Leukemia Research Vol, 9. No. 3, pp. 3 9 9 ~ 0 5 , 1985. 0145-2126/8553.b~) + .

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Printed in Great Britain. ~ 1985 Pergaruon Pre~s k t d

THE ROLE OF PLATELET-DERIVED GROWTH

FACTOR ON HUMAN PLURIPOTENT PROGENITOR
(CFU-GEMM) GROWTH IN VITRO

R1TA MICHALEVICZ, GILLIAN E. FRANCIS, GILLIAN M. PRICE

a n d A. VICTOR HOFFBRAND
Department of Haematology, Royal Free Hospital, School of Medicine, Hampstead, London,
NW3 20G, U.K.

(Received 14 February 1984. Revision accepted 13 August 1984)

Abstract--The effect of serum on the proliferation of human bone marrow pluripotent progenitor
cells (CFU-GEMM), was compared to that of fresh frozen plasma (FFP). Serum significantly in-
creased the number of mixed erythroid-granulocytic-megakaryocytic colonies (CFU-GEMM) and
erythrocytic bursts (BFU-E) in this assay system (p <0.01 and 0.02, respectively). Two possible ex-
plantations for this finding were considered: first the presence of citrate-phosphate-dextrose
(CPD) in plasma but not in serum, and second the presence of platelet-derived growth factor
(PDGF) in serum but not in plasma. CPD was indeed found to have an inhibitory effect on growth
of colonies of all types when added to serum-stimulated cultures. Nevertheless, when heparinized
plasma was compared to serum from the same donors, growth of CFU-GEMM and BFU-E was
higher in the serum-stimulated cultures (p<0.001 and p < 0 . 0 5 , respectively). PDGF at concentra-
tions of 120-240 pM was found to enhance the formation of CFU-GEMM and BFU-E by three-
and four-fold respectively when added to cultures containing FFP but not when added to cultures
containing serum derived from whole blood (WBS). Purified PDGF added at the same concentra-
tions, to cultures containing platelet-poor derived serum (PDS), promoted similar increases in
growth of CFU-GEMM and BFU-E but not of granulocytic-macrophage or megakaryocytic
colonies. Whether PDGF has a direct action on CFU-GEMM or its growth promoting activity is
via an interacting cell population is currently being studied.

Key words: Platelet-derived growth factor, human pluripotent progenitors, chronic

granulocytic leukemia.

INTRODUCTION
THE ADVENT of in vitro assays for pluripotent progenitor cells (CFU-GEMM) has allowed
a better understanding of the diverse regulatory mechanisms of human hematopoiesis.
Since the description of the method by Fauser and Messner [9], minor modifications have
been suggested that have improved the plating efficiency of the method [1]. Several centres
including our own [121 have introduced the use of fresh frozen plasma (FFP) rather than
fetal calf serum (FCS), as originally used by Fauser and Messner. We have now found that
the presence of normal human serum, either autologous or heterologous, promotes a
significantly better growth of CFU-GEMM and erythrocytic burst (BFU-E) than FFP in
these cultures. Platelet-derived growth factor (PDGF) is a known mitogen present in the c~
granules of circulating platelets. Smooth muscle cells, fibroblasts and a variety of other
mesenchymal cells require PDGF for optimum growth in vitro [4]. Dainiak et al. [5]
found that PDGF increased the growth of erythroid burst-forming units (BFU-E). The
present results suggest that platelet-derived growth factor (PDGF) in serum promotes the

Abbreviations: PDGF, platelet-derived growth factor; CFU-GEMM, colony-forming unit-

granulocyte-erythrocyte-macrophage-megakaryocytic; BFU-E, burst forming unit-erythroid; CFU-GM,
colony-forming unit-granulocyte-macrophage; CFU-Mk, colony-forming unit-megakaxyocyte; CFU-Eo,
colony-forming unit-eosinophil; CPD, citrate-phosphate-dextrose; FFP, fresh frozen plasma; PDS, serum
derived from platelet-poor blood; WBS, serum derived from whole blood; IMD medium, lscove's modified
Dulbecco's medium; FCS, fetal calf serum; CGL, chronic granulocytie leukemia.
Correspondence to: Prof. A. V. Hoffbrand, Department of Haematolngy, Royal Free Hospital, Pond Street,
Hampstead, London, NW3 2QG, U.K.

399
4OO RITA MICHALEVICZ el aL

growth of mixed erythrocytic-granulocytic-megakaryocytic colonies (CFU-GEMM) and

of BFU-E in this culture system. Citrate-phosphate-dextrose (CPD) was found to inhibit
growth of CFU-GEMM and the presence of CPD in FFP might also account for the dif-
f e r e n c e s i n p l a t i n g e f f i c i e n c y o b s e r v e d . A n a b s t r a c t o f t h i s w o r k h a s b e e n p u b l i s h e d [15].

MATERIALS AND METHODS

Samples
Bone marrow aspirates were obtained from healthy young adult volunteers after informed consent and with
the approval of the Royal Free Hospital Ethical Practices Committee. Samples were collected into preservative
free heparin (20 U ml-') and placed in Iscove's Modified Dulbecco's (IMD) medium (Gibco, Glasgow,
Scotland). Mononuclear cells were collected after centrifugation at 400 g for 25-30 min over lymphoprep
(Nyegaard & Co A/S, Oslo, Norway) and washed twice in IMD medium.
Normal sera were obtained after informed consent from laboratory workers or marrow donors. Samples of
venous blood were collected in glass tubes and allowed to clot at room temperature for 2-3 h. After centrifuga-
tion at 800g for 10 min, the sera were filtered through a 0.22-~tm filter (Millipore SA) and either stored at -20°C,
or used fresh. Plasma was prepared from blood collected in preservative-free heparin (10 U ml-~) from the same
donors.
FFP was obtained from normal human venous blood (AB Rh-) after removal of formed elements including
platelets by centrifugation in CPD sterile bags (Travenol Ltd, Norfolk, England). In order to evaluate the effect
of CPD (Travenol Ltd), 28 ~tl ml -~ were added to cultures containing serum and also to heparinized plasma
cultures.
Preparation of whole blood serum (WBS), platelet poor derived serum (PDS) and plasma
WBS (citrated), PDS and plasma from 6 normal individuals were prepared as described by Dainiak et aL [5].
Whole blood was anticoagulated with 1/10 vol of 3.13% trisodium citrate; plasma was obtained after cen-
trifugation; for WBS the citrated blood was recoagulated by addition of 1/50 vol of I M CaCI2. Platelet-poor
plasma was prepared by centrifugation of citrated whole blood at 1100 g for 15 min at 4°C, followed by cen-
trifugation of the plasma at 31,000 g for 30 rain at 4°C. PDS was prepared by coagulation of the platelet poor
plasma by addition of 1/70 vol of 1M CaCI2.
CFU-GEMM assay
Pluripotent hematopoietic progenitor cells were assessed using the Ash modification [1], of the method
described by Fauser and Messner [9]. 2 x 10S/ml nucleated bone marrow cells were incubated in IMD medium
containing 0.9*/0 methylcellulose, 10% FCS, 20% FFP AB Rh- group and 10% leucocyte-conditioned medium
from phytohemagglutinin-stimulated peripheral blood buffy coat cells prepared as described by Aye et aL [2].
5 x 10-~M 2-mercaptoethanol and erythropoietin (step III, Connaught Labs., Ontario), 1 U ml -~ were routinely
added to all cultures. Duplicate l-ml aliquots were incubated in 35-mm dishes (Nunc, Gibco Europe Ltd) in an
environment of 5*/o C02 at 37°C in humidified air. The cultures were examined with an inverted microscope
(Wild Heerbrugg, Switzerland), x 25 and x 40 magnification, after 14 days of incubation. Individual colonies
were removed from the plates by micropipetting and either applied to slides directly or cytocentrifuged. Colonies
were routinely stained with Giemsa or Wright's stain and also examined after chloracetate esterase, non-specific
esterase and acid phosphatase staining to confirm their identity.

Platelet derived growth factor (PDGF)

Porcine PDGF was kindly supplied by Speywood Laboratories Ltd, Nottingham and stored at -20°C. Ali-
quots were diluted into IMD medium according to the manufacturers' direction for use. The specific activity of
the PDGF supplied was approximately 1 U / I - 2 ~tg protein (1 U PDGF is equivalent to 14 ng of pure porcine
PDGF). Concentrations of PDGF from 0.25 to 0.75/ml units were added to either FFP or serum. In control
cultures, PDGF was replaced by diluent (1MD). Purified porcine PDGF kindly supplied by P. Stroobant and
M. D. Waterfidd (ICRF) was used in the same concentrations. Pure PDGF was added to cultures containing
platelet-poor derived serum (PDS). In the experiments using PDGF, 2-mercaptoethanol was not included in tests
or controls since PDGF is inactivated by reducing agents.
Statistical analysis
The mean -1-S.E.M. colonies formed in duplicate culture was determined, data sets were compared by Wilcoxon's
Rank test for unpaired samples and the Mann-Whitney U-Test [21].

RESULTS
C o m p a r i s o n o f colony g r o w t h in the presence o f F F P or serum
T a b l e 1 c o m p a r e s t h e r e s u l t s o f 2 0 e x p e r i m e n t s u s i n g F F P (2 b a t c h e s ) a n d 14 ex-
p e r i m e n t s u s i n g s e r u m . C F U - G E M M a n d B F U - E w e r e s i g n i f i c a n t l y i n c r e a s e d in t h e s e r u m
experiments 09<0.01 and p <0.02) respectively. No significant increase in the CFU-GM
a n d C F U - M k o r C F U - E o w a s f o u n d ( T a b l e 1). T h e r e s u l t s o f 4 i n d e p e n d e n t e x p e r i m e n t s
using plasma (heparinized) or serum from the same donor on the same target marrow are
 Role of PDGF on pluripotent progenitors 401

TABLE 1. COMPARISONOF COLONIESIN FFP vs SERUM

MARROWCULTURES

FFP(n = 20) Serum(n = 14)

CFU-GEMM* 7.5 + 1.4 12.2 + 1.4

BFU-Et 29.7 + 3.5 45.9 + 5.2
CFU-MkNs 21.8 + 1.0 29.6 + 2.6
CFU-GM Ns 82.2 :t: 2.0 77.3 + 5.2
CFU-EONs 12.1 :t: 1.4 14.6 + 1.4

The results are mean :l: S.E.M. from all donors in

each group reflecting the number of colonies/10 ~ cells.
*p<0.01 (Wilcoxon test).
tp<0.02.
NS : not significant.

uJ ,o 50 10' E~m 50
(J

l /
MJ 5" 25" 5 25"
Ey.,.~ll

CFU-GEMM BFU-E CFU-Mk CFU-GM

FIG. 1. Comparison of colony growth in cultures supplemented with heparinized plasma or serum
(S) from the same donors. Serum provides a significant increase in the number of CFU-GEMM
forming colonies (p<0.001) and of BFU-E forming colonies (/7< or = 0.05 - - Mann-Whitney
U-test). There is no significant difference in the number of granulocytic-macrophage or
megakaryocytic colonies. PiasmaD {2]; serum • ........ l .

s h o w n in F i g . 1. A s i g n i f i c a n t i n c r e a s e in t h e C F U - G E M M (p<0.001) and BFU-E

( p < 0 . 0 5 ) w a s a g a i n o b s e r v e d w i t h s e r u m p r e s e n t ( M a n n - W h i t n e y U-test). N o t o n l y t h e
number of colonies was increased when serum was used but the number of cells/colony
was higher (data not shown).
I n o r d e r t o test w h e t h e r t h e p r e s e n c e o f C P D in p l a s m a was a c o n t r i b u t o r y f a c t o r t o t h e
r e d u c e d g r o w t h w i t h C P D - p l a s m a , C P D w a s a d d e d in c u l t u r e s c o n t a i n i n g s e r u m . T h e ad-
d i t i o n o f C P D t o s e r u m w a s f o u n d t o i n h i b i t t h e C F U - G E M M a n d B F U - E by 40 a n d
402 RITA MICHALEVICZet aL

50%, respectively (mean of two experiments). Moreover, clotted citrate WBS also showed
inhibition of C F U - G E M M and BFU-E when compared with WBS (Fig. 2) in two ex-
periments.
Influence o f PDGF on colony growth
To assess whether the better support of growth obtained when serum was added to the
cultures was due to P D G F , we added P D G F over a range o f concentrations to FFP-
stimulated cultures. As shown in Fig. 3, the addition o f up to 0.66 U ml -' of P D G F
CFU-GEMM BFU-E
8a 4011

.=.

i ".1".'

°1 i
40 "'J"" -
- ,::[::
l[

~ ,,..,
~ .'.'.

o :?.i:.i
WBS W85 WBS IS
citrate citrate
FIG. 2. Comparison of CFU-GEMM and BFU-E growth using whole blood serum (WBS) and
citrated WBS in 2 individual experiments.

40. 2@

i 2c

o.'33 oJs6 o:~ o:~

PDGF UNfI"S/m!
FIG. 3. Dose-dependentincrease in colony formation of CFU-GEMM and BFU-E with the addi-
tion of P D G F (Speywood) to FFP cultures (results of two separate experiments). Results are mean
± S.E.M. obtained from duplicate dishes (assuming a Poisson distribution of clonogeniccells).
(o o) CFU-GEMM; (& A ) BFU-E.
 Role of PDGF on pluripotent progenitors 403
(Speywood) stimulated (in two experiments) CFU-GEMM and BFU-E formation. The
peak activity of P D G F in promoting the growth o f CFU-GEMM and BFU-E in both ex-
periments occurred at 0.33 U/plate. Higher concentrations of P D G F did not further in-
crease the growth. No significant increase was noted in CFU-GM, CFU-Eo or CFU-Mk
with F F P supplemented with PDGF. The addition o f P D G F to WBS in the same concen-
trations, however, did not promote a better growth than serum alone (data not shown).
Pure P D G F (0.66 U ml-') when added to PDS in 6 individual experiments (1 x 10' cells
ml-~), promoted a significant proliferation of CFU-GEMM as well as o f BFU-E (Table 2).

TABLE 2. EFFECT OF PURIFIED P G D F ' o N GROWTH OF

C F U - G E M M AND B F U - E IN P D S CULTURES IN 6 EX-
PERIMENTS

CFU-GEMM* BFU-E*
-PDGF +PDGF -PDGF +PDGF

1 5 14 25
2 6 12 32
5 11 37 76
6 9 34 81
2 5 16 26
2 4 11 29

Mean 3.0 6.5 20.6 44.8

Each result is a mean of duplicates p l a t e d a t 10 5

cells ml-t.
*p<0.05 (Wileoxon's Rank test).

DISCUSSION
The present results show that fresh human serum, when compared to flesh frozen
citrated human plasma, provides a stimulatory effect on the growth of human pluripotent
progenitors (CFU-GEMM), in vitro. This observation prompted the investigation of two
possible explanations for the difference: the presence of P D G F in serum and not in
plasma and the presence of CPD in plasma but not in serum. Our experiments show that
pure P D G F when added to platelet-poor derived serum has a stimulatory effect on growth
of CFU-GEMM as well as of BFU-E.
Cultures of many cells in vitro are stimulated by serum [3]. Vascular smooth muscle
cells require mitogens such as PDGF, fibroblast growth factor or serum to proliferate and
when exposed to plasma alone do not proliferate [11, 14]. P D G F is one of the most potent
mitogenic factors present in serum being released locally during the clotting process [14].
It is absent from plasma. Until recently, P D G F was known to stimulate proliferation of
many types of tissues and was not thought to stimulate hemopoietic cells [18, 20]. Dainiak
et al. [5], however, showed that P D G F stimulates proliferation of human BFU-E, its ac-
tivity being different from erythropoietin. Delwiche et al. [6] suggests that this is an in-
direct action via bone marrow stromal cells. Our data shows that the mixed
erythrocytic-granulocytic-megakaryocytic precursor, as well as unipotent erythroid
precursors are responsive to the stimulatory effect of PDGF. Surprisingly, no effect of
P D G F was found on the growth of granulocytic-macrophage and megakaryocytic col-
onies present in this assay. P D G F binds to specific saturable receptors on fibroblasts [13]
404 RITA MICHALEVlCZet al.

and stimulates a tyrosine specific kinase activity from human marrow [8]. It may be that
PDGF has a similar action on CFU-GEMM and BFU-E. It is also possible, however, that
the stimulation of proliferation of CFU-GEMM by PDGF is an indirect one on an interac-
ting cell population, e.g. stromal or adherent cell, rather than a direct action [6, 14].
Recent work has shown close structural and immunological homology between PDGF
and the protein p28s~ coded for the s/s-oncogene [7, 16, 23]. On the basis of these observa-
tions and the present data, Francis et ai. [10] hypothesized that the translocation of the s/s
oncogene from chromosome 22 to 9 in Philadelphia positive chronic granulocytic
leukemia (CGL) may lead to excessive expression of sis oncogene with consequent increased
autocrine stimulation by PDGF of a clone of marrow cells leading to excessive prolifera-
tion of cellular and/or stromal marrow elements in this disease. It is also possible that a
paracrine mechanism is involved [20]. Further studies ere now needed of the role PDGF
plays in vivo both in normal human marrow cell proliferation and differentiation and in
the excessive marrow proliferation that occur in CGL.

Acknowledgements--We thank Dr. F. Katz from the Imperial Cancer Research Fund for helping to
establish the CFU-GEMM assay. We are also grateful to Drs. P. Stroobant and M. D. Waterfield for kindly
donating pure PDGF and for helpful discussions. This work was supported by the Lewis Foundation (R. M.),
the Wellcome Trust (G.E.F.), and the Leukaemia Research Fund. We would also like to thank Miss J. Allaway
for typing the manuscript.

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