From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
A Cell
Line
Secreting
Stimulating
By
The
multipotent
growth
hemopoietic
requirements
glutinin-stimulated
been
for
used
of the
the
cell
has
fastidious
factors
hemopoietic
many
of which
fled.
The
usual
source
phytohemagglutinin-stimulated
medium
(PHA-LCM),
a way
conditioned
medium
closely
two
cobby
of a
identiis
by
in
necessitates
5637 conditioned
the stimulatory
medium
factors
is also
required
CFU-GEMM
from human
be used to replace
PHA-LCM
through
a 0.2-am
potency
YMIO
and stored
described
obtained
either
marrow
and can, therefore,
in these cultures.
Cell
Line
The
comparison
Cannon,3
was
Institute
was
for
kindly
with
line
provided
Cancer
passaged
mented
cell
by
routinely
every
calf
Dr
originated
J.
Walker
Research,
10% fetal
5637,
Fogh,
by
Sloane
Laboratory,
2 weeks
in
Dr
G.
NY,
alpha-medium
and
supple-
Conditioned
Medium
The
5637
cells
of either
From
the
Research
Cancer
Microbiology.
© 1984
Sutton,
Sept
6. 1 983;
reprint
requests
University
5323
Harry
by Grune
Hines
cm2 culture
on alpha-medium
and
Division
to
Blvd.
& Stratton.
Imperial
of Medicine,
Feb
Dr
Texas
Dallas,
Inc.
Myers.
Health
Cancer
Institute
TX
Department
Sciences
75235.
and
5637
used
were
culture
sources
of
Petni
dishes,
cells
were
was
in
series
1. Generally,
cells
in
cultured
calf
serum
fetal
of 0.8%,
and
i05
rapid
containing
Step
III,
from
Research
the
Terry
Fox
at 37#{176}C
in a humidified
all colony
types
marrow
marrow
(IMDM,
were
I 3-1 5 days
Gibco,
Down,
scored
and
addition
aliquots
were
of
placed
(either
Laboratories,
or Gibco
concentra-
20%
human
the
human
in 30-mm
Connaught,
British
Canada).
atmosphere
using
Paisley,
medium,
Sussex,
at a final
After
Vancouver,
or
gentle
bone
2-mercaptoethanol,
0.9-mi
by
of the
I U of erythnopoietin
Centre,
removed
hours
of I 0% conditioned
Cnawley
mL.
of 20%
four
washed
methylcellulose
of 2.7
mixing,
dishes
or
Lab,
by incubation
to some
i0
Medium
mol/i
volume
Petni
applied
in the presence
Scotland),
5 x
in a final
Iscove’s
(Sera
Glasgow,
also
for
2, all
presence
for
then
2, each
series
cells
in the
either
indepen-
senies
In
of adherent
Percoll
PHA-LCM
medium.
107/mL),
over
60%
performed
different
procedure
were
were
I and
x
nonadhenent
or
series
depleted
grade
hip replacement
designated
(<I
from
centnifugation
Norway)
Cultures
was
by aspiration
by
conditioned
The
Columbia
plates
containing
an Olympus
were
5%
invented
CO2.
micro-
of culture.
with
of
8, 1984.
CD.
Sweden).
of the
marrow
in 15
UK.
accepted
of
flasks
supplemented
Laboratory,
the
Surrey,
0006-4971/84/6401-0021$03.00/0
152
at 5 x I0/75
Immunology
London,
Research,
Address
seeded
TC199,
Membrane
Fund,
Submitted
Dallas,
were
Hams,
a
filtered,
a modification
undergoing
Oslo,
This
scope after
mi
Co.
used
and
Production
using
sterile
Bone
collected
samples
incubated
by filtration
volunteers
patients
were
and
the
The
Cancer
serum.
cells
concentration
in tissue
plasma
Kettering
Rye,
retained
4#{176}C.
Tenfold
at
Mass),
using
adult
or from
reagents
samples
cell
plasma
was
filtered
medium
stored
Messner.2
washing.
tion
carcinoma
consenting
crest
with
overnight.
10%
5637
bladder
and
investigators,
separate
Bio-Cult,
and
20#{176}C.
-
performed
Uppsala,
by two
mononuclear
METHODS
Media
cells,
debris,
Danvers,
were
(Nyegaard
(Pharmacia,
FCS
serum.
free-floating
and
was prepared
Fauser
iliac
iymphoprep
marrow
in
(Paisley,
Scotland).
was obtained.
The
conditioned
by
Mononuclear
at low
in
of this
PHA-LCM
without
when
cutures
from
the posterior
T.R.
that
at
the
Assays
method
using
This
weeks
Corp,
aliquots
CFU-GEMM
dently
some
medium
of
Bio-Cult
monolayer
remove
(Amicon
in small
surgery.
effective
in supplying
for the culture
of
AND
filter
those
cells
conditioned
Amicon
properties
medium
filter.
by
PHA-LCM
systems.
containing
of
the
mimic
to
a number
replace
that
Iscove’s
Millipore
for
conditioned
to
by Gibco
a confluent
centrifuged
Scotland)
MATERIALS
or with
medium,
supernatant
medium
culture
and serum
were supplied
After 8-1 5 days ofculture,
CFU-GEMM
conditioned
medium
has also
in stimulating
the growth
of
colonies
from
murine
and
report
calfserum
Culture
5637.
show
CFU-GEMM
concentrated
culture
of batches
in
activity
Stanley,
We
10% fetal
its
cells stimulated
medium
prepared
Patinkin,
ER.
observations).
separate
removed,
source.
The bladder
carcinoma
cell line, 5637,
produces
colony-stimulating
factor(s)
for myeboid
colony
(CFUGM) growth from both normal and malignant
(CML)
bone
marrow
(D.
Bradley,
unpublished
of
line.
progeni-
(CSA)
bone marrow.4
The 5637
been found
to be active
mixed
granuloerythropoietic
use
cell
required
yet
which
the
and
factors
conditioned
of a number
colony-stimulating
describe
carcinoma
cultures
by a 1-week
to variation,
and testing
a suitable
not
growth
leukocyte
mononuclear
Conditioned
is subject
the production
order
to obtain
these
produced
of peripheral
blood
phytohemagglutinin.
such
of
are
and J.L. MiIIar
We
CFU-GEMM
CFU-GEMM
HE GROWTH
OF multipotent
hemopoietic
nies (CFU-GEMM)
in vitro,
first described
Fauser
and Messner,”2
depends
on the presence
of factors,
G. Joshi,
tor.
has
T
number
for
bladder
medium
growth
multipotent
F.E. Katz,
phytohemag-
conditioned
undefined
human
Myers,
Traditionally,
leukocyte
to supply
culture
stem
in vitro.
CD.
Factors
Center
of
at
RESULTS
A number
of batches
of 5637 conditioned
medium
was tested,
two of them extensively.
Table
I shows that
both
of the main
batches
tested
contained
all the
stimulatory
factors
necessary
for
CFU-GEMM
growth.
However,
tently
performed
produced
better
control
PHA-LCM.
the batch
based
on TC- 199 consisbetter,
and,
in one series
of tests,
growth
of CFU-GEMM
than
the
The
Blood,
5637
conditioned
medium
Vol 64. No 1 (July). 1984: pp 152-155
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SOURCE
OF CSA
FOR CFU-GEMM
CULTURES
153
Table
1 . Colony
Growth
Using
1 0% 5637
Conditioned
Media
Colonies (% of Control)
Number
of Tests
CFU-GM
BFU-E
CFU-Meg
CFU-GEMM
Series 1
5637Alpha
5
87
5637TC199
5
92±
Series
±
18
42
17
62±
±
43
37
14
81 ±20
34
±
15
13
±
90±
60
2
5637
Alpha
4
90
±
15
53
±
5637
TC199
5
124
±
10
82
±
17
46
±
23
71
±
5
37
±
10
100
±
29
16
45
±
8
203
±
81
32
37
±
26
53
±
48
19
63
±
23
146
±
79
Overall
5637Alpha
9
89
±
5637TC199
10
111
±
Percentage
different
mean
results
for the number
investigators.
of duplicate
produced
of colonies
set of results
Each
grown
shows
using two
the mean
batches
and standard
of 5637
conditioned
deviation
for the number
medium
compared
of tests
to the normal
listed.
PHA-&.CM
The test and control
used by two
results
were the
plates.
good
numbers
of CFU-GM,
but
there
were
assay
using
5637
conditioned
medium
was
compared
usually
decreased
numbers
of BFU-E
and CFU-Meg.
Other
batches
of 5637 conditioned
medium
examined
showed
batch
variation
in stimulatory
activity,
although
the ratio ofcobony
types obtained
appeared
to
to that using traditional
PHA-LCM.
The results
show
that
the assay
is linear
between
0.5 and 2.0 x iO
cells/mL
plated
for all colony
types
(Fig
1 ). The
discrepancy
between
the megakaryocyte
colony
num-
be consistent
bers is due to the different
in the two series;
higher
(Table
conditioned
2). Two
medium
were
stimulate
erythroid
erythropoietin.
Both
using
the
throid
cultures
normal
or
batches
for
culture
of 5637
their
ability
differentiation
in the
of these preparations
conditions,
CFU-GEMM
lacking
exogenous
ther sources
med. GCT
further
tested
to
absence
of
were active
however
colonies
were
erythropoietin.
no ery-
observed
Two
of conditioned
medium
have been
conditioned
medium
(Gibco-Biocult)
in
furexamstim-
ulated
GM colonies
well but was a very poor stimulator
of other colony
types (C. Myers,
unpublished
observations).
The EJ bladder
carcinoma
cell line5 was unable
to stimulate
the growth
of GM
colonies
and
was
therefore
not examined
further
observations).
In another
series
of experiments,
(J. Millar,
the
unpublished
linearity
routinely
5637
less
between
titration
2.
Colony
Growth
Using
10 %
5%
of
However,
and 10%
PHA-LCM
higher
Series
BFU-E
CFU-Meg
1
88
48
25
5637Alpha
1
70
32
0
0
5637Hams
1
91
36
25
100
58
50
67
Series
120
the
tenfold
100
5637TC199
1
112
92
32
67
5637Alpha
1
169
162
112
267
5637Hams
1
155
33
56
batches
of 5637
to the normal
for the number
conditioned
PHA-LCM
results
shows
the
control
results
were
mean
medium
used by two
for
the mean
the
of colonies
(not shown
different
number
of duplicate
grown
in Table
investigators.
of tests
plates.
listed.
is similar
to the
observation).
of 5637
supernatant
in
concentrate
was
filter.
tenfold
The
maintained
by ultrafilpotency
of
after
freezing
not shown).
the
analysis
100
using
separate
1 ) compared
Each
The
test
traditional
of the growth
We have shown
results
at
of any exogenous
stimulator
is due
to the human
plasma
used in the assay.
The stimulating
effect
was
maintained,
though
not significantly
improved,
when
PHA-LCM,
2
Percentage
of the
conditioned
medium
used
in mixed
colony
cultures,
has the problems
of
being both tedious
and expensive
to prepare
and showing considerable
batch
variation.
Batches
are also
usually
of limited
volume,
making
the biochemical
Iscove
1
effect
when used
concentration
DISCUSSION
CFU-GEMM
5637TC199
(serum-free)
2). This
(unpublished
used
were
Media
1
5637
(Fig
sources
counts
were toxic to colony
growth
(Fig
level of CFU-GM
in the absence
(% of Control)
Colonies
CFU-GM
stimulatory
concentrations
Number
of Tests
2. The
was shown
to diminish
and
had
an optimal
the 5637 medium
was concentrated
tration
over an Amicon
YM1O
of the
Conditioned
5637
in series
the culture
mixture
2). The high residual
(data
Table
seen
supernatant
than
5%
human
plasma
megakaryocyte
set of
and
factors
difficult.
carcinoma
cell line
5637 will secrete
conditioning
factors
into its growth
medium
that can replace
PHA-LCM
in mixed
colony
cultures.
Although
there
are some obvious
differences
in the potency
of this preparation,
particularly
in its
ability
Meg,
balance
that the bladder
to stimulate
the
these differences
of the factors
growth
of BFU-E
and
are likely
to be due
rather
than
any specific
CFUto the
deficien-
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MYERS
154
ET AL
30
0
0
U
V
0
to
0
e
0
U
0
0
0
U
a
C
0
0
0
U
Percentage
Conditioned
Medium
Fig 2.
Titration
curve
for colony
stimulation
by 5637 conditioned
medium.
Results
are mean colony
counts
from duplicate
plates
of
cells in series
2. Similar
results
were
obtained
in
series 1.
Cells
300
U
cru-Gu
0
L
cFu-M.
plated
(x104)
growth
CFU-GEMM
BFU-.
0
than
most
each
3#{176}
separate
that
0
et al,6
20
activity
(x104)
it is clear
that
source.
The
provide
a better
5637
5637
does
added
conditioned
stimulatory
conditioned
medium,
not contain
erythrofrom
an exogenous
medium
source
(BPA).9
appears
for CFU-GEMM
to
However,
conditioned
of
CFU-
this does
medium,
not
as
been
largely
removed
by adherence
experiments.
various
Mouse
activity8
and
many
factors,
erythroid
Fig 1 .
The plating
efficiency
of colonies
in the CFU-GEMM
assay
when
stimulated
by (A) 5637
conditioned
medium.
(B)
PHA-LCM.
Panel A is mean ( ± standard
deviation)
from six experiments.
three
in each
of the
two
series.
Panel
B is mean
( ± standard
deviation)
for three experiments
in series 1.
in common
with PHA-LCM,
poietin,
and this must
be
have
of these
stimulating
produces
0
cy. However,
linearity
with
with
obtained
using
two
separate
The maintenance
of high levels
absence
of exogenous
growth
Cell lines secreting
described
previously.
t
plst.d
reported
assay
systems
does not agree
e
in many
Cells
who
up to 106 cells/plate.
to be due to the 5637
macrophages,
10
of our
plated
factors
suggests
that GM-CSF
is endogenous
in these
cultu-res.
This is probably
present
in the human
plasma
added,7
as the other
likely
sources,
monocytes
and
0
0
as
in
a
0
Jii’L
is the
similar
results
were
batches
of PHA-LCM.
of CFU-GM
in the
U
5O,’
this
medium,
cultured
GEMM
appear
U
100
that
systems.
range
of linearity
to number
of cells
of Ash
think
lO
200
150
We
feature
of this conditioned
other
colony
types
can be
assay
The
respect
25
250
PHA-LCM.
important
of the
Human
potentiating
growth
factors
have been
L cells produce
colonythe murine
WEHI-3
including
burst-promoting
lines
producing
factors’2”3
are
line
CSA’#{176}” and
also
known,
but
to date, only one line (Mo)
has been shown
to produce
all the growth
factors
necessary
to stimulate
human
CFU-GEMM
growth.’4
This and other similar
HTLV11-transformed,
factor-secreting
lines
are
but have the disadvantage
of secreting
active
available,
HTL V-I!
virions
The
(David
Golde,
personal
communication).
advantages
of a cell line secreting
these
is
convenience
the
conditioned
ited batch
medium
variation.
of
producing
quickly
This
large
factors
amounts
and easily
and
should
improve
with
both
of
limthe
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
SOURCE
OF CSA
long-term
FOR CFU-GEMM
reproducibility
of the
tate the biochemical
factors,
as previously
ity
CULTURES
to produce
155
assay
and
also
analysis
of early-acting
described.’5”6
Indeed,
growth
factors
fulfills
one of Stanley
for this type of analysis.
and
in serum-free
Guilbert’s’6
6.
facili-
tent
growth
the capac-
RC,
Detrick
7.
Das
5K,
criteria
of
three
subclasses
58:630,
Dr
Ray
their
Bradley,
help
and
Izaguirre
for
Dr
advice.
in setting
providing
program,
Richard
Mrs
and
Dr
the
assistance
acknowledge
up the CFU-GEMM
bone
and
We
Stanley,
marrow
Jackie
the 5637
Barbara
assay,
from
the
Needham
cell
line
10.
of
.
human
bone
52:1243,
2.
cell
marrow
cells
and
J:
tumor
tumors.
NatI
Messner
Holland
marrow
tumor
and
colonies
cord
Identification
in colonies
blood.
in
and
human
bone
erythroblasts.
human
cells
with
Cancer
JF,
Inst
human
GJ,
Fogh
cell lines.
CM,
emphasis
Monogn
Zinzar
J, Arlin
Lack
Exp
Povey
on
kidney,
49:5,
SN,
Z,
and
5,
bladdercancercelllines.
bladder
activity
IA,
BD:
P. Franks
Nature
I 5.
latory
stromal
Exp
1983
LM:
301:429,
Mann
CSF-producing
of human
7):76,
(abstr
Identity
1983
Human
bone
133)
of
BlOOd
1:65,
colony-stimulating
cell
species.
mega-
hydrophobicity
MA,
Blood
Speiser
Bi:
activity
51 :507,
the
and charac-
factor-producing
of the thyroid
for
1978.
N: Establishment
colony-stimulating
carcinoma
Molecules
1982
Lichtman
H, Oshawa
pro-
and
in size,
elaborate
and other
U:
macrophage,
Similarity
Suppl
macrophage
N, Guilbert
that
Ji,
ED:
Production
Golde
Kay
cell
DW,
NatI
Fauser
gland.
cell line
J NatI
line.
Acad
BlOOd
N,
Sci USA
Messner
cell
Ruppert
S, Lohr
activity
for
I 1:154,
ER,
GW,
human
Cancer
a human
Lusis
AJ:
Production
of
T-lymphoblastoid
cell
1980
iusis
AJ,
Golde
hemopoietic
Stem
Fauser
HS,
by a
1981
by
77:593,
Koren
factor(s)
Cells
AA:
pluripotent
DW:
progenitors
I :73,
Stimulatory
produced
Characterization
stem
cells
by
1981
of stimu-
(CFU-GEMM).
1983
Guilbert
and
57:170,
5G.
line.
T,
potentiating
Quan
HA,
plunipotent
T-lymphocyte
(CSF-l).
Earenfight-Engler
erythroid
activity
human
Stanley
NE,
of
Bersch
AA,
characterization
factor
Williams
granulocyte,
JK,
T, Nomura
1-lematol
16.
Resolution
1977
Brennan
Ascensao
for
a human
Svet-Moldavskaya
Clakson
8(suppl
Hepburn
and
14.
CA,
cell,
erythroid-potentiating
of
1978
of CSF-activity
Haematol
identification
testis,
LW:
1982
line. Proc
characterization,
58:309,
factors.
regulating
JF,
monocytic
13.
Blood
Forman
252:4305,
in culture:
of man
squamous
12.
marrow
U,
Factors
Chem
Physiol
lines
Inst69:1235,
Blood
of megakaryocytes,
of
granulocytes
Cultivation,
fibroblasts.
5. O’Toole
some
blood,
HA:
eosinophils
4. Svet-Moldvasky
human
Granuloerythropoietic
1979
Fogh
human
PE,
HA:
peripheral
neutrophilic
53:1023,
red
of a human
Zanjani
AA,
containing
3.
Messnen
marrow,
Fauser
Rotisch
early
Persio
Human
from
1978
macrophages
NN,
1 1 . Okabe
AA,
Fauser
plunipo-
Blood
radioimmunoassay:
colony-stimulating
PM:
J Biol
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Iscove
and charge.
transplant
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ER,
stimulating
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for
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Royal
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(CSF-1)
of
and growth.
duction
for providing
Studies
1981
9.
Dr J. Fogh
ED:
(CFU-GEMM)
ER,
factor
8. Stanley
to thank
Zanjani
cells
Stanley
colony-stimulating
ACKNOWLEDGMENT
We wish
RA,
stem
I981
medium
major
Ash
haemopoietic
target
J Immunol
U:
Methods
for the purification,
cell
binding
of
Meth
42:253,
1981
a colony
assay,
stimulating
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1984 64: 152-155
A cell line secreting stimulating factors for CFU-GEMM culture
CD Myers, FE Katz, G Joshi and JL Millar
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