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Separation
Progenitors
Marrow
of the Erythropoietin-responsive
BFU-E
and CFU-E
in Mouse
by Unit Gravity
Sedimentation
By D. S. Heath,
A. A. Axelrad,
D. L. McLeod,
The sedimentation
velocity
profiles
of the
entities
in mouse
bone
marrow
responsible
for erythropoietic
burst
formation
(BFU-E)
and for erythrocytic
colony formation (CFU-E)
have been studied
under conditions designed
to determine
whether
the
values
observed
are real or result
from
cell interactions
occurring
during culture of
the fractions.
Bone marrow
cells of adult
C3Hf/Bi
mice were subjected
to unit gravity sedimentation
in a bovine
serum
albumin
gradient,
and fractions
were
assayed in plasma
culture.
Because
it was
found that cell concentration
affected
the
efficiency
of erythropoietic
burst formation
in culture,
aliquots
were
plated
at two
different
cell concentrations,
as well as at
a fixed proportion
of each fraction.
The
modal sedimentation
velocity
of the BFU-E
population
averaged
3.9 mm/hr
and that
of the CFU-E population,
6.4 mm/hr;
both
were found to be independent
of cell con-
N MAMMALIAN
HEMOPOIETIC
CELL
ropoietin
(Epo),
colonies
develop
which
synthesizing
cells that mature
into non-nucleated
colonies,
In plasma
cultures
marrow
cells from
after
about
2 days
From
the
and
Division
Toronto,
Submitted
May
Supported
by
subsequently
within
they are
of Histology,
Ont.,
27,
the
of
up
to
of
disappear.
65
containing
erythof hemoglobinThese,
referred
to
benzidine-staining
cells.
Epo
and
seeded
with
bone
regularly
reach
peak
numbers
If cultures
are
initiated
with
a
and then
continually
fed with
the hormone,
crops
of new
about
6 days.
These
colonies
exist
in isolated
groups;
the
are relatively
few in number
and Poisson
distributed
in the
The colonies
colonies,
and
Toronto,
composed
CULTURES
are composed
red cells.”2
initiated
with
a low dose
adult
C3H
mice,
the colonies
high dose
of Epo
colonies
arise after
groups
themselves
cultures.
rocytic
are
M. M. Shreeve
centration
or method
of dividing
the fractions.
Cells
from
fractions
of different
sedimentation
velocity
were
mixed
with
one another
or with
unfractionated
cells.
No significant
inhibition
or stimulation
of
erythropoietic
burst formation
was seen.
We concluded
that
the observed
values
represented
the true modal sedimentation
velocities
of BFU-E and CFU-E in normal
mice. To determine
whether
a change
in
the physiologic
state of the animals
affected
the
sedimentation
velocities
of
BFU-E or CFU-E,
marrow
cells from mice
hypertransfused
with red cells were compared with those from controls.
The modal
sedimentation
velocity
of BFU-E was unaffected
by hypertransfusion,
nor
was
there any change
in the number
of BFU-E
under
these
conditions.
The number
of
CFU-E was substantially
reduced
without
a significant
change
in modal
sedimentation velocity.
I
as erythrocytic
and
Bone
the groups
composed
Department
are less discrete
of cells that vary
of A natomy.
Faculty
than
over
of
the 2-day
erytha larger
range
of
Medicine,
University
of
Canada.
1975;
Medical
accepted
Research
November
Council
17.
1975.
of Canada
and
by
the
National
Cancer
institute
of
Canada.
Address
versity
of
© 1976
Blood,
Vol.
for
reprint
Toronto,
by Grune
47,
requests:
Toronto,
No.
Ont.,
& Stratton.
5 (May),
A. A. A xelrad,
M. D..
(‘anada
M5S
1A8.
Ph.D..
Professor
of Anatomy
(Histology),
Uni-
inc.
1976
777
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778
HEATH
size
and
apparent
colonies
hemoglobin
“erythropoietic
content.
number
of
hypertransfused
trol mice, while
bursts
mice was
the number
at least as great
of erythrocytic
donors
marrow
of erythropoietic
entity
Epo”
the
called
such
large
was
cells
of erythropoietic
rise to erythrocytic
produced
by
as that
colonies
substantially
separated
burst-forming
peak
of erythrocytic
erythropoietic
burst
which
is responsible
new
for the formation
those
that gave
erythropoietic
from hypertransfused
in fractions
of bone
peak
have
AL.
groups
of
bursts.”3
The entities
responsible
to behave
differently
from
the
We
El
bursts
appeared
colonies.
Thus,
marrow
cells
produced
produced
lower
than
by unit gravity
activity
was
taken
“erythropoietic
clearly
burst-forming
concells
normal.
Moreover,
sedimentation,
the
separable
from
colony-forming
activity.
We therefore
concluded
is derived
from
an entity
distinct
from
the
for erythrocytic
colony
formation
and proposed
term
from
by cells from
by marrow
unit
that
the
that
the
CFU-E,”2
for this
responds
to
or BFU-E.3
In the
tion
present
contribution,
of BFU-E
from
we provide
CFU-E
in murine
further
bone
is presented
from
fraction-mixing
experiments
velocity
profiles
are the true profiles
of these
spurious
effects
of stimulator
or suppressor
show
that,
it has
murine
no
while
hypertransfusion
effect
marrow.
on
either
with
the
on
cell
the
physical
separa-
suspensions.
Evidence
that the observed
sedimentation
progenitors
and do not result
from
cells in bone
marrow.
We further
red
cells
sedimentation
MATERIALS
data
marrow
reduces
the
velocity
AND
or
number
number
of CFU-E,
of
BFU-E
in
METHODS
Mice
Eight-
to
ten-week-old
in the Division
male
C3Hf/BiUt
of Laboratory
mice
Animal
Science,
by
method
(descended
University
from
C3Hf/BiOci),
of Toronto,
were
bred
used
and
raised
in all experiments.
Hypertransfusion
Mice
were
packed
hypertransfused
red blood
Preparation
For
from
et
all
unit
to
bovine
Unit
Gravity
serum
were
Step
V)
used
the
was
out
100 ml
maining
of medium
contents
until
they
the
was
and
cells
(BSA)
Ct al.4
Injections
on each
a pool
a single
were
cell
of
of days
of
femoral
bone
suspension
resuspended
at a concentration
at 4’C
were
essentially
in
with
for
that
11.5
nonsterile
medium,
loaded
carried
(Coulter
Jacobson
administered
experiments,
pooled,
(diameter,
prepared
in
chamber
was
albumin
chamber
BSA
of
were
0.5
ml
of
0, 1, 2, and
washed,
6.
in
marrow
was
cells
prepared
supplemented
HMEM
of 4 x 106 nucleated
collected
as in
McLeod
containing
cells/mi.
Sedimentation
method
cylindrical
mice
sedimentation
mice
centrifugation,
0.33%
the
syngeneic
Suspensions
gravity
seven
After
The
from
of Cell
five
al.2
cells
of
with
Miller
sterilized
ml
3.5 hr. The
of
cell
chamber
by
HMEM
then
cultured.
Samples
of Canada
of each
Ltd.,
fraction
Mississauga,
by
One
(8 x l0
drained
The
apparatus
per
cent
dissolving
through
suspension
was
Phillips.5
base.
filtration
(i.e., volume
filling
the conical
of the chamber
were
collected
Electronics
and
a conical
supplemented
and
20
cm)
a
cells
at
a
were
taken
for
2%
powdered
O.45-
rate
total
and
of
BSA
The
sedimentation
15
ml/min.
nucleated
a
of
(Sigma,
filter.
was
The
was discarded.
and
maintained
and
of
solutions
Millipore
total)
base of the chamber)
in 15-mI
fractions,
Ont.).
consisted
and
first
The reat 4’C
cell
counts
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BFU.E
AND
Each
was
779
CFU-E
fraction
taken
were
was
to
remove
resuspended
and
medium,
however,
all
medium;
thus
at
medium
5 x 106
nucleated
at
cultured
for
fractions
or
culture
contained
nucleated
ml
10
the
mm
for
of
just
resulting
using
BFU-E,
to
the
were
portion
being
pellet.
In
In
in
and
care
experiments,
volume
below.
resuspended
ofa
cultured,
most
appropriate
as described
fractions
a constant
prior
cell
of
some
cells
collection
experiments,
a constant
volume
of
fraction.
Colonies
McLeod
cells/0.l
for
cells/mI,
pools
ofErythrocytic
of
g
from
and
each
method
160
CFU-E
cell
Cultivation
The
centrifuged
all excess
et al.2
culture
was
in
used.
medium
Cells
were
cultured
containing
0.25
of
nucleated
at
U/mI
a concentration
of
Epo
of
2.5
(Connaught,
x l0
Step
111)
for 2 days.
Cultivation
Cells
ofErythropoietic
were
cultured
creased
In
at
as used
composition
to
a concentration
for
erythrocytic
the
initial
experiments,
(containing
0.1
17-18
microcultures).
water,
for
7-9
and
0.1 U Epo
into
larger
from
Conn.).
The
NCTC-109
medium
containing
NCTC-l09
Fixation
the
and
Scoring
dried,
0.5
as isolated
nucleated
in
medium
concentration
in
large
addition
tray
incubated
petri
0.01
of
of
similar
Epo
was
in-
dishes
ml
and
time
clots
distributed
in
tray
containing
medium
0.1-mi
(yielding
a smaller
containing
of
cells
was
Linbro
(Disposo-Trays,
48 hr.
was
a microtitration
5%
dish
CO2
NCTC-l09
in
air
containing
96 hr.
containing
as above
of
atmosphere
of
medium
cells
wells
a humidified
at 24, 48, 72, and
of incubation,
M
with
benzidine-peroxide
fed
At
distributed
Chemical
by
the
72 and
addition
96
in 0.5-mI
Co.,
hr,
of
the
Inc.,
0.05
Haven,
of
medium
ml
cultures
aliquots
New
were
fed
fixed
with
with
phosphate
and
of
at
each
were
(pH
turned
7.0-7.2).
reagents
and
out
The
onto
slides
slides
were
counterstained
and
stained,
with
5%
after
Harris’
being
hematoxylin
Bursts
clot
was
nucleated
groups
buffer
et al.2
plasma
cells,
For
the
placed
in
the
culture
0.01
Colonies
entire
bursts.
the
containing
into
U Epo at 24 and
(0.05 mI/well).
only
in McLeod
benzidine-positive
were
were
appropriate
in
as described
The
cells/mi
that
Staining
glutaraldehyde
washed
by
clot
the
medium
37’C
fed
a titration
cultures
and
After
except
culture)
were
at
ofeach
experiments
wells
ml
cultures
were
to the surface
culture
Epo/0.l
incubated
Cultures
later
the
U
The
were
days.
In the
5 x l0
colonies,
I .0 U/mI.
aliquots
of
Bursts
least
small
some
experimental
scored
cells
colonies
of
for
were
or
which
point,
colonies
counted
as
were
10-12
of
or
as
bursts.
single
large
0.1-mi
groups
colonies.
colonies
benzidine-positive,
the
All
erythrocytic
of
eight
composed
were
cultures
or
or
Aggregates
of
scored
4-5
of
as
the
more
appearing
50
or
more
erythropoietic
0.5-mI
cultures
counted.
RESU
Appearance
of Erythropoietic
Mouse
bone
plasma
culture
marrow
medium
LTS
Bursts
cells
were
containing
plated
Epo at
at 5 x l0
1 U/mi,
and
additional
Epo.
Initially,
large numbers
of scattered
benzidine-positive
cells formed
in the cultures,
but
these colonies
had lysed and new groupings
of cells
of either
an isolated
group
of
large mass of cells (Fig.
1) and
considered
both
as representing
small
colonies
contained
up
erythropoietic
nucleated
subsequently
in
with
discrete
colonies
of 8-40
by 7 days
the majority
of
appeared.
Each
consisted
or a single,
to 1000 cells
bursts
cells/mi
fed
because
irregularly
or more.
shaped
We have
morphologically
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HEATH
780
*
El
AL.
I.
‘S.
--
-J
II”
0
4
,
8’
%5
S
-
j
C
‘
I:.
s,lp_-.
I.
S
I
50jm
‘.
B
Fig. 1.
t
#{149}IlL
Photomicrographs
of plasma
cultures showing
the appearance
I
of erythropoietic
bursts.
The cultures were harvested at 7 days and fixed and stained with benzidine-peroxide,
hematoxylin. (A) Three erythropoietic
bursts are visible. The burst on the left is composed of groups
of small colonies; the one in the upper right is more compact. (B) A single erythropoietic
burst
at higher
magnification.
Benzidine-positive
cells were
in the minority
in this burst.
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BFU-E
AND
781
CFU-E
z
6.0
0
0
‘0
c
5.0
4.0
0
0
0
I
0
I
CFU-E
62
mm/hr
BFU-E
RBC
3 7 mm/hr
2 1mm/hr
z
2000
UU-
w
15005
U-
U-
0
1000
Cell
Input
Z
500
Band
10
15
FRACTION
Fig. 2.
Unit gravity
in vitro. Fractions were
sedimentation
assayed
20
25
NUMBER
profiles of hemopoietic
cells that respond to erythropoietin
x
cells/0.1
ml culture for
at fixed cell concentrations:
2.5
erythrocytic colonies and 2.5 x 10 cells/0.5 ml culture for erythropoietic bursts. Horizontal
bars
indicate fractions pooled. The closed circles connected
by a solid line indicate
mean
numbers
of
CFU-E/fraction,
i.e., total number
of CFU-E per poo1 assayed at 2 days. The solid triangles connected
by a broken
line represent
mean numbers
of erythropoietic
bursts per fraction
at 7 days.
Vertical
bar indicates
intermediate
not
±
forms
distinguish
bone
marrow
duced
the
1 SE.
were
between
that gave
large
groups
demonstrable
this apparently
hemoglobin,
less mature
out
few
Epo:
very
bursts
not
the
uncommon
unit
in the
gravity
rise to the single
of small
colonies.
and often
type. No
developed
cultures
sedimentation
and
because
profiles
large cell masses
Bursts
contained
of the
were
not
could
in
and those
that
prosome
cells with
no
a majority
of the cells in the bursts
bursts
formed
if cultures
were set
if cultures
we
entities
fed with
additional
were of
up withEpo.
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782
HEATH
Table 1. Summary
of Modal
Sedimentation
Velocities
of CFU-E
El
AL.
and
BFU-E in Various Experiments
Modal
.
Exp.
Experimental
No.
Conditions
Se dimentation
Velocitie
RBC
CFU-E
s (mm/hr)
BFU-E
39
Standard
1.9
6.4
-
40
Standard
1.8
6.4
3.7
41
Standard
2.0
6.4
-
45
Standard
2.0
6.2
3.7
49
Mice
2.2
5.7
4.1
59
Standard
2.2
6.6
-
75
Standard
2.1
6.2
3.7
78
Standard
79
Lower
hypertransfused
than
standard
cell concentration
2.1
-
2.1
7.0
4.0
2.2
6.6
4.1
1.8-2.2
5.7-7.0
3.7-4.1
2.06
6.39
3.88
3.9
used
for erythropoietic
burst
culture
Constant
80
of each
portion
fraction
cultured;
variable
cell concentration
Range
Average
Effect
of Variation
ofEr,yihropoietic
The
that
relation
subsequently
in Number
Bursts
ofCells
between
the
developed
Plated
on Number
number
of cells
was nonlinear.
plated
Few
and the number
of bursts
no bursts
formed
at cell
or
concentrations
less than
l0 cells/0.5
ml culture,
and a fall-off
in burst-forming
efficiency
occurred
when
cells
were
plated
at concentrations
above
5 x l0cells/0.5
ml culture.
A cell concentration
of 2.5 x l0 nucleated
cells/0.5
ml
culture
was
found
formation,
and
fractions
for their
Unit
Gravity
Bone
in each
produce
ability
at 2 days
2 and
sedimentation
to form
bursts.
erythropoietic
1. The
velocity
ranged
oferythrocytic
sedimentation
formation
velocities;
a broad
had
and
red
consistently
was
Marrow
were
subjected
were assayed
experiments.
The majority
between
5.7
of erythropoietic
most
concentration
ofBone
and
Table
the
this
Sedimentation
marrow
cells
of the fractions
colonies
in Fig.
to
therefore
blood
Cell
at
cell
peak
was
sharply
1.8
and
2.2
developed
assay
7-9
sedimentation,
to produce
bursts
the sedimentation
peak,
with a modal
efficiencies
to
of
marrow
burst
cell
Suspensions
to unit gravity
for their
ability
between
colonies
high
used
days.
The
in cultures
profile
sedimentation
7.0 mm/hr
in different
experiments.
bursts
developed
in cultures
of more
for
and
cells
erythrocytic
results
are
defined;
its
mm/hr
in
of cells
modal
different
with
erythrocytic
velocity
that
In contrast,
the
slowly
sedimenting
shown
high
colony
ranged
majority
cells;
the sedimentation
profile
of these cells had a sharp
peak,
with a modal
sedimentation
velocity
that ranged
between
3.7 and 4.1 mm/hr
in different
experiments
and a small
shoulder
that
sloped
downward
toward
higher
sedimentation
ye-
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BFU-E
AND
CFU-E
783
Table
2.
Effect
Different
of Plating
Sedimentation
Cell Concentrations:
Velocity
Fractions
Experiment
No. of Erythropoietic
No. of Cells Plated
2.5x
1.25
locities.
the
This
10
0
12.4 ± 2.1
shoulder
CFU-E
Sedimentation
Concentrations
a consistent
of the high
ofthe
rapidly
burst-forming
jority
of the
x
numbers
20.8
corresponded
with
rapidly
The cells
in fraction
a small
peak
in
Fractions
no or very
velocity
i0
cells/0.5
few bursts
fractions
ml
of erythropoietic
thus
seemed
sedimenting
of fractions
culture,
could
of the
and
bursts
cells,
to
exist
cells.
1-8, although
13, proliferated
of these
which
in the
formed
in the
plated
at
produced
sheets
or
either
cell concentration.
cell concentration
did
not
In the
result
slowly
in an
be cultured
gradient.
half
by
that
each
next
velocity
laboratory.
from
A pool
cell
con-
were
then
determined
the
under
were
cells/0.5
In the
ml culture).
was
concentrations
tained
cells
resuspended
thus
of high
same
colonies,
did
not
stain
other
whether
of the
or to other
the
pH
of
the
is currently
in-
of cells
in
No.
80),
a
constant
each
under
capable
fraction
volume
of
the
also
were
in the
same
forming
of these
(1.25
or pool
of
(Exp.
x I0
of frac-
medium;
culture
to culture.
In those
fractions
velocity,
the total
cell counts
were
plated.
The results
for burst-forming
they
Most
benzidine.
inhibitory
effect
in the cultures
of
factors
velocities
(Exp.
culture
as those
density.
with
two sets of assay
conditions.
In one
plated
at a fixed low cell concentration
varied
from
sedimentation
and
cell
cells, there
was a noticeable
drop
in the
This did not occur
when
the cells of
cell concentration
or in fraction
13 at
sedimentation
for
contained
concentration
a higher
so only low cell concentrations
were
The modal
sedimentation
velocities
experiments,
that
sedimenting
fraction
13, reduction
in the
increase
in the number
of erythropoietic
fractions
erythropoietic
bursts
No. 79), all fractions
suspension
to reach
bursts
that
were
produced.
The question
of
high cell concentrations
is due to a lowering
high sedimentation
vestigation
in this
cell
at the
cultures
Concomitantly
with the growth
of these
pH ofthe
cultures
(phenol
red indicator).
fractions
1-8 were
plated
at the lower
two
± 4.1
From
the results
in Table
2, it can be seen that the pool of rapidly
fractions
1-8 gave rise to no bursts
when
plated
at the cell concen2.5 x l0 cells/0.5
ml culture.
However,
when
the cell suspension
to 1.25 x l0 cells/0.5
ml culture,
bursts
developed.
Some
in-
influence
hibitory
tions
13
28.0±2.3
profile
Velocity
finding
that
sedimentation
of 2.5
The
determined.
sedimenting
tration
of
was diluted
We
Fraction
sedimenting
fractions
1-8 (previously
shown
to have
little or no
activity)
and the slowly
sedimenting
fraction
13 (with
the maburst-forming
activity)
were
separately
plated
at the
routine
concentration
centration.
the
BFU-E
ml Culture
profile.
It was
cells
cell
in the
Bursts/0.5
1-8
x i#{248}
Effect
ofPlating
at Dtfferent
Cell
the
Fractions
at
No. 78
that
low,
are shown
in Table
activity
were
similar
range
as
those
found
cell
conand
1.
in
in
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
784
HEAIH
earlier
experiments.
entities
responsible
Thus,
the
for
modal
even when
the fractions
were
hibitory
effect
was eliminated.
concentrations
differed
slightly
at higher
cell
forming
fractio
n
burst
in that
could
be
at
detected
low
in
Fractions
Obtained
at Unit Gravity
Two experiments
cells were fractionated
different
cell
from
fractions
activity;
C were
and
bone
dium
and
at
for
(up
cells
then
to
6.25-25.0
2.4
x
l0
CFU-E
4.7-fold
was
ber of CFU-E
the fractions,
sents
a CFU-E
recovered,
was 1.4
recovery
total
in
3.5
1.3
x
l0
number
x
i0
BFU-E
enrichment
and
of erythropoietic
thus,
no reliable
from
results
low
ml
at day
experiment
which
had
x
realized
the
culture.
8. As
bone
marrow
way, and three
The
control,
cultures
a sample
provided
a measure
been loaded
into the
nucleated
cells.
in 2.9
x
In
the
gravity
marrow
loaded
cells.
In
were
of the
me-
bursts
figure
106
nucleated
into
the
these data.
in Fig. 3 show
cell
that
concentrations
the
7.2 x
of ap-
The
num-
4#{176}C
without
the
separation
BFU-E
cells.
The
sedimentation
fraction,
total
for the
fact
the number
recovery
of
of
erythropoietic
in all three
suspensions,
or
that
was
was
5.2
there
were
of
BFU-E
number
from
all
represent
to
efficiency
total
chamber
peak
except
proportional
enrichment
sedimenta-
there
were
enrichment
sedimentation.
at
of BFU-E,
was not
for either
pooled
surviving
cham-
together
the numbers
from
all
l0 nucleated
cells;
this
repreexperiment,
it was shown
that
by adding
together
the numbers
x l0 nucleated
cells.
This
would
an 8l#{176}
recovery
of the
separation
CFU-E
concentration,
cells.
Thus
a CFU-E
by unit
original
of BFU-E
at low
l0
the peak
nucleated
nucleated
calculated
x l0 in 3.2
was
in which
in the usual
burst-forming
activity;
(C)
unbone
marrow
cell suspension
kept
In Exp.
83, cells of suspension
A
culture,
and cells of suspensions
B
calculated
by adding
x l0 CFU-E
in 3.2 x
of 57#{176}c.
In a separate
of CFU-E
from
order
of 50#{176},,.
l0
obtained
The
burstvelocity
in a tube to sediment
at 4#{176}C
in the same
assayed
at the same
time
as the sedimentation
in 3.5
proximately
recovered,
was 4.2
some
10 hr).
included
x 106
The
the inlow cell
assayed
left
and
that
in 2.2
loss
the
same,
of
10” cells/0.5
bursts
was
mixed
tion fractions
l0 CFU-E
tion
x
data
obtained
in this
of CFU-E
in marrow
in the
where
at
were
sedimentation
by Sedimentation
erythropoietic
marrow
was
fractions
the
the
concentrations
higher
(Nos.
83 and 84) were
performed
by unit gravity
sedimentation
plated
scored
same
ber:
for
remained
suspensions
were prepared:
(A) a pool
of rapidly
sedimenting
cells
1-8, which
were expected
to possess
little
or no burst-forming
a pool of more
slowly
sedimenting
cells from
fractions
13 and 14,
(B)
The
number
cell
the
expected
to include
the peak
erythropoietic
fractionated
cells from
the same
original
at 4’C during
the sedimentation
procedure.
were plated
at 3.1-12.5
x l0 cells/0.5
ml
fed
obtained
AL.
.
Effect
ofMixing
Marrow
Cells
and
velocities
formation
assayed
at low cell concentration
The sedimentation
profiles
obtained
from
those
in which
the fractions
concentrations,
activity
sedimentation
erythropoietic
El
x
the fractions,
a threefold
the
number
of cells
plated:
BFU-E
could
be
burst
and
formaincreased
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
BFU-E
AND
785
CFU-E
#{149}
Suspensan
#{149}
Suspension
‘Suspenscn
A
B
C
0
U)
I-
I
Fig. 3.
Titrations
unfractionated
bone
sions for
poietic
fractions
their
of fractionated
and
marrow
cell suspen-
ability
to form
erythro-
Suspension
A-a
pool of
expected
to contain
little
erythropoietic
burst-forming
activity
and
including
most
CFU-E
responsible
for
erythrocytic
colony
formation.
Suspension
B-a
pool of those fractions
expected
to
contain
the major part of the erythropoietic burst-forming
activity.
Suspension
Cunfractionated
cell suspension.
with
bursts.
1-8,
increasing
concentration
unfractionated
again
seen
contained
cells
in the
the
(C).
the
suspension.
of the suspensions
erythropoietic
bursts
bers
expected
Note
that
pected
cultures,
Results
produced
numbers
sion
A or
suspension
However,
cells
(A).
were
the
by
B. A mild
when
cells
from
(B)
and
Suspension
activity,
plated
either
mixtures
each
alone
suspension
B, which
a titra-
suspension
sedimentacapacity
or
mixed
in
to-
alone
burst
B alone
with
the
num-
and additively.
of bursts
than
exwere
cells (5000
to cultures
of
the
was
yielded
compared
are
suspension
augmentation
of
of erythropoietic
bursts
the observed
numbers
of
had acted
independently
gave higher
numbers
bone
marrow
C were added
#{176}
concentration
of the whole
unfractionated
cell
cells separable
by unit gravity
of erythropoietic
burst-forming
by
irradiated
suspension
cell
burst-forming
in each pooi
the mixtures
produced
one experiment,
heavily
from the unfractionated
observed.
cells
higher
and the effect
on the number
are shown
in Table
3, where
if the cells
in all instances
if the
of
sedimenting
the
OF CELLS PLATED
sedimenting
effect
of erythropoietic
in shape
to that
that no inhibitory
the expression
gether
in the
was determined.
slowly
inhibitory
curve
similar
C. This suggested
tion were affecting
unfractionated
Cells
from
each
NUMBER
of rapidly
cultures
majority
tion
of
An
25
12.5
formation
and
additive.
rads
3l5
of either
from
In
“y-rays)
suspenwas
suspension
again
C
alone
were
cultured
at twice
the usual
concentration
(i.e.,
5 x 105/mI),
the
number
of bursts
which
subsequently
formed
also more
than
doubled,
as was
already
noted
in the titrations
depicted
in Fig. 3. It was thus evident
that,
while
the efficiencies
of erythropoietic
burst
formation
were
cell
concentration-
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
HEATH
786
Table
Cells of Different Sedimentation
3. Effect of Mixing
on Erythropoietic
Rapidly
Sedimenting
(1-8)
83
6.2
Velocities
Unfractionated
-
-
-
2.0
-
-
-
3.0±1.0
-
-
13.8±2.2
-
-
38.6±2.5
-
1.25x105
1.25x105
-
2.5
6.2
84
1.25
x
x
i0
1.25x105
-
2.5
1.7±0.6
-
-
36.8±2.6
-
4.8
10
-
2.5
x105
x
-
x
1.25
10
x
6.2
10
-
15.8 ± 3.0
2.7
-
± 0.6
-
24.0±1.8
-
-
-
-
± 0.5
0.8
-
6.2
-
-
±
-
x
x 10
40.8
-
1.25
1.25
10
-
-
x
x
-
-
6.2
Expected
0.8
38.2±4.4
1.88
x 10
±
7.8±1.6
-
10
-
-
-
x 10
2.5
Observed
5
1.25x10
-
x
Mixture
x i0
-
6.2
.
No. of Erythropoietic
Bursts per Culture
Slowly
Sedimenting
(13. 14)
x 10
AL.
Burst Formation
No. of Cells Pla ted per Culture
Exp.
No.
El
1.88
x
10
31.7
±
2.8
± 0.8
2.5
1.25
x 10
1.88
x 10
20.8
±
1.3
5.6 ± 0.8
1.25
x 10
2.50
x 10
32.2
±
1.3
6.5 ± 0.8
1.25
x 10
1.88
x 10
3.0 ± 0.6
0.8 ± 0.5
x 10
7.8 ± 1.2
1.7 ± 0.6
(irradiated)
x 10
1.25
-
1.25
x 10
2.50
(irradiated)
dependent,
cells
the
at
respective
Effect
effects
increased
of cell
of their
We
nonspecific
in the
erythropoietic
burst
sense
that
formation,
ir-
source.
have
previously
the
shown
of BFU-E
mice
and
treated
of
similar
that
in the
of marrow,
at unit gravity,
CFU-E.
same
CFU-E
and
to those
ofCFU-E
The
way.
normal
dependent
per
fraction
nor
the
of mice
were
number
BFU-E
Although
done
in Fig.
the
reduced
mice
was
cells
were
a pool
4, the
marrow
from
of
this
found
to be similar.
of marrow
BFU-E
days,
Thus
was
blood
from
cells
to
normal.
upon
was
made
absolute
neither
the
conveloci-
mice
The
un-
normal
sedimentation
hypertransfused
marrow.
the
affected
marrow
modal
normal
comparison
red
from
hypertransfused
assayed
for their
of
as compared
was superimposable
on different
with
of BFU-E
of marrow
affected
the sedimentation
marrow
fractions
control
from
and
mice.
separations
bone
and
As seen
BFU-E
of CFU-E
was substantially
BFU-E
from
hypertransfused
file from
hypertransfusion
number
ofCFU-E
but leaves
the number
To determine
whether
hypertransfusion
velocity
of the BFU-E
mice were sedimented
ties
were
facilitated
of Hvpertransfusion
reduces
changed.3
tent
concentration
concentration
total
were
number
The profile
the BFU-E
between
for
pro-
two
numbers
of BFU-E
sedimentation
velocity
in-
by hypertransfusion.
DISCUSSION
The
sedimented
erythrocytic
with
colony-forming
a modal
velocity
activity
ranging
of
C3H/Bi
from
5.7
mouse
to
7.0
mm/hr,
bone
marrow
while
the
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
BFU-E
AND
787
CFU-E
CFUE
5 7-6.0
mm/hr
BFUE
RBC
37-40
20-22
mm/hr
mm/hr
I
I
8000
z
0
I0
6000
z
UJ
0
0
a
0
400
4000
Cell
Input
Band
2000
z
LU
0
A
0
5
10
15
FRACTION
20
o
25
z
NUMBER
8000-
6000-
z
4000-
4O0
Cell
Input
2000
--- -
.----
B
0
5
10
15
FRACTION
Fig. 4.
A comparison
of the sedimentation
hypertransfused
(B) mice. Solid line: colonies
ture. Broken line: erythropoietic
bursts formed
modal
sedimentation
tween
3.7 and
entities,
velocity
4.1
separable
However,
mm/hr.
on
before
the
this
other
explanations
Worton
et al.6
or inhibit
difference
basis
of size,
could
for the observed
have pointed
out
colony
assay
cells
number
tation
velocity.
profiles
of CFU-E and BFU-E from normal
per fraction
assayed
at 2.5 x 1O cells/O.1
per fraction
assayed
at 5.0 x iO cells/O.1
burst-forming
suggested
were
be
that
responsible
drawn
with
might
for
plated.
in our
When
An
inhibitory
cultures
from
the
concentration
effect
on
burst
containing
of these
cells
be-
different
two
activities.
a number
of cells
of
is frac-
different,
and this
Cells that stimu-
in particular
of the true profiles
This factor
could
fractions
the
to be considered.
a population
be concentrated
lay
distinctly
confidence,
of cells in each fraction
may become
of assays
based
on colony
formation.
formation
(A) and
ml culml.
activity
two
fractions,
of those
entities
that
be especially
relevant
of erythropoietic
bursts
from
fractionated
because
the number
of bursts
was not directly
of cells
demonstrated
0
25
NUMBER
profiles
had
that whenever
and thus interfere
with determination
were actually
giving
rise to the colonies.
to the
marrow
2
Hb00
20
of erythropoietic
This
conclusion
tionated,
the spectrum
could
affect the efficiency
late
UI
a
Band-200
-
populations
proportional
formation
cells
was
was,
of higher
reduced
of
in
bone
to the
fact,
sedimenin the
cul-
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
788
HEATH
tures,
however,
modal
the
inhibitory
sedimentation
same
as in earlier
Similarly,
experiments
the
represent
with low
with
sedimentation
also
observed
were
mixed
when
a stimulatory
burst
ofburst
formation
tions
of both
addition
profile
tion
These
for
“BFU-E,”
the
entity
BFU-E
is the
to
would
be
tween
BFU-E
for
cell
cells)
erythropoietic
the
formation
and
CFU-E
Three
facts argue
cultured
from
colonies
that
tion
could
the
would
of
cultured
the
greatest
Finally,
below,
present
it should
conclude
progeny
fewer
and
that
cells
that
we
a single
initial
dose
of Epo
the
expect
a
same
that
to
be
to
taken
artifact
an
experiments
exceeded
and
dif-
interaction
beof
the
the number
number
of
the
if the
BFU-E
BFU-E
peak
formation
cell
BFU-E
or
that
possibility
Second,
burst
of
Epo,”
mean
replicates
the
the
burst
distribu-
an
responds
from
if the
true
merely
self-replication
on burst
formation
There
appeared
cells when
they
that
on
sedimentation
the
from
fraction.
titra-
that
another
result
cells
in
observed
sustained
of
efficiency
and
in culture,
in stimulating
be noted
that
effect
However,
the unfractionated
did cells from
the
would
experiments
is itself
recognize
Recent
work
in other
tions.
Iscove
and Sieber,7
plated
from
numbers
when
fracadded
suspension
contained
peak
fraction.
How-
by cells from the BFU-E
to be as much
“stimulawere mixed
with unfrac-
BFU-E
were
merely
a stim-
expect
the efficiency
of burst
formation
to decrease
cells from hypertransfused
animals,
because,
as dis-
CFU-E
previous3
the BFU-E
that
could
burst.
from
effect
ulator
of CFU-E
one would
in cultures
of bone marrow
cussed
unit
might
resulted
concentrations
not
term
that
supported
greater
represented
was
was
velocity
populations,
the
a
effect
that
stimulatory
and
induces
would
cells because
of CFU-E
than
cells.
when
cell
that
cells
in cul-
velocity,
observation
low
mild
The
a burst
no specific
stimulatory
action
fractions
could
be demonstrated.
by the more rapidly
sedimenting
tionated
the
that,
of
against
this. First,
in most
the BFU-E
peak
fraction
one
have
stimulation
cell
gradient
which
CFU-E,
to unfractionated
higher
numbers
ever,
peak
tion”
be
the
activity
entity.3
an
CFU-E.
of bursts
stimulated
this
actually
be concluded
fractions
the
at
a
not
the
in certain
could
sedimentation
presence
indicated
(or
create
that
than
burst-forming
is proposed
be
taken.3
stimulatory
in the
by
had
in the
system.
“erythropoietic
ferentiates
high
strongly
been
sedimentation
high
that
of the
burst-forming
of a burst-forming
of
unfractionated
cells
findings
erythropoietic
the experimental
The term
appear
at
irradiated
high
it could
supported
and
not
the
to
concentrated
a similar
AL.
conditions,
found
activity
Therefore,
effect
greater
had
were
concentrated
was
fractionated
heavily
formation.
view
was
the
of
was
This
of
of cells
protective,
cultures.
cells
However,
It would
perhaps
in the
with
cells.
population
was
burst-forming
concentrations
formation.
a nonspecific,
cells
low
these
cell. When
the fractions
containing
(about
3.9 mm/hr)
were
mixed
noted.
unfractionated
cell
maximum
or
in fact,
Under
activity
precaution
cells
for
stimulator
velocity
cells
was,
with
this
stimulatory
profile
unfractionated
effect
burst-forming
in which
of
the profile
of the
modal
sedimentation
stimulatory
disappeared.
for
if a population
fractions,
ture
effect
velocity
El
be
available
for
have shown
the entity
which
in culture
as an
stimulation.
erythropoietic
laboratories
has confirmed
and
using
methylcellulose
cultures,
at high
concentrations
The
results
that this did not occur.
gives
rise to the mass
of
We
of
burst.
extended
our observahave shown
that with
(3 U/mi),
burst-like
colonies
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
BFU-E
AND
CFU-E
developed
(up
789
in
to
l0
relatively
cells)
maker
et al.8
sponse
of CFU-E
The
at
have
cell
Sieber,
required
found
and
increase
marrow
large
10 days
and
With
distinct
of
reached
the
differences
BFU-E
in efficiency
number
numbers
of culture.
in mice
exposed
in our
raises
the question
for the expression
the
burst
kinetics
with
as in those
that with improved
culture
conditions,
marrow
cell numbers,
making
such
necessary.
This
of
formation
is necessarily
determine
whether
A small
which
or not
peak
was
corresponded
5 mm/hr
(Figs.
mouse
seen
in
or
small
4).
velocity
been
its
II) from
at
unit
marrow.
were
be actively
strongly
their
mm/hr,
at
represented
rather
of fraction
I in the
with
believed
mature
rat
BFU-E
a bovine
slightly
were
serum
2
days
or
past
separate
either
in
of
two
burst
in the medium.
could
have
of
another.
Epo
cell
by
Epo
the
Further
fraction
to
be
has
a
BFU-E
work
populations
will
with
by
cells.
be comparable
These
with
in that
considered
to
of
hemoglobin
latter
features
CFU-E.
I and
about
velocities
their
The
tar-
al)#{176}used
heme-synthesizing
were
sedimentation
Epo.
one
et
velocity
cells
nonproliferating;
stimulated
than
(fractions
produce
these
II,
more
McCool
sedimentation
to
vinbiastine,
density
around
exists
erythropoietic
system.
I, with
of
detected
to
CFU-E
at
out
component
that
two
offraction
albumin
of
there
of Epo
population
to one
required
profile
that
carrying
is
in
burst
be
profile
of
hemopoietic
erythroid
may
will
possibility
a minority
in the
to
and
possibilities.
stimulated
Cells
only
the
identical
sensitivity
were
most
of
increasing
of Iscove
erythropoietic
BFU-E
concentration
of the CFU-E
these
to
Cells
proliferating.
was
With
gravity
that
in the
at
the
re-
burst
numbers
a hypothesis
un-
sedimentation
peak
capable
mammalian
rat
From
parable
being
suggested
in the
the
raises
that
between
imply
experiments
formation
to
two
repeatedly
mm/hr,
progeny.
the
action
adult
l.3-3.9
population
to distinguish
sedimentation
thesis
cell
similar
without
It has
6.6
finding
colony
necessary
for
This
at 8 days,
depending
on
a minority
component
population,
get
is the case.
a minority
sedimentation
be
that
not
direct
repeatedly
functions-erythrocytic
formation
Alternatively,
does
more
to a shoulder
2 and
marrow
course,
clonal:
the
interaction
between
two cells
capacity.
However,
recent
work
our laboratory9
indicates
become
proportional
to
finding,
Wageof
hypoxia.
formation
as well
not
system,
to intermittent
experiments,
of whether
or
of burst-forming
dimensions
culture
between
of erythropoietic
seen
macroscopic
same
must
synthus
suggested
However,
have
that
cells
no cells
com-
study.
gradient,
Clissold”
recently
separated
two classes
of Epo-responsive
cells in anemic
rabbit
marrow
depleted
of hemoglobin-containing
cells by immune
lysis. Cells of the two classes
differed
with
respect
to their size and the time course
of their response
to Epo in vitro,
which
was measured
in terms
of 59Fe incorporation
into heme.
However,
only one of
the two
classes
it difficult
Stohlman
erythropoiesis
committed
was
detected
in similarly
erythroid
cells
(previously
cally recognizable
proerythroblasts
ferentiated
erythroid
cells from
erythroblasts
treated
normal
rabbit
marrow,
making
to relate
her results
to those
in the present
work.
et al.,’2 on the basis
of experiments
in vivo, proposed
a model
in which
Epo acts not only
on morphologically
unrecognizable
(to
increase
their
shown
to
under
its
proerythroblasts
rate
of hemoglobin
differentiate
influence’3),
to early
synthesis).
into
of
morphologi-
but also on the
polychromatophilic
A similar
dif-
model
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790
HEATH
has
been
the
responses
used
to
account
to
Epo
differences
at
to
sensitivity
and
They
the
observed
48
greater
between
(“late”)
hr
after
intensity
of
response
ad-
administered
in fractionated
doses,
as opposed
to a single
dose.’5’6
the notion
that there may be more than one class ofcells
within
the
of Epo-responsive,
committed,
but morphologically
unrecognizelements
of mammalian
marrow
seems
to have
originated
with
co-workers.’7”8
explain
at
to Epo when
However,
compartment
able erythroid
his
and
drug
(“early”)
of
and
hormone,’4
in
24
AL.
ministration
Reissmann
the
for
measured
El
showed
that
fan was given
to hypertransfused
mice erythropoiesis
several
days but afterward
returned.
Regeneration
siveness
to
Epo
were
to the
Epo
dose
Epo
had
sive
cell
cells
was
An
age
also
their progenitors
In all these
from
and
pended
not
an
at the
appeared
to
the
the
assay
of cells
behavior
method
stem
signal
of
cells
Epo-responsive
at
the
com-
proliferation
cells
themselves.
dif-
cell
descendants
to
postulated
through
differentiated
incorporation
changes
was
increased
stem
proerythro-
which
Epo-responsive
radioiron
sensitive
into
cells
Epo,
for
that
Epo-responerythroid
stem
the
had
to
de-
formed
level,
red
rather
than
level.
present
data,
entities
obtained
have
by direct
distinctive
lower
modal
sedimentation
CFU-E.
They
also have
quiring
a higher
the CFU-E
properties
in-
these
they
newly
population
be
of
Furthermore,
into
of
assay
at the
cellular
level
in vitro,
pro-
vide further
evidence
in favor
of the concept
of two different
Epo-responsive
cell populations
within
the compartment
of committed
but morphologically
unrecognizable
erythroid
cells of mammalian
bone marrow.
The data show
these
of
related
experiments
cells
agent,
from
of the
Epo-responsive
of
same
the
these
busul-
doses
was
ofcommitted
these
erythroid
the
provide
large
recovery
from
of
inhibited
for
for respon-
unrecognizable)
committed
this compartment.
the properties
on
on
concluded
of
outflow
measurements
cellular
The
the
ofthe
ofproliferation
Thus
dose
totally
capacity
Relatively
magnitude
transformation
responses.
within
instances,
responsive.
the
(morphologically
ofthe
two
studies
on
cells,
the
increased
partment,
cells,
of
distribution
for
ferentiation
the
stimulation
stimulation
Epo
and
It was
within
compartment:
blasts.
to be
effect,
administered.
actions
to account
found
for this
of Epo
two
and
ferred
itself
required
if a single
was
of the
physical
velocity
distinctive
concentration
the capacity
in culture
the
(and therefore
smaller
physiologic
characteristics,
of Epo
for erythrocytic
that
are compatible
(1) BFU-E
has
period
of time
characteristics,
colony
with
for
erythropoietic
formation.
its being
to produce
than
CFU-E.
BFU-E
diameter5)
the
burst
than
BFU-E
formation
The
BFU-E
the progenitor
that
having
a
the
rethan
also
has several
of the CFU-E.
more
numerous
progeny
(2) Each
erythropoietic
over
burst
a longer
is com-
posed
of groups
of colonies
which
morphologically
resemble
erythrocytic
colonies that arise
from
CFU-E.
This situation
is what
would
be expected
if each
BFU-E
produced
a number
of CFU-E.
(3) Although
precise
estimates
of frequency
await the development
of a more
reliable
quantitative
assay
method
for
BFU-E,
there appear
to be many
more
CFU-E
than BFU-E
in the normal
marrow
and
spleen
ber
of CFU-E,
the
number
of the
mouse.
in agreement
or modal
(4)
with
sedimentation
Hypertransfusion
previous
velocity
markedly
findings,
of BFU-E.
but
has
Finally,
reduces
no
the
num-
on
either
effect
we have
shown
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
BFU-E
AND
791
CFU-E
that, when
marrow
cells
of high Epo concentration
bursts,
new
capacity
CFU-E
of the
from hypertransfused
which
would
favor
arise
in
trypsinized
concentration
in
the
secondary
the absence
of Epo
vivo or in vitro.
In conclusion,
the
rise
basis
these
could
to produce
culture.3
to give
on
cultures;
cultures
mice are grown
the production
the
demonstrated
erythrocytic
Thus
BFU-E
to CFU-E
of
be
available
later
we
Epo
available
to
believe
the
at low
remain
exposed
data,
by
colonies
could
when
under
conditions
of erythropoietic
Epo,
that
in
either
what
in
has
in
the past
been
termed
the (morphologically
unrecognizable)
“erythropoietinresponsive
cell” compartment
of mammalian
hemopoietic
tissue
may be considered
to be composed
of at least two Epo-responsive
subcompartments,
each
of which
can now
would
correspond
suggested
progenitor,
be assayed
separately
in vitro.
In this model,
to the immediate
precursor
of proerythroblasts,
by Gregory
et al.’9’2#{176}
The
the committed
erythroid
this hypothesis
BFU-E
and
i.e., whether
to a clone
BFU-E
stem
would
represent
cell. While
the
is strong,
direct
experiments
will
CFU-E
stand
in progenitor-progeny
the BFU-E
spontaneously
or under
of CFU-E,
and
if the
BFU-E
its Epo-responsive
evidence
in favor
be necessary
relationship
the influence
is capable
the CFU-E
as has been
of
to establish
if the
to one another,
of Epo gives
rise
of self-renewal.
ACKNOWLEDGMENT
We acknowledge
with
thanks
the technical
assistance
of Mr.
Bruce
Smith.
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1976 47: 777-792
Separation of the erythropoietin-responsive progenitors BFU-E and CFU-E
in mouse bone marrow by unit gravity sedimentation
DS Heath, AA Axelrad, DL McLeod and MM Shreeve
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