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Proliferation
and
Maturation
Eitan
By
Fibach,
Hemopoiesis
is studied in vitro
where
hemopoietic
progenitors
of Human
Daphna
Manor,
Erythroid
Ariella
mainly in semisolid
culture,
develop into discrete
cob-
Progenitors
Oppenheim,
and
Eliezer
in Liquid
Culture
A. Rachmilewitz
blood (PB) of patients
with -thalassemia
in liquid medium
in the presence
of conditioned
medium
from human bladder carcinoma
cells. Seven days later, RBCs, normoblasts,
antihuman
lymphocyte
antibodies
(CAMPATH-1
) and
complement.
Reculture
of the remaining
cells in liquid
medium
supplemented
with
recombinant
erythropoietin
(EPO) resulted
in the exclusive
development
of erythroid
cells, with myeloid cells reduced
to <2%. Stainable
hemoglobin
(Hb) appeared
on day 3. with
over 85% of the
population
containing
hemoglobin
by day 1 1 and the cell
number
increasing
from 0.2 x iO’ to 3 x 10/mL.
By
granulocytes.
permitting
nies.
We
describe
proliferation
tors.
a liquid
and
We
culture
maturation
seeded
system
of
mononuclear
and
cells
monocytes
that
human
supports
erythroid
from
the
disappeared,
the
progeniperipheral
and
the
number
of lymphocytes
dropped
considerably.
In contrast,
crythroid
colony-forming
cells increased
fourfold
to tenfold.
The next step entailed
the removal
of colony-stimulating
factor
(CSF) and CSF-secreting
cells. the exclusion
of
macrophages
by harvesting
nonadherent
cells. and the
lysis of T lymphocytes
by treatment
with monoclonal
rat
H
EMOPOIESIS
is studied
in vitro
culture
medium.
In this system,
specific
growth
develop
outcome
into discrete
colonies.’
of the proliferation
and
ticular
cell,
hemopoietic
factor
in the
making
out
progenitors
colony
is the
of one par-
and
analysis
immunologic
loping
cell.
In the present
study
that supports
the growth
progenitors.
This
of progenitors
fact that the
in
cells
semisolid
medium
results
in several
it technically
difficult
or impossible
quantitative
biochemical
erythroid
hemopoietic
Since
each
differentiation
the number
and frequency
tissue can be measured.
The
are immobilized
disadvantages,
to carry
added,
mainly
in semisolid
depending
on the
of growth
kinetics,
characterization
and/or
of the
deve-
we describe
a liquid
culture
system
and maturation
of human
erythroid
system
may
prove
useful
for
studying
cell development.
MATERIALS
AND
METHODS
Peripheral
blood (PB) from patients
with fl-thalassemia
was
drawn into heparinized
tubes. After centrifugation
at 800 rpm for 10
minutes,
the platelet-rich
plasma
was discarded
and the pellet
resuspended
in four volumes
of culture
medium
and layered on a
gradient
of Ficoll-Hypaque.2
After centrifugation
at 1,200 g for 20
minutes, the interphase
layer was collected and the cells washed and
cultured
at a density
of - 10’ cells/mL
in alpha minimal
essential
medium supplemented with 10% fetal calf serum (FCS) (both from
Gibco, Grand
Island,
NY) and 10% conditioned
medium
(CM)
collected
from cultures of the 5637 bladder-carcinoma
cell line.3 The
cultures
were incubated
at 37#{176}C
in an atmosphere
of 5% CO2 in air
From
the
Hospital.
Devartment
Supported
in part
20. 1988;
ofDimes
Address
by Basic
Birth
reprint
Deftc:s
requests
Hematology,
P0 Box 12.000,
salem,
iI-91
Israel,
The publication
This
article
in accordance
accepted
August
Research
Grant
24. 1988.
No.
1-lO73from
the
Foundation.
to Eitan
Fibach.
Hadassah
article
must
with
this fact.
© I 989 by Grune
& Stratton,
0006-4971/89/7301-0055$3.00/0
100
of culture
the
cell
yield,
conditions
the
liquid
and
com-
system
facilitate
with extra humidity. Following a 1-week incubation
in these primary
cultures,
the nonadherent
cells were harvested
and washed
and the
lymphocytes
removed
by treatment
with monoclonal
rat antihuman
lymphocyte
antibodies
(CAMPATH-l),4
followed
by fixation
of
human
complement
derived from fresh serum. The remaining
cells
were then recultured
in alpha medium
containing
(unless otherwise
specified)
30% FCS, 1% deionized
bovine serum albumin
(BSA),
l0 mol/L 2-mercaptoethanol,
1.5 mmol/L
glutamine,
10’ mol/L
dexamethasone,
and 0.2 to 2 U/mL
recombinant
erythropoietin
(EPO, Genetics Institute).
These secondary
cultures were incubated
at 37#{176}C
in an extra-humid
sealed incubator
flushed with a mixture
of
6% 02, 7% CO2. and 87% N2. Viable cells were counted
after
staining
with trypan
blue. Hemoglobin
(Hb)-containing
cells were
scored with use of the acetic acid-benzidine
peroxide
reaction.5
Cellular
morphology
was assessed
by preparing
cytocentrifuge
(Shandon,
Cheshire,
UK) slides stained with alkaline benzidine
and
Giemsa.
Cloning
in semisolid
culture
was performed
in alpha
medium
containing
0.9% methylcellulose
(Fisher Scientific
Co. Fair Lawn,
NJ) and supplemented
with FCS, albumin,
2-mercaptoethanol,
and
glutamine
at the concentrations
mentioned
above, and 2 U/mL
EPO. The cultures
were incubated
in a sealed incubator
as described
above.
Erythroid
colonies,
identified
by their red color when
unstained or by their blue color after staining
with the acetic
acid-benzidine
peroxide
reagent,
were scored with the aid of an
inverted microscope.
The mean number
of cells per colony was
determined
as follows: First the number
of colonies
in a plate was
counted.
Then a known volume of medium
was added
to the plate
and mixed thoroughly
in order to disperse
the colonies
into single
cells, which were counted
in a hemocytometer.
The number of cells
per plate
was divided
by the number
of colonies
in the same
plate.
University
PhD.
University
Department
Hospital,
of
Jeru-
/20.
costs ofthis
payment.
“advertisement”
indicate
Hadassah
manipulation
increasing
RESULTS
June
charge
Hematology,
and
Jerusalem.
Submitted
March
of
the
may
quantitative
analysis of growth
kinetics
as well as
biochemical
and immunologic
characterization
of the developing erythroid
cell.
C 1989
by Grune & Stratton,
Inc.
ponents
Inc.
were defrayed
therefore
18 U.S.C.
in part by page
be hereby
section
1734
marked
solely
to
After
seven days’
liquid EPO-deficient
incubation
of PB mononuclear
primary
cultures,
the number
herent cells
the plastic
by 30% to 60%. Monocytes
where
they
differentiated
decreased
surface,
cells in
of nonadadhered
to
into macro-
phages.
Normoblasts,
which
are quite
frequent
in blood
obtained
from
splenectomized
thalassemic
patients,
and
granulocytes,
which
despite
the Ficoll-Hypaque
separation
procedure
were
always
present
in small
numbers
at the onset
of primary
culture,
were
not found
after
seven
days
culture.
The bulk of the cultured
cells were lymphocytes,
myeloblasts,
and
intermediate
Blood,
Vol
myeloid
73. No 1 (January).
precursors.
1989:
Early
pp 100-103
of
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ERYTHROID
CELL
as well
erythroblasts
observed.
No
staining,
were
The
DEVELOPMENT
this
cells
the
were
in liquid
their
deficient
population.
liquid
medium
of smaller
was
of
associated
as
well
I), but
the time
shortened.
After
the
one
to
a
ent
unit-erythrocytes
in human
PB.6
bone
Since
0
cloned,
I week
after
the
they
primary
0
contained
progenitors
liquid
in
which
not
of
culture
and differentiation
Further
proliferation
tion
are
appearance
in liquid
an
are
normally
in
CFUe-like
progenitors
by prolifera-
BFUe.
and
maturation
of these
also
in
mented
cells
were
as described
composition
presented
in
recultured
and
in
fresh
and
in Materials
Methods.
The
In the
presence
the
1 . The
of EPO
Culture
of Preculture
on the Number
in EPO-Deficient
and Characteristics
Direct
Ptl
No.ofcolonies/mL
Liquid
of Erythroid
The
Pt2
164
Pt3
270
112
10
Time
cloning
PB mononuclear
after
(dl
cells
days
14
from
14
three
Primary
Pt2
Pt3
1,030
semisolid
of preculture
medium,
are the mean of two plates.
calculated
Abbreviation:
per milliliter
Pt, patient.
either
in EPO-deficient
The number
of the primary
culture.
erythroid
and
in these
cultures;
of the cells,
removal
followed
of
by reculture
culture
are
presented
ber and
after
cells
percentage
lOto
in
Fig
started
3.
In
the
second-
presence
to appear.
of
EPO
A maximum
of benzidine-positive
cells
were
numachieved
12 days.
Table
2.
Cellular
Compositi
on of the Second
(%)
My&oid
ary Cultures
(%)
Lymphd
(%)
1 .5
2.0
1.2
EPO
95.8
±
1.4
3.7
±
1.0
0.5
±
0.4
175
252
70
EPO + CM
42.2
±
4.4
57.0
±
3.2
0.8
±
0.2
99.2
±
2.8
0.8
±
0.4
7
with
both
Cloning
1,620
16
patients
CM,
the
consid-
cellular
Ptl
12
whereas
decreased
and the absence
of CM,
the total number
of cells did not
change
during
the first five days,
but the percentage
of
myeloid precursors decreased,
whereas
that of erythroid
cells
increased.
Two to five days after
exposure
to EPO,
the first
1,180
2,520
cells,
cells
and
required
washing
CM
in EPO-containing
seven
The results
were
1.477
of erythroid
8
7
f9-thalassemia
were
directly
primary
of
calculated
culture.
ary
of hemoglobiniza-
tion after
cloned
11
1,370
1 5 days after initiation
resulted
in cell death
(data
not shown).
of erythroid-cell
development
in the
kinetics
No. of subcolonies/
colony
i)
whereas
CM alone
stimulated
the
cells exclusively.
The continuous
Erythroid
No.ofcells/colony
primary
(#{149}.
0. and
supple-
Colonies
Indirect
Cloning
in the
lymphoid
of EPO
was
by
in its absence,
of these
secondary
cultures
after
1 1 days
is
Table
2 and Fig 2. In the presence
of EPO, most
Effect
progenitors
and
presence
benzidine-positive
Table
8
three patients
in EPO-deficient
consisted
developed,
of myeloid
compound
The
medium,
6
Culture
data are presented
as the
cells per milliliter
of primary
of myeloid
EPO-
complement.
remaining
population
occur
ment
of erythroid
cells from
the liquid
culture.
The
number
of colony-forming
myeboid
cells
development
cells were
harremoved by treat-
antibodies
Kinetics
percentage
CFUe-like
normoblasts
could
week
of preculture
4
in Primary
culture.
with
-thalassemia
were cultured
liquid medium.
and
at indicated
intervals
various
portions
of the culture
were
harvested,
washed, and cloned in EPO-containing
semisolid
medium.
erably.
of the
CAMPATH-1
Fig 1 .
PB mononuclear
of the
deficient
liquid medium,
the nonadherent
vested and most of the lymphocytes
were
with
2
Days
The number of colonies was determined
pres-
found
can be explained
into Hb-containing
culture.
After
one
.C
cells increased
fourmaximal
size and Hb
no CFUe
were present
in the original
PB cell
as no such colonies developed
when the cells were
population,
directly
but
C
0
hemoglo-
in the
(CFUe),
marrow
E
II.
C.)
V
average of 140 cells arranged
in only one to three subcolonies
(Table
1). These
erythroid
colonies
were similar
to colonies
that developed
from the more differentiated
progenitors,
the
colony-forming
C
0
EPO-
gradual
for their
week
when
days,
in
I,
0
the
required
content
nine
were
total
number
of
colonies
that developed
of colony-forming
colonies reached
seven
cells
with
as
culture,
the number
fold to tenfold.
The
after
1 ). These
PB
E
colonies
(BFUe)-derived
cobthe frequency
of BFUe in
Preculture
and
size,
was
binization
(Table
represented
increase
in the frequency
colony-forming
cells (Fig
were
produced
0
Hb content
on day 14, when
and contained
thousands
of
unit-erythroid
cell
0
on day
cloned
medium)
and
macroscopic
number
of erythroid
Cells
in five to 20 subcolonies
original
also
phase was studied
in the primary
culture,
medium.
size
maximal
burst-forming
nies, and
were
by benzidine
differentiation
periods
semisolid
arranged
typical
various
preculture
colonies
and
EPO-independent
after
cells,
achieved
the
as measured
0
of proliferation
during
(ie, without
blasts
unidentified
cells,
101
CULTURE
present.
in EPO-containing
that
as some
Hb-containing
extent
progenitors
by cloning
IN LIQUID
or indirectly,
liquid
of colonies
medium.
presented
0
Mononuclear
5637
cells were
CM for 1 week
U/mL),
5637
stained
with
of erythroid,
myeloid,
the
±
means
patients.
in liquid
and then recultured
CM (10%),
alkaline
cultured
followed
and lymphoid
SD of the
results
in the presence
in the presence
or EPO plus CM.
benzidine,
medium
After
1 1 days
by Giemsa,
in cultures
of
EPO (1
the cells were
and the percentages
cells were determined.
obtained
of either
The values
of cells
from
are
six
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102
FIBACH
Fig 2.
Morphology
for one week and then
(A) or alkaline
benzidine
cells were cultured
in liquid medium
in the presence
of 5637 CM
12 days the cells were stained with either May-Gr#{252}nwald Giemsa
of cells in the secondary
cultures.
PB mononuclear
recultured
in the presence
of EPO (1 U/mL).
After
(B).
3
DISCUSSION
In the
:
l.
:
E
0
present
ment of the
tors (BFUe)
study
we have
erythroid
series
into Hb-containing
demonstrated
from
,
tors,
9-thalassemia
:
BFUe,
namely
the
early
BFUe.7
contains
a
(b)
high
The
antibodies
and complement.
and seeded at a density
of
with
BFUe.6
(c)
culture
depends
of 0.4 x 10’ cells/mI
in liquid medium
supplemented
with 1 U/mI
EPO. Viable
cell number
(S--)
was determined
after
staining
with trypan
blue. and the percentage
of Hb-containing
cells was
determined
(O--O)
after
staining
with
the benzidine
peroxide
reagent.
progenibe accom-
in contrast
to the late CFUe
progenitors,
can survive
for several
days
in the absence
of EPO
when
supplied with a source
of burst-promoting
activity
(BPA).8’9
in
The development
(multiplication
CFUe requires
EPO.’#{176}” (e) The survival
colony-forming
units
into granulocytes
Fig 3.
Cellular kinetics in the secondary
cultures.
PB mononuclear
cells from
a patient
with
-thalassemia
were
cultured
in
liquid
medium
in the presence
of 5837
CM. After 1 week of
incubation,
the nonadherent
cells were
harvested,
treated
with
develop-
PB of patients
frequency
#{163}(d)
Days
that
early committed
normoblasts
can
plished
in liquid
culture.
The experimental
procedure
is
based on several
known
observations:
(a) In contrast
to BM,
PB contains a homogeneous
population
of erythroid
progeni-
g
-
C
CAMPATH-1
ET AL
on the continuous
presence
and
maturation)
and
of
of
development
and
ofCSF.’2
macrophages
(f) Activated
T
lymphocytes
and monocytes/macrophages
serve as sources
of CSF in vitro.’’6
In the procedure
described,
the culture
was divided
into
two
phases.
In the first
phase,
or the primary
culture,
peripheral
fl-thalassemic
mononuclear
cells were seeded
in
liquid
Under
decrease
medium
supplemented
of erythroid
absolute
number
Analysis
of colonies
gested
their
appearance
by
with
these
conditions,
1-week
in total
cellularity
but
proliferation
of these
and
by
these
to
CFUe-derived
CFUe-like
progenitors
differentiation
CM.’7
progenitors.
colony-forming
developed
similarity
BPA-containing
cultures
showed
a sharp
a significant
increase
in
of
progenitors
sug-
colonies.
can
the
The
be explained
BFUe
in
the
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ERVTHROID
CELL
EPO-deficient
DEVELOPMENT
liquid
culture.
amounts
EPO
is
highly
required
sensitive
undergo
We regard
the primary
culture
exclusively.
phase,
of EPO
indeed
103
CULTURE
although
it is conceivable
that
are present in the FCS. However,
if
as an EPO-independent
trace
IN LIQUID
to
at this
phase,
losing
EPO,
differentiation-a
the
this
most
cells
should
as
sensitivity
be
they
sequence
unlikely
of
18
Before
the second phase,
were removed;
nonadherent
the CSF and CSF-secreting
cells were harvested,
cells
washed
thoroughly,
monoclonal
specific
against
This
and
treated
human
with
T lymphocytes,
antibodies
and
lysed
removed
all the monocytes
the lymphocytes
but had
procedure
most of
erythroid
progenitors.
tured
under
conditions
by complement.
and macrophages
no effect
on
erythroid
cells: the presence of EPO, a high concentration
FCS, and the addition
of albumin,
2-mercaptoethanol,
tamine,
and
dexamethasone,
with
incubation
resulted
in the disappearance
and the development
of erythroid
precursors
of
glu-
under
oxygen pressure. This
myeloid
the
The remaining
cells were then reculconducive
to the development
of
low
of the
cells
When
the secondary
cultures
were
CSF instead
of EPO, myeboid
cells developed.
Similar
liquid cultures
were also established
bone marrow
that cultures
thalassemia
liquid-culture
procedure
studying
erythroid
development:
affords
(a)
several
separation
independent
and EPO-dependent
phases;
of culture
conditions
and
components
without
termination
of the culture;
and
per culture,
molecular
from
and PB (data
not shown).
However,
obtained
from
PB cells of patients
were superior
in terms of erythroid-cell
purity.
The
yield
supplied
facilitating
with
normal
we found
with $yield and
advantages
of the
in
EPO-
(b) manipulation
at various
stages
(c) improved
cell
immunologic,
biochemical,
and
analyses.
ACKNOWLEDGMENT
We thank Professor
S. Slavin of the Hadassah
University
Hospital for providing
the CAMPATH-l
antibodies,
Genetics
Institute
for a generous
gift of EPO, and A. Treves for her expert
technical
assistance.
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1989 73: 100-103
Proliferation and maturation of human erythroid progenitors in liquid
culture
E Fibach, D Manor, A Oppenheim and EA Rachmilewitz
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