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Identification
Resembling
By K. A. DiCke.
of Cells
in Primate
the Hemopoietic
in the
Mouse
M. J. van
Noord.
B. Maat.
The colony-forming
unit culture
(CFU-C)
in the thin-layer
agar colony
technique
is
considered
to be representative
for hemopoietic stem cells (HSC),
according
to our
studies
in mouse and monkey
bone marrow. Using this in vitro assay as a guide,
stem cell concentrates
were prepared
from
monkey
and human
bone marrow
by repeated density gradient
centnfugation.
The
number of CFU-C
could be enriched
up to
70-100-fold.
In such concentrated
CFU-C
suspensions,
a cell, morphologically
identical with the hemopoietic
stem cell in the
mouse (MSCLC,
mouse stem cell-like
cell)
was frequently
observed,
using a May-
M
ANY
cell
ATTEMPTS
have
(HSC)
by morphologic
to enumerate
this cell
as well as in the field
been
Bone
Stem
U. W. Schaefer.
and
Marrow
Cell
D. W. van
Gr#{252}nwald-Giemsa (MGG)
and electron
microscope
MGG-stained
preparations,
superficially
resembles
Bekkum
staining
method
techniques.
In
the
MSCLC
the
small
lympho-
therefore,
a staining
method
has
been described,
the polychrome
procedure,
by which
both cell populations
could be
clearly
distinguished.
Since
a fair correlation
exists
between
the
number
of
MSCLC
and the number
of CFU-C
in a
variety
of primate
hemopoietic
suspensions, we concluded
that the MSCLC
might
be a good candidate
for being the HSC in
monkeys
and man.
cyte;
reported
criteria.”3
to identify
the
The possibility
is of obvious
importance
in many
of bone
marrow
transplantation,
hemopoietic
to recognize
hematologic
since
by
stem
and
disorders,
extrapolation
from
lines
extensive
mouse
data
it is held to generate
all different
hemopoietic
cell
including
the lymphoid
line. None
of the above-mentioned
investigations,
which were carried
out in rodents,
has led to a convincing
identification,
since
quantitative
sults
correlations
of functional
HSC
tered in its identification
highest
concentration
between
tests
was
of
numbers
were
not
its low
HSC
as
of candidate
provided.
The
concentration
calculated
for
stem
greatest
cells
and
difficulty
in hemopoietic
mouse
bone
the
re-
encountissues.
marrow
The
is
0.4%-0.6%,
which
percentage
has been
estimated
by the spleen
colony
assay
of Till and McCulloch.4
Spleen
colonies
arising
from
a single
cell have
been
shown
to produce
erythropoietic,
myelopoietic,
and thrombopoietic
cell populations.4
Moreover,
Trentin
and
Fahlberg5
demonstrated
that
in lethally
irradiated
lymphatic
From
mice complete
organs,
can
repopulation
be effectuated
the Radiobiological
Institute
R:jswijk (ZH).
Submitted
accepted
TNO
of the hemopoietic
tissue,
by descendants
from one
and
Institute for
Experimental
as well as of the
spleen
colony.
Gerontology
TNO,
The Netherlands.
September
January
29. /972; first revision December
5, 1973.
M.D.:
Radiobiological
3, /972;
second
revision January
2. 1973;
K. A. Dicke,
Institute
TNO, R:jsw:jk (ZH),
The Netherlands.
M.
Institute for Experimental
Gerontology
TNO, Rijswijk
(ZH),
The Netherlands.
B.
M.D.: Radiobiological
Institute
TNO, Rzjsw:jk (ZH),
The Netherlands.
U.
W. Schaefer,
Department
of Internal Medicine (Tumor Research). Clinicum Essen. Ruhruniversit#{228}t Bochem,
Germany.
D. W. van Bekkum,
M.D.:
Director,
Radiobiological
Institute
TNO,
Rijsw:jk
The Netherlands.
© /973 by Grune & Stratton,
Inc.
Noord:
Blood, Vol. 42. No. 2 (August).
1973
J. van
Maat,
M.D.:
Essen.
(ZH).
195
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196
DICKE
Recently,
using
an adapted
May-Grunwald-Giemsa
(MGG)
staining
ET AL
method
and electron
microscopic
(EM)
techniques,
we defined
the morphologic
characteristics
of a candidate
stem
cell in highly
enriched
stem
cell preparations.6
Such stem cell concentrates
were prepared
from
mouse
bone
marrow
by treating the donors
with a vinblastine-nitrogen
mustard
regimen
and by subjecting
the marrow
suspensions
to repeated
density
separation
over
albumin
gradients.”7
These
morphologic
criteria
have
been
applied
to enumerate
candidate
stem cells in preparations
varying
over a 100-fold
range
in their
CFU-content,
and a fair correlation
between
the two entities
was found.
From
these
studies
became
evident
tion of ultrathin
that
cross
it
criteria
determined
differentiated
the
MGG
all
magnification
cells could
cells allowed
In the primate,
general
by
from
to the high
ture of the
the morphologic
characteristics,
sections
by EM,
were
much
the
in that
cell
especially
types,
used in EM
be distinguished
a distinction
the existence
properties
preparations,
other
of the
studies
better;
as defined
by examinamore
exclusive
than
the
the
HSC
could
from
(l7,000x),
in addition,
be
the
system
do not
differ
Due
subcellular
struccross
sectioning
of
of the subcellular
organdIes.
of a HSC
is a logical
assumption,
hemopoietic
clearly
lymphocytes.
because
essentially
from
the
those
of rodents.
Strong
support
for this concept
would
be the finding
of cells with
similar
morphologic
properties
as the HSC in the mouse.
The frequency
of this
cell population
in the bone
marrow
of primates
is likely
to be less than
1% if
comparable
tion without
In the
concentrations
exist
prior concentration
present
study,
as in mouse
bone
would
be virtually
monkey,
as well
as
marrow,
impossible.
human,
bone
so that
their
marrow
detec-
suspensions
have
been
fractionated
by albumin
density
centrifugation.
To isolate
highly
enriched
stem
cell fractions,
the discontinuous
gradient
method
previously
developed
for primate
bone
marrow
with
the purpose
of separating
immunocompetent
cells from
HSC8’9
had to be adapted
to our recent
program.
For
this adaptation,
a quantitative
test system
for HSC was required
as a monitor.
In the primate
such an HSC assay
obviously
has to be an in vitro
method,
and extensive
showed
that
studies
in mice
and
the CFU-C
(CFU-C,
form
form
agar
tative
ing cells culture:
cells capable
of forming
colonies
culture
method,
developed
by us specifically
for
measure
for the HSC population.
paper,
tions
enriched
morphologically
in agar;
it will
in
these
be shown
cells
that
are
were
unit
ally
In this
colonies
monkeys
that
colony-forming
a subclass
in monkey
CFU-C,
high
concentrations
similar
to the mouse
HSC.
reported
culture:
and
of
In such
of
the
elsewhere’#{176}’2
cells that actuCFC-C,
colony-
in agar)
in the thin-layer
this purpose,
is a quantihuman
bone
marrow
cells
are
found
purified
fractions,
fracthat
are
quanti-
tative
morphologic
studies
using
the light
microscope
(LM),
as well
as the
electron
microscope,
revealed
a strict
correlation
between
the mouse
stem celllike cell (MSCLC)
and the number
of the CFU-C.
Moreover,
a polychromatic
staining
method
will be described,
by which
cross
sections
of cells of the hemopoietic
suspensions
the visability
of the
can be stained
and
subcellular
structures
examined
under
considerably.
LM,
which
increased
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IDENTIFICATION
OF
CELLS
IN
BONE
MARROW
197
MATERIALS
The
CFU-C
The
methods
short,
AND
METHODS
Assay
thin-layer
developed
bone
agar
technique’#{176}”3”4
is
by Pluznik
and Sachs’5
marrow
cells
are
cultured
in
based
and
on
agar
by Bradley
plastic
Falcon
colony
formation
and
Metcalf.’6
Petri
bottom
of which
0.5 x 106 embryonic
fibroblasts
are layered
case mouse
marrow
suspensions
are cultured.
When
monkey
kidney
tubules
were used
fibroblasts,
On
top
of the
Dulbecco’s
24 hr.
of adult
as feeder
kidney
feeder,
top
monkeys
In humans
from
adult
4.5
modified
the
rhesus
system.
tubules
ml
of
Eagle’s
layer,
a mixture
the
of
and
of 0.25%
bone
marrow
agar
agar
+
of the
dishes
ploying
0.5% agar medium.
The total
volume
of the top layer
was 0.2 ml. The
were kept
in 7%-lO%
CO2 in a gas-controlled
incubator
at 37#{176}C,ema 100% humidified
atmosphere,
for 7-10
days.
After
that
period,
dis-
Gradient
Albumin
technique
Density
for
ofthe
albumin
(bovine
albumin
concentrations,
monkey
cells,
were
a gradient
phate-buffered
of fractions
(PBS)
and
tion (35%
albumin)
from
1.0525(17%)
tube was centrifuged
Gradient
prepared
suspended
marrow
fractionation.
by the method
in 17% BSA
of
Tris
than
80 x).
50 cells
were
top
scored
Technique
19%, 21%,
Buffer.
The
was 340 milliosmols
and
to 1.0730(25%).
Following
at 1000 g (at the bottom
Using
the standard
setup,
it was
centration
of CFU-C
was found
that is located
in the 19% albumin
tion yielded
To further
bone
more
serum
albumin,
BSA) stock
as well as of the gradient,
The monkey
marrow
were to be separated
consisting
saline
containing
(magnification
suspended,
After
Dulbecco
was put on
+
serum)
Discontinuous
were
(agar
0.5%),
pipetted.
horse
tinct colonies
were visible.
Colonies
by the use of an inverted
microscope
cells
medium
were
in
chimpanzee
fibroblasts.
concentration
serum
In
the
fibroblasts
embryonic
embryonic
(final
horse
on
as a monolayer
cells were cultured,
chimpanzee
we used,
besides
donors
or human
medium,
consisting
in which
or embryonic
dishes
reported
consistently
solution
The
preparation
solution
and of the different
were described
previously.8
described
and were
23%,
and
osmolarity
elsewhere,8’9
layered
on
top
that
of
25% BSA in phosof the stock
solu-
the density
of the layers
varied
the introduction
of the cells, the
of the tube)
for 30 mm at 10#{176}C.
previously”
that
an 8-15-fold
conin fraction
2, the cell population
of specific
density
1.0575.
This frac-
2%-5%
of the total number
of cells and 40%-60%
of the stem cells.
concentrate
the CFU-C,
fraction
2 was refractionated
by using
a
0.5% step gradient
in the range
of 18%-23%
(density:
1.0550-1.0682).
The cell
populations
located
in the 19%, 19.5%,
and 20% albumin
solutions
were designated
as fractions
2/1, 2/A,
and 2/IB,
which
were found
to contain
the highest
proportion
of stem cells. The osmolarity
of the 35% BSA stock
solution
from
which
the refractionation
gradient
is prepared
was 350 milliosmols.
After
collecting
the fractions,
the cells were washed
in Hank’s
solution,
following
which
the cells were counted,
cultured,
or used for the preparation
of stained
preparations and electron
microscopic
pictures.
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1 98
DICKE
Gradient
technique
of the
discontinuous
routinely
employed
for
human
bone
albumin
density
to separate
HSCs
marrow
Technical
technique,
fractionation.
gradient
from
centrifugation
lymphocytes
in human
ET
AL
details
as is
bone
mar-
row, have
solutions
been described
The concentration
ofthe
various
albumin
of which
the gradient
consisted
was identical
to the routine
monkey
marrow
gradient.
In humans,
up to 50% of the total
number
of CFU-C
appeared
in fraction
3, located
in the 21% albumin
solution
and not in fraction
2
as is the case in monkeys.
This phenomenon
is due to the high osmolarity
of
the 35%
was
BSA
stock
prepared.
used
by
which
CFU-C
solution
(360
Furthermore,
in
fraction
3 was
preparations.
The
milliosmols)
the
from
human,
which
the human
a refractionation
separated
in
refractionation
order
to
gradient
gradient
procedure
obtain
consisted
was
highly
enriched
of the
following
albumin
solutions:
25%,
stock
solution
was 370
23%, 21%,
milliosmols.
bone
marrow
gradient
keys.
yields
This type of refractionation
is also used for preparative
purp554l7
and
fractions
3/2 and 3/3 located
in the 19% and 21% BSA solution,
respecwhich
contain
the majority
ofCFU-C
present
in the original
fraction
317
tively,
It appeared
high
a 2% step
that
Electron
to the
Microscopic
Small
cells,
instead
of CFU-C
number
ofCFU-C
of fractionated
as lymphocytes
of 0.5%
step
in the fractions
in the
human,
from
in a mixture
of glutaraldehyde
in uranyl
acetate.’8
The fixation
mols,
and the
a microcentrifuge
used
as in the mon-
3/2
unfractionated
pH
was adjusted
according
the
monkey,
thoracic
and osmium
had a total
to 7.3. After
to Malamed.’9
and
duct
mouse
bone
of C57BL
tetroxide,
osmolarity
fixation,
After
The standard
of cells by
smears
could
disintegration
methods
of avoiding
of
method
MGG
Polychromatic
Fixation,
carried
out
was
pelleted
with
in a graded
the material
was embedded
knife
and were collected
on
uranyl
acetate
and lead ci-
not be employed
because
as a result
of the smearing
this
complication,
which
preparations,
were
investigated,
described
stain
fixed
All data conserial
sections
sections
was
20 cells to be
(MGG)
analysis
the
were
field.
Mav-Grunwald-Giemsa
loss
was
followed
by immersion
of about
300 millios-
trate2’
and were examined
with a Philips
300 electron
microscope.
cerning
the ultrastructure
of the studied
cells were obtained
from
of multiple
cells.
Quantitative
analysis
of electron
microscopic
carried
out using
a magnification
of 3000x,
which
allows
about
in one
3/3
marrow
mice,
the cells were
dehydration
ethanol
series and treatment
with propylene
oxide,
in Epon.2#{176}Thin
sections
were cut with a diamond
uncoated
copper
grids.
Sections
were stained
with
examined
and
marrow.
Techniques
numbers
as well
was
the concentration
as compared
and 19%. The osmolarity
of the 35% BSA
Note
that
for refractionation
of human
by Sayk22
carried
Staining
pelleting,
according
was
out
for
and
Light
adapted.
described
Microscopic
dehydration,
to the
previously
and
would
there
is a substantial
procedure.
Various
jeopardize
and
finally
A modified
staining
in full
elsewhere.6
detail
the
the
qualitative
precipitation
procedure
using
Examination
embedding
described
in
Epon
procedure.
of
the
One-half
cells
were
micron
From www.bloodjournal.org by guest on March 4, 2016. For personal use only.
IDENTIFICATION
Epon
OF
sections
were
tion Paragon
C. Paragon
different
of whole
ofthe
IN BONE
stained
MARROW
with
1301 (Paragon
Co. Inc.,
Bronx,
that
the
in the
staining
with a mixture
borax
solution
stain,
199
the
commercial
multiple
N.Y.).2325
-
from
tissue,
covered
ofa 2%
CELLS
above-mentioned
method
was
(Merck)
was
staining
studies,
namely
modified
as follows:
of one part of this
and were stained
Entellan
polychromatic
solu-
stain for frozen
sections
PS 1301, C. and
Since
the material
to be stained
was
used
single
The
multiple
purpose
stain
for 60 sec at 75#{176}C.To
exclusively
cells
instead
sections
were
and two parts
prevent
fading
as mounting
medium.
RESULTS
Since concentration
results
of a single
as ofan
experiment
in Table
1. It can
fractions
2/1 and
factor
of 27
tration
traction
of CFU-C
is of crucial
importance
in these
studies,
the
experiment
of monkey
bone
marrow
fractionation,
as well
in which
human
marrow
was fractionated,
have
been listed
be noted
that in case of monkey
marrow
fractionation,
the
2/lA
and
factors
factor
56,
contained
the
respectively.
In
highest
concentration
several
of CFU-C
experiments
even
by
higher
a
concen-
were found,
namely
up to 70. In the human
gradient,
a conceiiof 50 was found
in fraction
3/2 in that particular
experiment
that
is representative
for
seven
other
experiments.
In other
factors
as high as 100 were achieved.
In the electron
micrographs
obtained
from
experiments,
serial
enrichment
ultrathin
sections
of
CFU-C-rich
monkey
and human
bone
marrow
fractions,
a morphologic
entity
similar
to the HSC
in the mouse6
was
frequently
observed
(magnification
17,000x).
No differences
in cell structure
were noted
between
the mouse
HSC
and
the
MSCLC
scored
in the
primate
marrow
fractions.
MSCLCs
from
primate
and mouse
marrow
fractions
have
Fig. 1. Besides
the close
morphologic
similarity
between
the
ferent
noted
species,
in Fig.
Table
detailed
ultrastructural
1. It is obvious
that
1.
Results
of Concentration
by the
characteristics
various
structures
CFUC
Monke
y Marrow
CFUCt
Unfractionated
in Monkey’
of CFU-C
Discontinuous
Albumin
suspension
of the
in the
and Human’
Concentration
Factor
Marrow
CFUCt
hO5
1
1
Gradient
Cells
83
Unfractionated
Fractionation
1200
12
85
700
Fraction
Refractionation
3 (5%)
Refractionation
Fraction
2/1
2736
27
50
4100
Fraction
Fraction
2/lA
(03%)
5600
56
18
1500
Fraction
3’3
Fraction
2/18
(0 3%)
2000
20
87
Fraction
3/4(2%)
Fraction
2/iC
(0 5%)
1000
10
Fraction
2/2
(2%)
500
Fraction
2/3
(3%)
300
Values
of one
Number
colonies
I
suspension
Fractionation
2 (5.5%)
Fraction
Figures
be
cy-
.
Human
99
can
and
Bone Marrow
Assay
Concentration
Factor
MSCLC
nucleus
in
dif-
Gradient
Gradient
/lO5Cells
Representative
been
depicted
cells from
the
(03%)
experiment,
of cells
or half
in
of
parenthesis
5
3
for seven
representative
plated
the
per
surface
dish
of
represent
are adapted
a dish
the
1
least
two
cell
yield
of
-
-
-
-
-
-
-
-
-
11 3%)
experiments
to the
At
3/2 (0 5%l
number
dishes
the
of CFUC
per
fraction
fraction
expressed
being
were
expected
Values
are obtained
by counting
used.
in percentage
ol the
total
unfractionated
material
at least
100
From www.bloodjournal.org by guest on March 4, 2016. For personal use only.
200
DICKE
ET AL
;‘:
‘
-‘
11
1l
____
la
t
‘r.
‘..L7
,
.;
‘,
lb
iii,,
.
.
ei’
#{149}#{149}.
.
..,,
.
.
,
Fig. 1.
Electron microscopic sections of the candidate stem cell of the human (A), mouse
(B). and monkey (C). Note resemblance
of cells in the diff#{243}rent
species:
Candidate
stem cell
is round and its diameter is 8-1 Ojs. Nucleus is round with indentations.
One or two large nucleoli
are visible, and chromatin
pattern in the nucleus is finely dispersed beside some small flocculent
aggregates
at the
nuclear margins. Cytoplasm
is narrow,
and the following
cytoplasmatic
organdIes
are absent: Golgi apparatus.
endoplasmatic
reticulum.
multivesicular
bodies, lysosomes, and clustered ribosomes. Several small mitochondria
are visible. An EM picture
of a small
lymphocyte from human marrow has been depicted in part D of the figure. Diameter of the cell is
S-6i. Nucleus shows large areas of densely aggregated
granular chromatin
and smaller light
regions. In this cell, nucleoli are inconspicious.
Cytoplasm
contains dense bodies and many
vesicules of different sizes. Golgi apparatus is well developed.
Many nbosomes
are clustered.
Mitochondna
are large. Nu, nucleolus; M, mitochondrium;
G, Golgi apparatus;
DB, dense body.
X 12,300.
From www.bloodjournal.org by guest on March 4, 2016. For personal use only.
IDENTIFICATION
See legend
toplasm,
OF
CELLS
IN BONE
MARROW
201
on facing page.
such
as
a Golgi
apparatus
and
endoplasmatic
reticulum,
which
are
apparent
in more
differentiated
cells,
are not present
in the MSCLC.
At low
power
magnification,
one may observe
a superficial
resemblance
between
the
morphology
of the candidate
stem cells and the various
examined
lymphocytes.
For this reason,
the ultrastructural
features
in serial
sections
of both
cell types
have been compared
extensively.
Lymphocytes
are generally
smaller,
present
more
densely
clumped
nuclear
chromatin,
and
frequently
display
in their
cytoplasm
a Golgi
apparatus,
a
centriole,
profiles
of endoplasmatic
reticulum,
dense
bodies,
multivesicular
bodies, and lysosomes-organelles
that have
not been
observed
in our candi-
From www.bloodjournal.org by guest on March 4, 2016. For personal use only.
202
DICKE
Table
2.
Comparison
With
of the Percentage
the Percentage
MSCLC
No
Fraction
MSCLC
CFU-C
Determined
by Electron
#{149}
in Monkey
Marrow
ET AL
Microscopy
Suspensions
CF U-C Assayt
-
Counted
%
CFU.C/105
Cells
Ratio
%MSCLC/%CFU-C
%CFU-C
2/lAf
49(267)
18
5600
5.6
3.2
2/lA
64(395)
16
5700
5.7
2.8
2/1
56(347)
16
5500
5.5
2.9
2/1
16(140)
11
5000
5
2.2
2/1
16(224)
7
4700
4.7
1.5
2/iC
15(300)
5
1440
1.4
3.6
2/3
13(319)
4
1000
1
4
7(290)
2
1000
1
2.4
2/iC
Mean
MSCLC.
fraction
mouse
stem
cell-like
cell.
Figures
in parenthesis
represent
the
total
2.8
number
of cells
per
counted
t 0.1-1
X iO
cells
half of the surface
Fraction
per
dish
of the dish.
number.
The
were
plated.
At
least
Duplicate cultures were
fractions
used
were
100
colonies
per
dish
were
counted.
or
one-
used.
obtained
from
gradients
in
prepared
different
ex-
periments.
date stem cell. It should
be reminded
that lymphocytes,
by their response
to P1-IA in vitro,
are absent
from our
After
identification
of the MSCLC
in the ultrathin
tion of 17,000 x, quantitative
centrates
were performed
sults
of the examination
listed.
A striking
fraction
CFU-C
These
ism
morphologic
studies
of the
using
a magnification
of 3000x.
of eight
monkey
CFU-C-rich
parallelism
is again
evident.
between
Table
in CFU-C-concentrated
results
resemble
those
between
the
Table
3.
With
the
number
3 presents
entity
of
of the Percentage
the Percentage
from
monkey
the
MSCLC
Fraction
No
%
20(181)
3/3
11(124)
3/3
19(240)
3/3
3/2
and
CFU-C
and
numbers
human
with
marrow
regard
to
and
the
in
by Electron
Marrow
of
suspensions.
the parallelvitro
assay.
Microscopy
Suspensions
CFU-C/1
11
0
Cells
Ratio.
% MSCLC/%
% CFU-C
CFU-C
4800
4.8
9
3000
3
3
8
2000
2
4
21(300)
7
6000
6
1.2
11(180)
6
2000
2
3
3/3
16(257)
6
1700
1.7
3.5
3/3
13(250)
5
2000
2
2.5
2.3
Mean
#{149}
MSCLC.
fraction
mouse
stem
cell-like
cell.
Figures
in
parenthesis
represent
the
total
number
2.8
of
cells
per
counted.
t 0.1-1
half
per
CF U.CAssayt
Counted
3/2f
MSCLC
Determined
in Human
primate
CFU-C
conIn Table
2, the refractions
have
been
of MSCLC
MSCLC
MSCLC
CFU.C
of
counts
fractions
found
in the
morphologic
Comparison
functionally
determined
stem cell fractions.68
sections
at a magnifica-
of the
FractIon
X
i#{248}cells
surface
number.
of the
The
per
dish.
dish
were
Duplicate
fractions
used
plated.
cultures
were
At
least
100
were
used.
obtained
from
colonies
gradients
per
prepared
dish
were
in different
counted
or
experiments
one-
From www.bloodjournal.org by guest on March 4, 2016. For personal use only.
IDENTIFICATION
Note
%
that
OF
in the
MSCLC/%
Under
tative
in Fig.
light
BONE
monkey,
CFU-C
the
enriched
morphologic
IN
CELLS
MARROW
as
well
is 2.8 (Tables
microscope,
203
as
in
the
2 and
3).
MGG-stained
mouse,
monkey,
2 and
showed
the
eter, the diameter
rather
homogenous
and
following
was estimated
structure,
studies,
preparations
fractions
of monkey
and human
criteria
listed
by van Bekkum
of the
human
marrow
et al.6
human
candidate
characteristics:
HSC
cells
have
size,
to be 8s; shape,
round;
round
or kidney-shaped;
mean
of
revealed
MGG-stained
similar
the
the
CFU-C-
fulfilling
the
cells represen-
been
using
nucleus,
cytoplasm,
ratio
presented
a micromlight purple,
pale
blue,
no granules.
Using
enriched
the criteria
listed
above,
quantitative
monkey
bone marrow
fractions
were
compared
with the number
tions.
As can be observed
of MSCLC
out and these
of CFU-C
determined
per
in Table
4, the ratios
consisting
a
b
c
d
Fig. 2. May.GrUnwald-Giemse-stained
row (C). Stained preparation
of small
X 1370.
counts
carried
l0
cells
of the
in CFU-Ccounts
were
in these
percentage
fracof
HSC of human (A). monkey (B. arrow). and mouse marlymphocyte
is depicted
in lower right part of figure (D).
From www.bloodjournal.org by guest on March 4, 2016. For personal use only.
DICKE
204
Table
4.
Comparison
With
of the Percentage
the Percentage
MSCLC#{149} Determined
in Monkey
CFU.C
MSCLC
Fraction
No.
by Light
Marrow
Microscopy
Suspensions
CFU -CAssayt
Counted
%
CFU-C/l0
Cells
%CFU-C
Ratio.
%MSCLC/%CFU-C
2/lAf
59(532)
ii
5600
5.6
2
2/iA
64(594)
11
4000
4
2.7
2/i
42(427)
10
4700
4.7
2.1
9
3700
3.7
2.4
2/lB
109(1100)
2/iC
52(651)
8
2700
2.7
3
2/lB
32(626)
5
2000
2
2.5
2
27(670)
4
1200
1.2
3.3
2
8(395)
2
1000
1
2
0.1
7
Unfractionated
8(1170)
0.7
100
Mean
MSCLC.
.
ET AL
fraction
mouse
stem
cell-like
cell.
Figures
in
parenthesis
represent
the
total
3
of cells
number
per
counted.
t 0. 1 -1
X
cells
10
half of the surface
t Fraction
per
dish
of the dish.
number.
The
were
plated.
Duplicate
fractions
At
cultures
used
least
were
were
1 00
colonies
per
dish
were
counted
or
one-
used.
obtained
from
gradients
prepared
in
different
ex-
periments.
MSCLC
and the percentage
mean
ratio
of % MSCLC/%
MSCLC
centrated
counts
CFU-C
sistent
ratio
It can
cyte
have been
fractions
can again
be seen
is superficial,
2 that
it still
by applying
preparations,
Table
compared
obtained
be noted.
in Fig.
that
the ultrastructure
stained
preparations.
tated
of CFU-C
per fraction
are highly
constant.
The
CFU-C
is 3.0 (n = 9). In Table
5, the results
of
The
might
confusion.
of the
of the Percentage
the
Percentage
with
This
staining
method
is strikingly
Determined
Marrow
MSCLC
due
lymphoto the
fact
MGGfacili-
sections.
identical
by Light
#{149}
in Human
is 3.1.
small
visible
in the
types
has been
in cross
concon-
fractions
the
is mainly
MSCLC
MSCLC
CFUC
in highly
A very
for six different
resemblance
nucleus
is not clearly
between
both
cell
polychromatic
Comparison
ratio
the
cause
of the cell
Differentiation
the
mean
although
the appearance
5.
with the number
of CFU-C
from
human
bone
marrow.
In these
to that
Microscopy
seen
With
Suspensions
CF U-CAssayt
Ratio:
Fraction
No.
Counted
%
CFU -C/l05
Cells
%CFU-C
% MSCLC/%CFU-C
3/2f
49(615)
8
4000
4
3/2
90(1474)
6
4000
4
1.5
3/3
30(61
5
2000
2
2.5
3/3
23(456)
5
2400
2.4
2.1
3/3
30(732)
4
1100
1.1
3.6
3/4
12(1400)
0.85
120
0.12
7
5)
‘
2
Mean
#{149}
MSCLC.
fraction
of the
* Fraction
periments.
stem
cell-like
cell.
Figures
in parenthesis
represent
the
total
3.1
number
of cells
per
counted.
t 0.1-1
half
mouse
X 10
cells
surface
of the
number.
per
dish.
The
dish
were
Duplicate
fractions
plated.
At
cultures
used
were
least
were
100
colonies
per
dish
were
counted
or
one-
used.
obtained
from
gradients
prepared
in
different
ex-
From www.bloodjournal.org by guest on March 4, 2016. For personal use only.
IDENTIFICATION
OF
CELLS
IN
BONE
MARROW
205
“b.
;;#{216}
a..
‘A
Fig. 3.
(A) Polychrome-stained
(0.5k ) cross
section
of a CFU-C-enriched
monkey
marrow
fraction.
Arrow
indicates
a MSCLC.
Note the nucleoli
and the chromatin
structure
in nucleus.
Light microscope.
X 1 750.
(B) Electron
miprograph
of an ultrathin
cross
section
of the same
CFU-C-enriched
monkey
marrow
fraction
as presented
in Fig. 3A. Cell indicated
by arrow
is the
same
MSCLC
that is depicted
in Fig. 3A (cell indicated
by arrow).
Note
identical
morphologic
appearance
of the same
cell in the polychrome-stained
preparation
and in the electron
micrograph. X 4200.
in electron
micrographs
morphologic
(Fig.
appearances,
chrome-stained
3).
Figure
especially
MSCLC
and
4 illustrates
in
nuclear
the lymphocytes
the
marked
structure,
that
differences
between
underwent
in
the
the same
poly-
staining
procedure.
DISCUSS
ION
The resemblance
between
the candidate
stem
cells
in human
and
monkey
marrow
with those
described
in the mouse
is striking,
as evidenced
by the electron
microscopic
pictures
presented
in Fig. 4. Moreover,
the electron
microscopic
appearance
tinguished
from
of the
cell
MSCLC
types
is so characteristic
belonging
to one
that
of the
four
it can
hemopoietic
easily
be
cell
dislines,
specifically
the myeloid
and lymphoid
ones.
Due to its outspoken
morphology,
quantitative
analysis
of the electron
microscopic
sections
is a reliable
measure
for the number
of MSCLC
in a hemopoietic
suspension.
Just as in the mouse,
light
microscopic
examination
using
the May-Grunwald-Giemsa
staining,
revealed
a superficial
resemblance
between
the MSCLC
and the lymphocyte.
Although
some
of the
found
by the
electron
terms,
which
in MGG-stained
studies
cells,
in our
the
was
characteristics
microscope
helpful
for
preparations,
opinion.
structures,
specific
By
especially
of the
could
differentiating
the
the polychrome
the
latter
in the
ultrastructure
be “translated”
MSCLC
method
procedure,
nucleus,
are
of the
into
light
MSCLC
microscopic
from
the lymphocyte
is preferable
for LM
which
uses
clearly
visible,
cross
sections
and
the
of
simi-
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206
DICKE
ET AL
Fig. 4.
(A) Polychrome-stained
cross section
of a mouse
thoracic
duct cell suspension.
Small
lymphocytes
that are predominant
in this preparation
are smaller
than the MSCLCs.
Nuclear
chromatin
structure
of the lymphocyte
differs
from that of the MSCLC.
X 1750.
(B) Polychromestained cross section
of CFU-C-enriched
monkey
marrow
fraction.
Arrows
indicate
the MSCLCs
present
in this preparation.
X 1750.
larity
in morphology
of the polychrome-stained
striking
between
(Fig.
3).
MSCLC
Moreover,
the
and lymphocytes
analysis
by light
microscopy
cedure
should
be less
polychrome
more
can
be performed
time-consuming
and
the number
of HSC than the CFU-C
to stain
cross
sections
was that the
embedded,
inhibited
why
the
dala
presented
stain
in this
paper
much
less
enter
underscore
into
with
the
small
lymphocyte
cells
our
previous
compartment
the
which
belongs
our MSCLC,28
MSCLC.
Moore
et al.29
cells with
to transitional
centrated
tunately,
cell type
morphologic
this
the
that quantitative
a staining
profor
determining
was not
fixation
It is not
these
is
differences
yet clear
conditions.
statement6
used
were
The
that
the
dif-
and the various
lymphocytes
cells cannot
easily
be associbone
marrow.
introduction
which
was
Although
Some
con-
of the term
“transiheld
to represent
a
one of the cell types
to the lymphocyte
transitional
compartment,27
might
the majority
of transitional
lymphocytes
is larger
than
described
colony-forming
spond
closely
under
picture
the
laborious
in the
fusion
has been caused
in the literature
by the
tional
lymphocytes”
by Rosse
and
Yoffey,26
primitive
hemopoietic
precursor
cell population.
described,
resemble
50
Such
the cells.
the
ferences
between
the mouse
HSC on the one hand,
on the other
hand,
are so manyfold
that the stem
ated
out
routinely.
of MGG
does
to its EM
assay.
The reason
that MGG
Epon
in which
the cells after
the penetration
polychromatic
MSCLC
method
brings
clearly
(Fig.
4),
hemopoietic
cells
morphologic
lymphocytes
with
the aid
description
or,
to
characteristics
with basophilic
of
continuous
of these
be
more
that
precise,
in ‘vitro
they
hold
to correcytoplasm.
They con-
albumin
cells
was
gradients.
Unforderived
only
from
From www.bloodjournal.org by guest on March 4, 2016. For personal use only.
IDENTIFICATION
OF
CELLS
Giemsa-stained
IN BONE
MARROW
preparations
207
studied
by
light
microscopy.
In our
opinion,
difficult
to compare
the results
of Moore
et al.29 with
our own
other
studies
because
of the absence
of EM data.
The same
term,
lymphocytes,”
has been used by others
to designate
characteristic
precursors.3032
The unique
morphologic
appearance
of dispute.
Although
there
identical
to the pluripotent
is no
HSC,
the parallelism
of its frequency
poietic
suspensions
in mice are
of MSCLCs
correlate
with
suspensions
strongly
supports
of the
pluripotent
Now
that
HSC
with the
striking.6
the
pluripotent
capacity
hypothesis
of the
forms
a matter
stem cell is
entity
and
of a variety
of
mammalian
description
HSC
that our candidate
as one morphologic
number
of CFU-S
in a variety
of hemoThe fact that in monkeys
the numbers
restorative
our
in these
a detailed
of the
absolute
proof
its appearance
it is
or any of the
“transitional
lymphocyte
a single
of hem opoietic
morphologic
entity
species.’2
morphologic
properties
of the
candi-
date stem cell is available,
these
cells can be enumerated
in various
abnormal
states
of the hemopoiesis
in human
patients.
Obviously,
such
analysis
will be
facilitated
by concentrating
these cells, e.g., with the density
gradient
technique.
It is to be expected
that the results
of such studies
in human
bone
marrow
will
eventually
provide
confirmation
or dispute
of the
conclusions
presented
here.
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From www.bloodjournal.org by guest on March 4, 2016. For personal use only.
1973 42: 195-208
Identification of Cells in Primate Bone Marrow Resembling the Hemopoietic
Stem Cell in the Mouse
K. A. Dicke, M. J. van Noord, B. Maat, U. W. Schaefer and D. W. van Bekkum
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