0.05). The effect of
Flt-3L was demonstrated in the TPO + IL-1␤ group without
PDGF (P = 0.039). Again, PDGF significantly enhanced
the expansion of CFU-MK in most cytokine combination
groups (Figure 1). In the four treatment groups with three
to five cytokines, mean CFU-MK were expanded by 122–
409-fold and the addition of PDGF improved the yield by
30–134%.
Ex vivo expansion of cord blood CD34+ cells
T
T+1
T+1+F
T+6+F
T+3+6+F
T+1+3+6+F
Day 7
Total cell count
(×105)
−P
+P
2.59 ± 0.64
4.03 ± 0.94*
4.16 ± 0.74
5.39 ± 1.03
9.27 ± 1.66
13.3 ± 1.85*
9.31 ± 2.17
12.6 ± 2.93*
15.4 ± 2.43
22.1 ± 3.30**
19.8 ± 2.86
27.6 ± 3.02*
CD34+ cells
(×105)
CD61+CD41+
cells (%)
−P
+P
−P
+P
0.32 ± 0.08
0.47 ± 0.10*
55.6 ± 3.20
54.9 ± 4.35
0.38 ± 0.08
0.48 ± 0.07
51.7 ± 4.35
56.7 ± 4.17
1.34 ± 0.29
1.97 ± 0.34*
25.7 ± 3.27
27.8 ± 3.53
2.07 ± 0.47
2.93 ± 0.58*
15.5 ± 2.21
14.1 ± 1.97
2.55 ± 0.45
3.82 ± 0.63*
24.1 ± 3.07
23.6 ± 3.56
3.01 ± 0.69
4.19 ± 0.69*
25.0 ± 3.54
27.1 ± 4.29
CD61+CD41+
cells (×105)
CFU-MK
(×103)
−P
+P
−P
+P
1.49 ± 0.41
2.27 ± 0.60*
1.26 ± 0.31
2.00 ± 0.39*
2.26 ± 0.53
3.19 ± 0.73
1.96 ± 0.44
3.21 ± 0.51
2.60 ± 0.68
3.93 ± 0.85*
5.03 ± 0.87
9.41 ± 2.18*
1.64 ± 0.49
1.98 ± 0.54
6.64 ± 1.07
8.64 ± 0.98**
4.06 ± 1.12
5.78 ± 1.15*
8.40 ± 1.35
14.1 ± 2.20**
5.15 ± 1.05
8.11 ± 1.84*
14.7 ± 1.85
23.1 ± 3.69*
Day 14
Total cell count
(×105)
−P
+P
13.4 ± 5.22
15.8 ± 5.87
27.9 ± 13.7
44.3 ± 16.9*
81.8 ± 15.0
120 ± 13.9**
102 ± 16.3
134 ± 18.7*
164 ± 57.4
215 ± 66.5*
179 ± 41.7
223 ± 55.3
CD34+ cells
(×105)
−P
+P
0.13 ± 0.09
0.23 ± 0.16
0.30 ± 0.12
0.47 ± 0.12*
2.40 ± 0.73
3.59 ± 0.85**
9.84 ± 2.49
14.1 ± 3.50*
8.88 ± 2.44
12.9 ± 2.82
5.13 ± 1.11
6.90 ± 1.44
CD61+CD41+ cells
(%)
CD61+CD41+
cells (×105)
−P
+P
−P
+P
75.9 ± 4.82
74.1 ± 6.99
10.5 ± 4.28
12.3 ± 4.83
75.7 ± 3.66
76.4 ± 4.01
21.1 ± 9.97
34.2 ± 12.5
27.1 ± 7.32
26.3 ± 8.10
21.7 ± 6.24
33.7 ± 12.3*
19.0 ± 4.25
20.6 ± 5.35
19.2 ± 4.92
26.9 ± 7.81
19.9 ± 6.03
23.8 ± 7.05
29.5 ± 10.2
43.0 ± 11.8**
20.5 ± 3.71
18.7 ± 3.70
32.8 ± 5.22
36.5 ± 6.30
CFU-MK
(×103)
−P
+P
2.19 ± 0.63
2.93 ± 1.01
9.16 ± 2.42
15.0 ± 4.21
51.9 ± 15.8
97.2 ± 35.4*
56.5 ± 18.0
103 ± 30.1*
135 ± 47.2
183 ± 54.0*
159 ± 58.8
221 ± 83.5
The cell populations were derived from the cultures of 4 × 104 CD34+ cells at day 0. Parameters in cultures with and without PDGF were compared.
−P = without PDGF; +P = with PDGF; N = 7.
*P ⬍ 0.05; **P ⬍ 0.01.
Bone Marrow Transplantation
PDGF enhances ex vivo expansion
RJ Su et al
1078
*
T+1+3+6+F
100
**
**
T+6+F
10
Ploidy (%)
T+3+6+F
*
T+1+F
T+1
-P
*
T
0
10
+P
1
0.1
0.01
20
30
40
50
60
70
0.001
Fold expansion of CFU-MK at day 7
1
2
3
4
5
T+1+3+6+F
*
T+6+F
>=16N
*
T+1+F
*
T+1
-P
+P
T
0
100
200
300
400
500
600
700
Fold expansion of CFU-MK at day 14
Figure 1 Ex vivo expansion of cord blood CD34+ cells to CFU-MK
CFU-MK were expanded in the presence of TPO (T); IL-1␤ (1); IL-3 (3);
IL-6 (6); Flt-3L (F) and PDGF (P). The data represent the mean (s.e.m.)
of seven independent experiments and were calculated as the fold increase
of the starting CFU-MK values on day 0. Paired tests were used to compare the treatment groups with and without PDGF. *P ⭐ 0.05, **P ⬍ 0.01.
Polyploidisation of megakaryocytes
CD61-FITC
FL1-H\FL1-Height --->
8
R2
R3
R4
8N
9
10
4N
11
12
2N
4N
8N
>=16N
2N
Figure 3 Effect of cytokines and PDGF on megakaryocyte ploidy distribution. The ploidy status of CD61+ cells was analysed by two-colour flow
cytometry using CD61-FITC/propidium iodide staining after 14 days of
culture with T = TPO; 1 = IL-1␤; 3 = IL-3; 6 = IL-6; F = Flt-3L; P =
PDGF. Treatment 1 = T alone; 2 = T+P; 3 = T+1; 4 = T+1+P; 5 = T+1+F;
6 = T+1+F+P; 7 = T+6+F; 8 = T+6+F+P; 9 = T+3+6+F; 10 = T+3+6+F+P;
11 = T+1+3+6+F; 12 = T+1+3+6+F+P. Seven independent experiments
were performed and no significant differences were observed between
treatment groups with or without PDGF.
affected by cytokine treatments or the presence of PDGF
(Figure 3). Morphological features of these cells and the
presence of polyploid nuclei were confirmed under
microscopy (Figure 4).
Discussion
At day 14, polyploid cells (4N, 8N, 16N, 32N) were present
in the CD61+ population but not among CD61− cells
(Figure 2). The majority of CD61+ cells were 2N (mean
82.5–87.1%) and 4N (11.2–14.8%), with a consistent but
low proportion of 8N (1.38–2.39%), 16N (0.16–0.27%) and
32 N (0–0.01%) cells. The distribution of ploidy was not
103
7
*
T+3+6+F
104
6
R5
Bertolini et al3 demonstrated the safe administration of ex
vivo expanded megakaryocytic progenitors from autologous
peripheral blood progenitor cells to 10 cancer patients and
observed that platelet transfusion support was not required
in two of the four patients receiving the highest doses of
expanded cells. Paquette et al4 reported that the infusion of
R6
102
101
PI
100
103
FL2-H\FL2-Height --->
104
Figure 2 Dot plot analysis of DNA ploidy. Analysis of ploidy was performed by two-colour flow cytometry using CD61-FITC/propidium iodide
staining after 14 days of culture. Cell multiplets were gated out by the
CellFIT software. R2 = 2N, R3 = 4N, R4 = 8N, R5 = 16N, R6 = 32N.
Bone Marrow Transplantation
Figure 4 May–Grünwald-Giemsa staining of megakaryocytes. CD34+
cells were cultured in the presence of TPO, IL-1, IL-3, IL-6, Flt-3L and
PDGF. Diploid (a) and polyploid (b) megakaryocytes were observed under
light microscopy on day 14. The photographs were taken at 1000× original magnification.
PDGF enhances ex vivo expansion
RJ Su et al
a high dose of expanded peripheral blood to breast cancer
patients reduced the duration of thrombocytopenia by 1 day
(median) when compared to that in patients who received
fewer cells. However, significant improvements in platelet
recovery were not demonstrated in other trials of the
infusion of expanded cord blood,28,29 bone marrow5 or
mobilized peripheral stem cells,30 in spite of the reduction
in post-transplant neutropenia. An efficient system for the
ex vivo expansion and a resultant high cell dose of megakaryocytic progenitor cells could be critical factors for producing the desired clinical outcomes.
In this study, we assessed the efficiency of early-acting
cytokines Flt-3L, IL-1␤, IL-6 and IL-3 in combination with
TPO on the expansion and maturation of CD34-enriched
cells into the MK lineage. In addition, we investigated the
effects of PDGF on the expansion systems. Our data demonstrated that culture for 14 days produced moderate
expansions of CD34+ cells and high increases of MK progenitors (CFU-MK) and CD61+CD41+ cells. The presence
of TPO alone resulted in a high proportion of CD61+CD41+
cells to over 70% but low total cell counts and high cell
death, leading to an inferior expansion overall. The addition
of IL-1␤ maintained the proportion of these cells in the
culture and significantly increased total cell number. This
observation supported the proposal that IL-1␤ might play
an important role in the MK lineage9 and expansion.8,31 The
number of dead cells was low in cultures with three or
more cytokines in spite of the high cell density, possibly
contributed by the anti-apoptotic effect of Flt-3L.32 Consistent with our previous report,14 Flt-3L significantly
increased CFU-MK. IL-3 increased CFU-MK and
CD61+CD41+ cells on day 7 but not on day 14. In general,
the treatment groups with three to five cytokines produced
efficient expansions of CFU-MK up to 400-fold with the
highest yield being observed in the presence of TPO, IL1␤, IL-3, IL-6 and Flt-3L. With these cytokine combinations, CD34+ cells were moderately expanded by five- to
22-fold.
PDGF improved the expansion of all cell types. The
maximal increases of CD61+CD41+ cells, CFU-MK and
CD34+ cells on day 14 were 101%, 134% and 70%, respectively, when compared to those in cultures without PDGF.
The marked expansion of CFU-MK and CD41+CD61+ cells
suggested that a highly proliferative compartment of MK
was responding to the various cytokine combinations. We
evaluated the DNA content of CD61+ cells on day 14 and
demonstrated that a small number of polyploid cells was
observed among the CD61+ cells. Their proportions were
not affected by PDGF or other cytokines. This observation
was in agreement with that of Dolzhanskiy et al,10 who
demonstrated that TPO alone significantly increased MK
cell ploidy but neither SCF nor IL-3 enhanced this effect.
The mechanism of PDGF on the expansion to MK lineage
is unclear. Since PDGF ␣- and ␤-receptors are expressed
on human bone marrow MK and several human MK cell
lines and platelets,25 PDGF might exert a direct stimulating
effect on this lineage. However, we cannot rule out the
possibility of additional indirect effect of PDGF such as
activation of the secretion of growth promoting factors by
other haematopoietic cells or stromal cells in the culture.33,34 Our data revealed that polyploid nuclear formation
was observed after 14 days of culture, indicating a normal
developmental process for the MK lineage. PDGF might
act on the early stage of differentiation and proliferation
without stimulating polyploidisation and terminal maturation.
In conclusion, we have reported for the first time that
the simultaneous use of multiple MK-promoting cytokines
and PDGF resulted in a marked ex vivo expansion of the
MK lineage from cord blood CD34+ cells. This represented
a highly efficient system for expanding the MK lineage
while CD34+ cells were also effectively expanded. PDGF
did not appear to affect the in vitro maturation process, with
respect to polyploid nucleus formation. PDGF might be a
suitable growth factor to enhance the ex vivo expansion of
MK progenitors for clinical applications.
1079
Acknowledgements
We thank Ms Cecilia Mei Yan Chui and nurses of the Labor Ward
for cord blood collection and the Industrial Support Fund
AF/203/98 (Industry Department, the Government of the Hong
Kong Special Administrative Region) for financial support.
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