From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Human
Cell Lines
That Elaborate
Colony-stimulating
Activity
for the Marrow
Cells of Man and Other
By John
F. Di Persio,
James
K. Brennan,
We have established
two human
which elaborate
colony-stimulating
ity
(CSA)
for
at
least
four
Marshall
cell lines
activ-
species:
A. Lichtman,
and
cell-line-conditioned
5-200
chromatography
man,
maximum
mouse,
isolated
Species
Burton
1. Speiser
medium
by Sephacryl
indicated
that the
activity
of
the
CSA
for
human
rabbit,
and dog. One, GCT, was
from a lung metastasis
of a fibrous
histiocytoma;
the other, RC4, from a monocyte-enriched
haction
of normal
blood.
Medium
conditioned
by either GCT or RC4
cells was more potent
in stimulating
hu-
marrow
cells is eluted
within
the range of
30,000-40,000
daltons.
These
cell lines
provide a continuous
source of large quantities of conditioned
medium
for purification of CSA.
Moreover,
the
invariable
growth-supporting
activity
for all species
man
tested
marrow
growth
in
monocyte-conditioned
leukocyte
T
feeder
layers.
HE CLONAL
colony-stimulating
serum
and
vitro
urinary
cation
GROWTH
activity
CSA
for
of
and
and
and
cells that
growth
do
not
elaborate
CSA
From
the
Departments
School
Mar
Supported
by Grants
9. 1977;
12790,
and a contract
versity
of Rochester
cell-line
elaboration
granulocytic
evaluate
amounts
steady
of colony-forming
for CSA
embryo
cells,
quantifistudies.
kidney
cells’3
monocytes,’7”8
macrophages,’9
cells and Ieukocytes
have
“conditioned”
is due,
cells
finite
by leukocytes
has
in part,
to the admixture
that
release
state
the role of CSA
of human
CSA
inhibitors
and
and
life
marked
with
of
colony
leukemic
monocytes,
have
been
We have also observed
that cells
may be a potent
source
of CSA.
than a few weeks
in culture.
Medicine
and
accepted
CH.71
from
and
the
and
Dentistr1’,
September
IN-18-R
and
Radiation
Biologb’
and
Biophysics.
Universits’
of
N. Y.
22, 1977.
from
U. S. Energy
Biomedical
of
Rochester,
shown
to release
from acute
myeloThese
cells, how-
the American
Research
Environmental
and
Cancer
Society.
Development
Research
Project
USPHS
Administration
and
has
been
Grant
CA-
to the
Uni-
assigned
report
UR-3490-1134.
Address
for
of Rochester
© 1978
Blood,
of
of Medicine
Submitted
No.
of
its
of physiologic
by human
or with
of
2I
Leukemic
cells,
probably
CSA in short-term
culture.22
monocytic
leukemia
patients
ever, do not grow
for more
Rochester
of the
implementation
medium
variability
potency
progenitors
in culture
requires
mice,
appropriate
variations
in
the bioassay
radioimmunoassay)
by human
leukocytes,’4’6
specifically
mitogen-stimulated
lymphocytes.2#{176} Kidney
high
studies
effects.
function.8’2
To
source
of large
is elaborated
spans
in culture.
Moreover,
variability
in potency.
This
biologic
perturbation
urine,
the
facilitates
of granulocytic
(CSA).’7
In
would
facilitate
methods
(e.g.,
marrow
and
CSA
a regulatory
a convenient
blood
human
was
human
following
Such
a source
of improved
in human
CSA
or
Fractionation
suggest
that it may have
in human
granulopoiesis,
is needed.
development
than
medium
reprint
School
by Grune
Vol. 51,
requests:
Dr.
of Medicine
& Stratton.
No. 3 (March),
and
Inc.
1978
James
K.
Dentistry,
JSSN
Brennan,
Assistant
Rochester.
0006 -4971/78/5103-2
Professor
of
Medicine,
Universits’
N. Y. /4642.
003$02.00/0
507
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
508
DI
In this
report
species,
we describe
including
two
human
cell
1, M.P.
with
with
packed
left
lobe.
An
tissue,
right
fibrous
refractory
coincident
metastases
to
with
were
Case2,
The
plateletpheresis
donor
was
weekly
By
streptomycin
UR-HCL-1
medium
direct
dispersed
in
cultured.
After
had
when
patient
in
of
cell
The
a
variant
monocyte
died
of
dis-
count
to
examination
duodenal
the
from
blood
Rochester
Division,
no history
ofinfectious
M.P.
10%
were
showed
ulcer.
The
of
an
in
December
spleen
individual
under1975.
The
mononucleosis.
migrating
and
Fragments
by
trypsinization
For
bovine
serum,
to 60
x 15 mm
CO2
in air.
were
supplemented
with
tissue
Medium
had
dishes
in
cells
at
confluent
consecutive
were
serum,
and
made
a
passed
thawed
bovine
in
and
were
formed
successfully
fetal
culture
exchanges
studies,
l0
resuspended
U penicillin/mI
100
fragments
present
the
a metastatic
were
fetal
from
stored.
pneumonectomy,
saline.
transferred
week,
cells
underwent
normal
with
dishes
of
Clone
the
A
addition
of
obtained
was
l.6
w/v
of a micropipette
means
standard
medium,
concentration
adapted
penicillin,
and
attached
transferred
of
by
to
to
5 x 105/ml
culturing
GCT
methylcellulose.23
a
was
A
cells
reached,
mm
the
standard
was
a micromanipulaton.
12 x 75
in
colony
removed
The
clone
polypropylene
suspension
was
tube,
was
used
and
to
seed
culture.
(RC4).
centrifugation
A residue
rich
in
Further
in metnizoate-Ficoll.25
to surfaces.25
lymphocytes
enrichment
Monocytes
Combining
these
and
can
can
be
monocytes
achieved
be separated
procedures
yields
can
be
obtained
by equilibrium
from
a cell
as
density
lymphocytes
population
a by-
gradient
by differential
comprised
of
more
90#{176}
monocytes.’8’25
Ten
ml ofresidue
ml
of
from
32.8#{176} w/v
iS.
donor
in medium
metnizoate:9
sodium
preparation
to
supernatant
vested
into
fraction
was
removed
and
a centrifuge
tube
containing
washed
sion
She
washed
medium
of plateletpheresis.24
adherence
left
1974
neutrophil
Postmortem
a perforated
isolated
of 5%
(GCT-C).
by
a cell
UR-HCL-2
product
A
by
I ml
volume
as
giant
patient
in
reduced
April
recur.
the
increase
death.
in the
medium).
viscous
vision
Cross,
atmosphere
frozen
5A
clone
made
a larger
from
were
(standard
was
aliquots
fourth
removed
McCoy’s
line
supplemented
the
Samples
in
1974,
Five-mI
Cells
to growth
well
Red
female.
and
a humidified
in
intervals.
monolayer.
Ten
In April
medium
37’C
subcultures.
An
his
not
a
Lines
streptomycin/mI.
at
white
minced,
removed,
essential
cultured
American
44-yr-old
(GCT).
minimum
as
to
did
and
mass
In
as the
later
he
Absolute
histiocytoma
persisted.
and
months
radiation.
prior
kidney
(RC4)
the
of Cell
was
remitted
and
fibrous
reported
when
normal.
cells/zl),
anemia
was
Fourteen
noted
and
UR-HCL-2
a healthy
UR-HCL-1
was
liver,
at
Establishment
nodule
peritonitis
and
neutropenia
otherwise
malignant
white
1974,
normal.
J.S.
going
under
several
a 29-yr-old
of
February
a circumscribed
revealed
histopathology
persisted.
lung
(260-300
showed
Neutnopenia
to chemotherapy
lung,
brain,
marrow
anemia
x-ray
mass
the
until
was
count
Chest
the
The
and
monocyte
subtype.
from
health
examination
axillary
and
isolated
in good
Physical
of the
histiocytoma.
was
was
present.
performed
normal
line
He
a normal
were
biopsy
was
cell
mass.
fibroxanthoma
disease
900O/tl
than
axillary
excisional
remained
seminated
(GCT)
cells/xl),
lobectomy
of malignant
100 g
for
CSA
METHODS
AND
histiocytoma.
(36#{176}-4l)
atypical
lower
and
a
cell volume
count
UR-HCL-l
fibrous
(l6l0-l900
neutropenia
soft
The
malignant
presented
lower
elaborate
AL.
Data
Case
male
that
ET
man.
MATERIALS
Clinical
lines
PERSIO
twice
were
tightly
create
in
M-l99
transferred
a
stepgnadient.
containing
After
the
glass
(M-199)
Ficoll
was
(1:2.4
centnifugation
upper
M-199
20#{176}fetal
to a 250-mI
199
w/v
bottle,
calf
transferred
v/v)
at
500
third
of the
at 4’C.
Cells
serum.
which
was
to a centrifuge
injected
g
for
30
mm
at
metnizoate-Ficoll
were
sedimented
Approximately
was
then
200
gassed
with
tube.
beneath
the
cell
25’C,
the
layer
was hanat 160 g and
ml
7%
of
cell
CO2
in
suspenair
and
capped.
Following
containing
10 volumes
a 16-hr
incubation
nonadherent
cells
of phosphate-buffered
in a Brunswick
(chiefly
lymphocytes)
saline
(PBS).
shaking
was
Cells
water
bath
decanted
adherent
(20
and
to
the
oscillations/mm),
the
bottle
bottles
was
(chiefly
medium
washed
monocytes)
with
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
HUMAN
were
CELL
LINES
removed
by exposure
of standard
medium
policeman.
The
cell
clumps.
Cell
monocytes,
then
made
and
at
were
had
observed.
creasing
Two
GCT,
weeks
GCT-C,
suspension
Monolayer.
cells
Counter
with
calculated
at
one
mission
M
and
acetic
acid
exclusion
10,000
ultrafiltration
against
rabbit
Surface
(I
with
hr.
sheep
2 x
106/ml
coated sheep
for
ingested
Studies
Wright’s
medium.
fresh
rounded
of
were
proliferation
done
Subcultures
medium
removed
Fc
drying,
with
were
and
de-
made
at
transferring
for
coli.
Bacillus
terminated
at
period
ofexponential
in
with
dishes.
the
2-day
Coulter
time
was
growth.
and
Cloning
ZBI
Doubling
medium
dishes
At
a model
2 wk.
standard
containing
cultured
1.6#{176},,
for
efficiency
‘y,
was
14 days.
expressed
from
at
I
(2.0-Mm
subtilis,
were
ic
x
l06/ml
transwith
swollen
of
for
a
with
ethanol
mm
30
the
ofJondal
were
cell
The
radial
and
proportion
of
antibodies.28
polysterene
or
Neisseria
gonorrhea.
Candida
at intervals
ofO-
cells
with
an
polyvinyl
forming
equal
and
cells
cul-
rosettes
of
spon-
volume
toluene),
and
were
concentrations
number
albicans),
120 mm
from
tested
immunodiffusion.
cell
The
were
(nominal
were
residue
equivalent
et al.29
incubated
filter
concentrate
plateletpheresis
to
cultures
10
fractionation
utilizing
diameter
removed
8 hr.
PM
sulfate.
from
adjusted
as
method
Amicon
sulfate
chains
erythrocyte
by the
for
a solution
re-
slides
volume
acetoorcein
ammonium
prepared
determined
for
growing
an
ammonium
and
prepared
with
exponentially
a, A, and
anti.sheep
measured
2,
glass
examined.
0%-805
j,
with
were
onto
median
colcemide
twice
monolayer
6 (2)
their
fixed
through
substratum,
were
1.0 iM
stained
were
or monocytes
with
Samples
stains,
and
in
elleted
for
were
from
(I)
analyzed
of
media
cells
were
(3)
slides,
with
growing
histochemical
ultrafiltration
human
RC4,
suspended
enythrocytes.
mm
were
presence
glass
precipitate
coated
Cells
or
slides
by
from
was
60 x 15
a 48-hr
Samples
metaphases
receptors
cells
replicate
microscope.
and
Supernatant
specific
particles
were
of
microbes
antibodyexamined
particles.
of CSA
Preparation
were continuously
at 4-day
subcultures
flasks
exchanges
originally
islands
enumerated
cells/mI.
onto
either
GCT,
latex
(Escherichia
The
Medium
cells,
Local
of
Exponentially
l0
with
resuspended
erythrocytes
taneously
rosetting
Phagocytosis.
flasks.
air.
culture,
60 x 15 mm
inverted
or precipitation
the
x 106 cells/mI)
of
suspended
microscopy,27
After
daltons)
antisera
48
culture
in
and
to
volume.
consecutive
receptors.
for
were
an
5 x
sedimented
100-fold
and
ml
in
transferred
with
secretion.
over
106
92#{176}c
cultured.
treated
6 mm,
Immunoglobulin
20
during
104/ml
were
electron
hundred
concentrated
start
Further
cultured
number
and
(3: 1 v/v).
One
tissue
8#{176}
CO2
in standard
in
resuspended
with attached
Channelizer.
Cells were cultured
in the
for
250-mI
arrangement.
divided.
Experiments
of
and
KCI
mounted.
were
2 x
ml
to cells
scanning
Coulter
Counter
Kar;’otype.
0.075
at
a concentration
and
x
showed
flask.
in cell
histochemistry.
to
I
established.
cells
were
determined
in a cytocentnifuge
tured
culture
Three
was
Morphology,
suspended
the
maintained
attachment.
ofcolonies
ratio
ml
disperse
of
count
10
a rubber
to
concentration
Differential
of
in
were
were
sample
Cells
number
as the
after
syncitial
5 x l0
increment
methylcellulose.
Colony
of two
atmosphere
was
2 x l04/ml
Channelizer
the
vortexed
of
with
of Cells
Cells
from
to each
weeks
line
cells
tissue
from
Colonialgrowth.
(w/v)
RC4
Characteristics
intervals,
added
cultures
the
resuspending
to a 1000-mI
Growth
and
a final
exclusion.
addition
bottles
tube
obtain
dye
by
of the
Lines
and
by
to
95#{176}c
by
and
later
a centrifuge
added
a humidified
Four
until
ofCell
intervals
in
followed
scraping
granulocytes.
fusiform
ofcells
Maintenance
2
was
intervals.
become
innocula
2-wk
and
4 mm,
gentle
to
was
than
for
and
transferred
greater
at 37’C
weekly
discrete,
0.25#{176}ctrypsin
of trypsin
was
suspension
incubated
of
medium
was
of cell
509
action
standard
lymphocytes,
ml
4 ml
the
suspension
viability
8
Twenty
were
cell
CSA
to
to stop
Sufficient
cells/mI.
were
ELABORATING
intervals
of
conditioned
cultured
and
the
media.
Confluent
in 50 ml of standard
conditioned
media
monolayers
(medium.
Complete
were
pooled.
I
x
l0
cells)
in 1000-mI
medium
exchanges
were
flasks
made
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
510
Dl
Fractionation
limits
of conditioned
of l0
(XM
daltons
were
within
certain
tioned
medium
pH
7.4,
2 x
used
106
to
were
of
ferential
sedimentation
removed.
conditioned
and patients
medium.
50
was
in
also
similar
was
l0
was
marrow
to
cells/mI
that
were
ColonT’
morphology.
medium.
the
medium
pipette,
feeder
by
via
cultured,
of standard
and
using
obtained
an
leukocytes
trocar
and
marrow
and
(3) the
was
transferred
slowly
was
injected.
slide,
(I)
The
GCT
and
large
of Cell
were
of GCT
large
3-4 nucleoli.
multinucleated
crest
of
healthy
obtained
by
fetal
serum,
conditioned
certain
were
marrow
bovine
or
Colonies
control
greater
than
experiments
marrow
mice
McCoy’s
adherent
cells
with
McCoy’s
medium.
were
medium.
Culture
not
of
removed,
and
was filled
a colony
using
the
from
matrix
an
with
inverted
were
0.05-0.
1 ml
microscope
aspirated
into
of Monocytes
Growth.
trast,
GCT
Monocytes
and RC4
efficiency
quired
serum.
Volume.
about
the
LTS
and
RC4
by
light
The
was
observed.
microscopy
nucleus
is shown
contained
basophilic
However,
in
lacey
Fig.
more
in GCT
uniform
was
and vacuolated.
transmission
not due
than
GCT.
to
1.
chromatin
Apparent
electron
miwere
were
fea-
multiple
cell types.
RC4
Their
nuclei
had
thicker
strands.
Comparison
cloning
4 x
7 days.
stained.
pleomorphic.
The
cytoplasm
cells were
hence,
pleomorphism
were
larger
and
chromatin
both
(2)
croscopy
showed
these
to be a cluster
of individual
cells
since
nuclei
separated
from each
other
by a double
plasma
membrane.
Viral
particles
not observed.
The progeny
of a single
GCT
cell (GCT-C)
showed
similar
tures;
cells
dif-
cells
Lines
appearance
cells
lysozyme
marrow
10 days.
DBA/2J
into
RESU
Morphology
of
PBS,
(MW)
Nonadherent
to a micromanipulator
loosened
and
and
iliac
20’
In
in
column.
were
methods.iS30
in
was
lowered
Cells
dried,
the
cells
for
GCT-condi-
of CSA.’4
of
interval
attached
micropipette
to a glass
that
culture
A micropipette
The
femurs
x l0),
adherent
microscope.
resuspended
except
from
containing
as a source
the
6.8
concentrations
invented
05)
weight
ofthe
Nucleated
dishes
exclusion
media
(molecular
(MW,
previous
varying
flushing
dextran
AL.
102 (UM
equilibrated
standardization
medium
5 x
of
column
experiments
of
5A
having
and
10 ultrafiltration
obtained
in 35 x 12 mm
with
of human
for
10),
ET
monocyte-conditioned
5-200
hemoglobin
certain
and
(PM
and
Blue
markers
McCoy’s
membranes
l0
PM
mI/mm.
human
For
cultured
obtained
of 0.5
a modification
in
enumerated
was
from
Sephacryl
CSA.
Marrow
was
diagnostic
aspiration.
by
were
cm
weight
gravity.14
1 x
RC4-,
retentate
120
antibiotics,
in agan
marrow
Rabbit
x l0),
immobilized
were
cultured
Mouse
1.23
unit
aliquots
size
rate
media for
undergoing
methylcellulose,
One-mI
cells
a flow
was cultured
were
1 .6#{176}c
(w/v)
at
as molecular
at
Marrow
(2 x l0)
x
30),
GCT-,
The
a 2.5
ultrafiltration
(PM
concentrate
to
(MW,
used
Diaflo
3 x l0
ranges.
PBS
a-casein
x l0)
Assas’
volunteers
cells
weight
with
50),
and
applied
eluted
Amicon
(XM
fractionate
was
daltons],
2.4
media.
5 x l0
molecular
and
(MW,
100),
PERSIO
1000
and Cell
did
had
following
( Table
I)
not divide
in liquid
or semisolid
doubling
times
of 55 and 60 hr,
ofO.2%-O.5
Monocytes
cu zm
Lines
had
in methylcellulose.
a median
macrophage
volume
Growth
of 500 cu m,
transformation.
medium.
respectively,
in both
median
volumes
of 2000 cu tm and 3000 cu tm, respectively.
Karyotype.
Monocytes
and macrophages
were
presumably
and RC4 cells had a chromosome
number
that ranged
from
35
modal
number
of approximately
70 in each line.
Surface
receptors.
Neither
monocytes
nor cell lines
formed
conand a
instances
which
GCT
In
and
re-
increased
to
RC4
cells
had
diploid.
to 300
GCT
with
a
spontaneous
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HUMAN
CELL
LINES
ELABORATING
CSA
511
Fig. 1.
Comparative
morphology
of GCT (A) and RC4 (B) cells
OCT cells are more pleomorphic
than RC4. Wright’s stain. x2400.
Table
1 . Compariso
n of GCT and
cultured
in liquid
medium.
RC4 C elI Lines to Monocytes
GCT and
Property
Monocytes
GCT-C Cells
RC4 Cells
Growth
Doubling
Cloning
time
(hr)
efficiency
-
(%)
-
55
60
<0.5
<0.5
Structure
Volume
(cu m)
Chromosome
Surface
500
number
(modal)
46
2000
-
70
3000
-
70
receptors
Sheep
red blood
Fc
cells
-
-
-
+
-
-
Histochemistry
Peroxidase
-
-
-
PAS
+
+
+
NASDA
+
+
+
NASDA+NaF
-
-
-
Function
Adhesion
+
+
+
Phagocytosis
+
-
-
IgG secretion
-
-
-
CSA elaboration
+
+
+
The
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
DI PERSIO
512
rosettes
with
rosettes.
Approximately
sheep
erythrocytes,
90%
whereas
50%
of monocytes
had
of
indicated
by rosetting
with antibody-coated
sheep
formed
rosettes
with sensitized
sheep
red cells.
Histochemistry.
Monocytes
and cell lines were
periodic
acid-Shiff
positive;
however,
GCT
and RC4 cells. All were stained
the reaction
was inhibited
by sodium
blood
lymphocytes
demonstrable
formed
Fc
erythrocytes.
receptors
Neither
peroxidase
ET AL.
as
cell
negative.
All
line
were
staining
was more
intense
and clumped
in
by napthol
AS-D
acetate
(NASDA)
and
fluoride
(NaF)
in each.
Inhibition
by NaF
was
less in RC4 than in GCT
or monocytes.
Adhesion.
Monocytes
and cell lines adhered
and
surfaces.
Phagocytosis.
Monocytes
were
able
to ingest
spread
latex
on
glass
and
particles,
plastic
antibody-
coated
erythrocytes,
E. coli, B. subtilis,
N. gonorrhea,
and C. albicans.
Greater
than 8O
of monocytes
ingested
these
particles.
Neither
GCT
nor RC4
cells
phagocytized
inert particles,
sensitized
sheep
erythrocytes,
or microorganisms.
Immunoglobulin
secretion.
Neither
monocytes
nor cell lines
released
immunoglobulins
into
their
culture
medium
as measured
by immunodiffusion
techniques.
Elaboration
ofCSA
Monocytes,
days.
mouse,
by Monocytes.
GCT,
Media
and
and
RC4
conditioned
rabbit
marrow.
by
GCT,
cells
these
were
cells
and RC4
Cells
incubated
were
at
tested
S x
for
lO
CSA
cells/mI
against
for
4
human,
Human
marrow
growth.
Figure
2 depicts
the mean
and standard
error
of
colony
number/2
x lO marrow
cells cultured
in three
separate
experiments
as
the proportion
ofconditioned
media
was increased
from O, to 3O, (v/v).
When
medium
incubated
in the absence
of cells was added,
no growth
was observed.
Increasing
the proportion
of monocyte-conditioned
medium
resulted
in a linear
increase
in colony
number
to 62 ± 11 per 2 x l0 marrow
cells at 30 ml/l00
ml. GCTand RC4-conditioned
mation
(95 ± 8 and 96 ±
medium
stimulated
14) at a concentration
of
near maximal
5 ml/lOO
ml.
Fig. 2.
Stimulation
growth
by cell-line-.
medium.
monocytes
days.
Equal
were
Supernatant
colony
forAt concen-
of human
marrow
colony
or monocyte-conditioned
numbers
cultured
medium
of GCT,
RC4,
and
in monolayer
for 4
was
filtered
and
assayed
for CSA in cultures
of normal
marrow.
Incubated
medium
without
added
cells served
as a control.
To minimize
endogenous
colony
stimulation,
the adherent
cells of human
marrow were
removed
prior
to culture.
Colonies
were
enumerated
on day 1 0. RC4, GCT,
and
0
05
CONDITIONED
(0
(5
MEDIUM
20
25
ADDED(ml/ml)
30
monocyte
refer to the cells from
ditioned
medium
was derived.
three experiments.
which
Mean
the con±
SE of
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HUMAN
CELL
LINES
ELABORATING
CSA
53
RABBIT
MOUSE
210
Fig. 3.
Mouse and rabbit
marrow
colony
growth
by cell-lineor monocyte-conditioned
medium.
The
experiments
ulation
show
of mouse
and
the
stim-
rabbit
mar-
row colony
formation
tioned
medium
derived
by condifrom GCT
cells, RC4 cells, or monocytes.
herent
moved
marrow
prior
mouse,
to
cells were
culture.
endogenous
150
120
90
Ad-
not reIn the
growth
Q 180
was
nil, i.e., there
were
no colonies
without
added
conditioned
medium.
In the rabbit,
endogenous
growth
was
present.
Colonies
were enumerated
on day 7.
60
c::
?
MONOCYTE
i’
..)V
INCUBATED
MEDIUM
01
1
0
“‘T’
I
0.1
0.2
0.3
CONDITIONED
MEDIUM
0
0.1
0.2
0.3
ADDED(mI/mI)
trations
greater
than
20 ml/IOO
ml of GCTand
RC4-conditioned
medium
colony
growth
decreased
(84 ± 4 and 86 ± 16).
Animal
marrow
growth.
Figure
3 illustrates
the results
of representative
experiments
stimulate
in which
4 x l04/ml
monocytemouse
or cell-line-conditioned
or rabbit
marrow
cells.
medium
was
Cell-line-conditioned
used
to
me-
.s
.
I.
1
I
‘C
-#{193}
Fig. 4.
Cell morphology
from human
marrow
colonies
that were grown
in the presence
of GCTconditioned
medium.
Single colonies were removed
from a 10-day
culture.
Cells were dispersed
on
a slide, stained,
and examined.
Both granulocytes
(A) and macrophages
(B) were present.
Wright’s
stain.
x2400.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
514
DI PERSIO
dium
stimulated
greater
colony
numbers
these
species.
The difference
was
lating
to the greater
endogenous
to human
marrow,
no inhibition
at higher
concentrations
cell-line-conditioned
of conditioned
medium
was also
conditioned
monocyte-conditioned
in stimulating
medium
in rabbit
marrow,
in this species.
or rabbit
marrow
medium.
In experiments
found
to be more
potent
in
perhaps
reIn contrast
was noted
than
not shown,
monocyte-
cell morphology.
Most
human
colonies
contained
and
macrophages
(Fig.
4). Rarely,
colonies
contained
granulocytes.
Mouse
colonies
contained
predominantly
developing
macromacro-
medium
Colonial
granulocytes
phages
or
than
less noticeable
colony
formation
of either
mouse
ET AL.
dog
marrow.
phages.
This
finding
was independent
of conditioned
medium
concentration
and culture
time.
Macrophagic
maturation
was
also
preeminent
in rabbit
colonies.
Some
cells in this species
had the features
of plasma
cells with eccentric nuclei
and deeply
basophilic
cytoplasms.
Further
efforts
to characterize
rabbit
colony
cells were not made.
Comparison
on Human
ofGCT-conditioned
Marrow
Growth
GCT-conditioned
and
to
Medium
medium
those
stimulated
Table
was
by
2.
compared
1 x
Comparison
and
Leukocyte
and Leukocyte
106
of the Effect
Feeder
to
blood
Feeder
cultures
leukocytes
without
in
of GCT-conditioned
on Human
Marrow
Colonies/2
and Subject
Unstimulated
added
CSA
two-layer
agar
Medium
Growth
x lO5CeIIs
Feeder
GCT-conditioned
Medium (0.15 mI/mI)
110
Leukocyte
Diagnosis
the
Normal
P.R.
30*
105
1.1.
20*
87
88
J.P.
10*
110
140
Hypoplasia
C.P.
(severe)
0
0
0
AG.
(mild)
0
22
32
Neutropenia
D.A. (congenital)
2*
17
24
P.C. (congenital)
2*
14
50
Preleukemia
E.C.t
0
R.H4
100*
N.T.
2*
Leukemia
0
880*
19
60
I
B.C. (AML)
0
0
0
R.M. (AMML)
0
0
0
30
0
58
EL. (AMoL)
( <50
*Clusters
tRefractory
tSubsequently
cells).
sideroblastic
died
developed
IIAML,
myelogenous
acute
anemia
with
increased
myeloblasts.
of AML.
§Subsequently
leukemia.
0
450*
AMML.
leukemia;
AMML,
acute
myelomonocyte
leukemia;
AMoI,
acute
monocytic
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
HUMAN
CELL
method.’4
neutropenia,
were
not
LINES
ELABORATING
Marrows
from
preleukemia,
removed
prior
medium
growth
was equal
of normal
formed
colonies
to or greater
than
marrows.
In those
(A.G.,
increase
greater
D.A.,
medium
Fractionation
ofCell-Line
N.T.,
by 20 ml/lOO
100
and
Ieukocyte
medium
E.L.)
stimulated
GCT-conditioned
feeder
cells.
and leukocyte
less
growth
ml oftest
retained
fractions
cluster
In
than
sample.
by
marrow.
in which
Unfractionated
x lO5cells,
cells.
The
an
derived
or colony
3.
200
XM
XM
rabbit
100
50
from
the
formation
Ultrafiltration
of Medium
Human
Added
(20%
to Culture
XM 100
retentate
XM 100
filtrate,
Colonies/2
v/v)
XM 50 retentate
medium
an
Table
marrow
stimulated
a
instance
where
feeder
x
lO5CeIIs
and
also
50 filtrate
inhibited
3)
stimulated
colony
of CSA.
10 filtrate-
Marrow
x lO4Cells
(n = 2)
(140-260)
Mouse
Colonies/4
Morrow
x lO4CeIIs
(n = 9)
94
(80-1
12)
200
102
(86-1
20)
178 (1 17-240)
291 (145-380)
310(160-400)
170(100-210)
86
(70-100)
137 (85-190)
32t
(28-38)
47 (30-65)
0
32 (20-45)
0
221 (10-46)
UM 05 retentate
Cell-free
incubated
0 (0-1)
0
26t
(1 6-45)
40 (20-60)
0
23t
(10-40)
50 (30-70)
0
1 t (0-5)
medium
Data represent
the mean and range of three studies.
* Desalted
and concentrated
five-fold by ultrafiltration
1’Clusters
Not
(<50
concentrated.
cells).
of cluster
Mono-
by GCT Cells
PM 10 filtrate,
UM 05 fiItrate
medium.
molecular
Inhibitory
by the absence
05 retentate.
PM 30 filtrate,
PM 10 retentate
against
310
of
were
devoid
by the PM
Rabbit
Colonies/4
stimu-
l0 cells, and
that
portion
with
RC4 conditioned
suggested
that
the
than
50,000
daltons.
Conditioned
weight
assayed
medium
x
XM 50 filtrate,
PM 30 retentate
cells.
3 depicts
the mean
cells were stimu-
conditioned
colonies/4
retentate
membrane
XM
was
Marrow
(n
Unfractionated*
medium
preleukemic
pacells stimulated
leukocyte
activity
in the 500-10,000
dalton
range
was suggested
formation
in cultures
containing
the PM
10 filtrate-UM
cyte CSA was also retained
by an XM 50 ultrafilter.
Medium
one
feeder
the
who
medium
into
various
molecular
were
concentrated
fivefold
and
UM 05 retentate.
Similar
results
were obtained
The retention
of CSA
by an XM
50 ultrafilter
weight
of cell-line
CSA(s)
might
be greater
Table
in stimulating
abnormalities
CSA
colonies/2
x lO
filtrate
growth.
All
Endogenous
feeder
cells
with marrow
human,
rabbit,
and mouse
number
in experiments
lated 94 human
mouse
colonies/4
XM
leukocyte
patients
fractionated
GCT-conditioned
by ultrafiltration.
All fractions
for CSA
against
and rangeofcolony
lated
P.C.,
than
patients
with
marrow
aplasia,
were
studied.
Adherent
cells
in Table
2, GCT-conditioned
in clusters;
however,
GCT-conditioned
number
ofclusters.
We have not encountered
GCT-conditioned
We
ranges
515
healthy
subjects
and
and
acute
leukemia
to culture.
As shown
stimulated
greater
growth
tient (R.H.),
GCT-conditioned
only an
twofold
CSA
a UM 05 membrane.
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Dl
516
22
Vo
BLUE
PERSIO
ET AL.
HUMAN
DEXTRAN
HEMOGLOBIN
LYSOZ
YME
100
CASEIN
18
80
14
60
1.0
40
0.6
20
0.2
0
70
50
90
FRACTION
Fig. 5.
Elution
profile
medium
was ultrafiltered
of GCT
through
Sephacryl
column
equilibrated
were
assayed
for CSA using
120
NUMBER
cell-line
human
CSA
a PM 10 membrane.
from
Sephacryl
The retentate
in PBS at pH 7.4 and eluted
human
marrow.
The left-hand
5-200.
GCT-conditioned
was applied
to an 5-200
with PBS. Five-mI
ordinate
shows
fractions
of eluate
the absorbance
of
eluate at 280 nm. The right-hand
ordinate
shows colony
number
per 2 x 10 human
marrow
cells
in the presence
of 0.2 mI/mI
of each fraction.
For reference,
the elution
peaks
of blue dextran
(MW,
2 x 106 daltons),
a-casein
(MW
1.23 x 10),
human
hemoglobin
(MW,
6.8 x 10),
and
lysozyme
(MW, 2.4 x 10) are shown.
To evaluate
medium
was
further
the
concentrated
the retentate
was
Five-ml
fractions
human
various
marrow.
molecular
eluted
CSA
over
applied
of the
of cell-line
over a PM
CSA,
GCT-conditioned
10 membrane.
Five
ml of
to a Sephacryl
5-200
column
and eluted
with
PBS.
eluate
were collected
and assayed
for activity
against
The results
are shown
in Fig. 5. The elution
fractions
in which
weight
markers
were recovered
are indicated.
Cell-line
CSA
a molecular
eluting
molecular
nature
by ultrafiltration
with
weight
an apparent
range
of
molecular
30,000-90,000
weight
daltons
of -40,000
with
maximal
daltons.
DISCUSSION
Several
features
of GCT
exhibit
similar
and
RC4
resemble
phages.
They
histochemical
do not
marrow,
Cell-line
secrete
immunoglobulins.
Moreover,
a property
associated
with human
CSA is similar
to monocyte
CSA
species
tested,
stimulates
granulocyte
an XM
50 ultrafilter.
The differences
however,
prevent
us from concluding
monocytes
or
ploidy,
capacity
macrophages.
for sustained
those
of monocytes
reactions,
adhere
and
to
surfaces,
macroand
they
elaborate
CSA
for human
monocytes’7”8
and macrophages)9
in that it supports
the growth
of all
and monocyte
colonies,
and is retained
by
between
the cell lines
and
monocytes,
with certainty
that they are derived
from
For example,
proliferation,
the
and
immature
absence
of
appearance,
Fc receptors
phagocytic
capability
are different
from
monocytes
and macrophages.
dase positivity
is sometimes
present
in monocytes,
whereas
it is absent
and RC4 cells. In morphology,31’32chromosome
number,3’
histochemistry,32
aneuand
Peroxiin GCT
and
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HUMAN
CELL
LINES
ELABORATING
CSA
517
growth
rate,31
OCT
and RC4
more
closely
resemble
cell lines
lymphomatous
effusions
than normal
monocytes
and macrophages.
We cannot
explain
the unique
conditions
which
have permitted
derived
ofcell
line RC4,
from
the blood
of a healthy
from
similar
monocyte-enriched
preparations,
rise to lymphoblastic
cell lines which
did not
such attempts
died
or gave
Cell-line-conditioned
medium
the
donor.
In 14 other
the cultures
either
elaborate
CSA.
provided
a reproducible
and
from
isolation
potent
source
of
CSA for animal
and human
marrow.
It was more
consistent
than
human
blood
leukocyte-conditioned
medium
or human
blood
mononuclear
cell-conditioned
medium.
It had a similar
potency
to leukocyte
feeder
cells when
normal
human
marrow
was studied.
In several
abnormal
marrows,
its potency
seemed
greater
than
feeder
cell systems
tioned
medium
was less
Thus,
cell-line
material
CSA
to assure
and,
potent
may
that
be
although
than
of critical
clinical
ditioned
their
jected
medium
has
studies
medium
to ammonium
apatite
marrow
traction
adsorption,
of -96,000,
of ultrafiltered
been
studied
mouse
have
exposed
tion,
adsorption
G-75.
They
to
isolated
curve.
the possible
most
performed
embryo
optimal
of CSA
and
in con-
co-workers.
blood
leukocytes
chromatography,
or
In
and subhydroxyl-
filtration
yielded
CSA
for human
daltons.33
Chloroform-methanol
exmedium
yielded
an additional
leukocyte-conditioned
purified
fractions
report,
kidney-conditioned
a 60,000-80,000
high-potency
in the
by Price
Although
unfractionated
and human
activity,35
phosphate
ceU-condicell-line
CSA.
heterogeneity
intensively
In a preliminary
calcium
mononuclear
system
or
in providing
being
and
Sephadex
G-150
35,000,
and
15,000
leukocyte-conditioned
marrow.33’34
human
are
conditioned
by unfractionated
sulfate
precipitation,
DEAE
CSA of about
1300 daltons.34
medium
possessed
both
mouse
stimulate
importance
studies
near optimal
range
of the dose-response
The molecular
nature
ofCSA
and
not shown,
feeder
cell
the
Walasek
medium
gel,
dalton
and
and
to
filtration
CSA,
PM
not
10 ultrafiltra-
through
which
did
colleagues36
Sephadex
retained
mouse
ac-
tivity
but had only
weak
human
activity,
and
a 30,000-40,000
dalton
CSA,
which
stimulated
only human
colony
growth.36
Studies
of the molecular
nature
of GCT
and RC4
cell-line
CSA
are in an
early
phase.
Our
findings
are in approximate
agreement
with
the above
two
studies
in that the maximum
human
CSA eluted
from
5-200
with an apparent
molecular
weight
of about
40,000
daltons.
The range
of activity
using
Sephacryl
gel filtration,
of our elution
however,
profile
6O,O00-80,000
weight
fraction
dalton
CSA of Walasek
of Price and co-workers.
The
requirements
raphy,
the difficulty
sulphate-polyacrylamide
ciation
of albumin
was broad.
Thus,
contained
another
for
bioassay
of
it is possible
molecular
and
elution
in bioassay
of fractions
gel electrophoresis
from
the
serum-fortified
that
species
colleagues
and
fractions
the
after
removed
fractionations,
culture
the skewed
analogous
from
medium
higher
portion
to the
molecular
CSA
chromatog-
sodium
dodecyl
and the close
assoto
the
-40,000-
dalton
human
CSA,
among
other
technical
problems,
makes
molecular
characterization
difficult.
Recently
we have
been
able
to recover
CSA
from
the
medium
of these
cells
when
incubated
in serum-free
medium
fortified
with
Ficoll.
These
adaptations
and the availability
of cell-line
CSA
will facilitate
further
biochemical
and
physiologic
studies
of these
molecules.
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518
DI PERSIO
ET AL.
ACKNOWLEDGMENT
expert
technical
1. Stohlman
F Jr:
The
assistance
of David
Nemchick
is gratefully
acknowledged.
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Cline
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EngI
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1972
Regulation
J Med
of
ditioned
of in
PL,
vitro
Rev
evaluation
Med
4.
Boggs
DR:
4:535-551,
4:509-533,
1975
and regulation
D:
Hansen
Morley
RK,
Nunna
Jr:
IV.
K,
factor.
Vogler
Smith
BA:
urine:
of
normal
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1978 51: 507-519
Human cell lines that elaborate colon-stimulating activity for the marrow
cells of man and other species
JF Di Persio, JK Brennan, MA Lichtman and BL Speiser
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