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Enhancement of Murine Blast Cell Colony Formation in Culture by Recombinant
Rat Stem Cell Factor, Ligand for c-kit
By Kohichiro Tsuji, Krisztina M. Zsebo, and Makio Ogawa
Mice with W mutations characterized by hypopigmentation,
sterility, anemia, and mast cell deficiency have abnormalities
in c-kit, a receptor with tyrosine kinase activity. Recently, the
ligand for c-kit was cloned by investigators in several laboratories. Zsebo et al identified and cloned a gene for a cytokine
termed stem cell factor (SCF) in the medium conditioned by
buffalo rat liver cells, and this cytokine proved t o be c-kit
ligand. We have examined the effects of recombinant rat SCF
(rrSCF) on colony formation from primitive hematopoietic
progenitors in culture. rrSCF and erythropoietin (Ep) supported formation of granulocyte/macrophage (GM) colonies
as well as a small number of multilineage and blast cell
colonies from marrow cells of normal mice. We then examined the effects of rrSCF using marrow and spleen cells of
mice that had been treated with 150 mg/kg 5-fluorouracil
(5-FU). Unlike single factors, combinations of factors such as
rrSCF plus interleukin-3 (IL-3). rrSCF plus IL-6, and rrSCF plus
granulocyte colony-stimulating factor (G-CSF) markedly stim-
ulated the growth of multilineage colonies. In contrast t o
these factor combinations and a combination of IL-3 and IL-6,
a combination of rrSCF and IL-4 did not support multilineage
colony formation. Mapping studies of the development of
multipotential blast cell colonies further indicated that rrSCF,
like IL-6, G-CSF, and IL-11, shortens the dormant period in
which the stem cells reside. When we tested the effects of
rrSCF using pooled blast cells, which are highly enriched for
progenitors and are devoid of stromal cells, rrSCF plus Ep
supported formation of only a few multilineage colonies,
indicating that rrSCF itself is ineffective in support of the
proliferation of multipotential progenitors. However, rrSCF
supported formation of a significant number of neutrophil
and neutrophil/macrophage colonies from pooled blast cells,
indicating that rrSCF is able t o support directly the proliferation of progenitors in neutrophiVmonocyte lineages. c-kit
ligand may play important roles in adult hematopoiesis.
o 1991 b y The American Society of Hematology.
M
marrow cells in the medium conditioned by buffalo rat liver
cells. The human gene was then cloned by virtue of its
nucleic acid homology to the rat gene. The rat cytokine,
termed stem cell factor (SCF), is the rat homologue of a
murine mast cell growth factor’ and c-kit ligand.”” Rodent
(rat and mouse) SCF appear to have broad effects in
hematopoiesis, including mast cell proliferation, and works
synergisticallywith other hematopoietic cytokines in progenitor proliferation in culture.” In our laboratory we have
examined the direct and synergistic effects of recombinant
rat SCF (rrSCF) on the early stages of hematopoietic
development, particularly in blast cell colony formation.
We have observed that SCF is a potent inducer of proliferation of dormant hematopoietic progenitors.
URINE BLAST CELL colony assay that was developed in our laboratory proved to be a useful tool in
elucidation of the mechanisms of proliferation of hematopoietic progenitors. The blast cell colonies were originally
defined as small colonies consisting of a few hundred blast
cells showing no signs of differentiation in situ and identified late in the culture period.’ In addition, the blast cell
colonies showed high secondary colony-forming ability,
including multilineage colony formation. Subsequent studies indicated that these characteristics of the blast cell
colonies are based on the fact that their progenitors are
dormant in the cell cycle and that blast cell colonies are
identified only a few days after the first cell division of their
progenitors. Blast cell colonies develop into granulocyte/
erythrocyte/macrophage/megakaryocyte(GEMM), granulocyte/macrophage (GM), and on occasion megakaryocyte
colonies when they are allowed to express their full lineage
potentials by continued incubation under permissive culture conditions.* By serial observations (mapping studies)
of blast cell colony development from cells of mice that had
been treated with 150 mgkg 5-fluorouracil (5-FU), we
concluded that interleukin-3 (IL-3) is a necessary factor in
blast cell colony formation but that it does not trigger cell
divisions of the dormant stem c e k 3Our data indicated that
IL-3 supports proliferation of multipotential progenitors
only after they exit from G,. Subsequently we found that
IL-6: granulocyte colony-stimulating factor (G-CSF)? and
IL-1 l6appear to shorten the dormant period in which these
progenitors reside. Studies of human blast cell colonies
indicated that these synergistic factors do not have the
ability to support blast cell colony formation by themselves
and that they act only synergisticallywith IL-3 in support of
blast cell colony formati~n.’,~
Recently, investigators in several laboratories identified
the ligand for c-kit, a tyrosine kinase receptor that is
encoded at the W locus in m i ~ e . ~Zsebo
. ’ ~ et al”,” identified
and cloned the rat gene for a cytokine that supports
macroscopic colony formation from post-5-FU murine
Blood, Vol78, No 5 (September I), 1991: pp 1223-1229
MATERIALS AND METHODS
Cellpreparation. Female BDF, mice, 10 to 15 weeks old, were
obtained from ARS Sprague Dawley (Indianapolis, IN). Cells were
obtained from marrow and spleens of normal mice as well as from
mice that had been injected with 150 mgkg 5-FU intravenously.
5-FU (Adria Laboratories, Columbia, OH) was administered
intravenously through the tail veins of mice at 150 mgkg body
weight.” Bone marrow and spleen cells were harvested 2 and 4 days
after 5-FU injection, respectively.43
From the Department of Medicine, Medical Universiy of South
Carolina and VA Medical Center, Charleston; and AMGen Corporation, Thousand Oaks, CA.
Submitted January 17,1991; accepted May 3, 1991.
Supported by National Institutes of Health Grant No. DK32294, the
Department of Veterans Affairs, and Kirin Brewery Co, Ltd (Japan).
M.0.is a VA Medical Investigator.
Address reprint requests to Mako Ogawa, MD, PhD, VA Medical
Center, 109 Bee St, Charleston, SC 29403.
The publication costs of this article were defrayed in part by page
charge payment. mis article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I991 by The American Society of Hematology.
0006-4971I91/7805-OO22$3.00/0
1223
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1224
TSUJI, ZSEBO, AND OGAWA
Factors. rrSCF was purified from Escherichia coli as described
previously.” Recombinant murine IL-3 was a medium conditioned
by Chinese hamster ovary (CHO) cells that had been genetically
engineered to produce murine IL-3 to high titer (approximately
30,000 UimL) and was a generous gift from Dr Tetsuo Sudo
(Biomaterial Research Institute, Yokohama, Japan). Human G-CSF
expressed in E coli was purified as described before.I6 Human IL-6
was also expressed in E coli. Human erythropoietin (Ep) was
obtained from AmGen (Thousand Oaks, CA). Purified recombinant murine IL-4 was purchased from Genzyme Corp (Boston,
MA).
Clonal cell culture. Methylcellulose cell cultures were established in 35”
Lux suspension culture dishes (#5221R, Nunc,
Inc, Naperville, IL). One milliliter of culture contained 2 x lo4
marrow cells from normal mice, 5 x lo4 to 1 x lo6 cells from
5-FU-treated mice, a-medium (Flow Laboratories, Inc, McLean,
VA), 1.2% 1,500 cps methylcellulose (Fisher Scientific Co, Norcross, GA), 30% fetal calf serum (Hyclone Laboratories, Inc,
Logan, UT), 1% deionized fraction V bovine serum albumin
(Sigma Chemical Co,St Louis, MO), 1 x lO-‘mol/L 2-mercaptoethanol (Eastman Organic Chemicals, Rochester, NY), and hematopoietic factors. Dishes were incubated at 37°C in a humidified
atmosphere flushed with 5% CO,. Except for megakaryocyte
colonies, colonies consisting of 50 or more cells were scored on an
inverted microscope on the specified day of incubation. Megakaryocyte colonies were scored when they contained four or more
megakaryocytes. Abbreviations for colony types are as follows:
GM, granulocyteimacrophage; Mast, mast cell colonies; E, erythroid bursts; M, megakaryocyte colonies; GMM, granulocyte/
macrophageimegakaryocyte colonies”; GEMM, granulocyteierythrocyteimacrophageimegakaryocyte colonies”; and B1, blast cell
colonies.’
Blast cell colony replating. The hematopoietic potentials of the
blast cell colonies were determined by blast cell colony replating.
Between days 5 and 15 of incubation, individual blast cell colonies
containing 50 to 500 cells were picked with an Eppendorf pipette
(Brinkman Instruments Inc, Westbury, NY) and replated in
secondary methylcellulose cultures containing 2 U/mL Ep and 200
UimL IL-3.
Blast cells were also used as pure target populations of hematopoietic cells to determine whether the observed effects of
rrSCF were direct efforts or due to the release of other factors by
coexisting stromal cells. One million day 4 post-5-FU spleen cells
were cultured in the presence of 200 U/mL of IL-3. On day 8 of
culture, individual blast cell colonies (between 50 and 500 cells)
were picked from cultures, pooled, washed twice with medium, and
replated in secondary cultures each containing different combinations of factors.
RESULTS
Colony formation from marrow cells of normal mice.
Analysis of colony formation from marrow cells of normal
mice is presented in Fig 1. rrSCF gave rise to colonies in a
dose-dependent manner and 100 to 1,000 ng/mL rrSCF
supported maximal colony formation. The analysis of the
types of colonies supported by rrSCF in the presence or
absence of Ep is presented in Table 1. The majority of the
colonies supported by rrSCF were GM colonies. In contrast, IL-3 supported formation of various types of colonies
as has been previously d ~ c u m e n t e dBoth
. ~ rrSCF and IL-3
supported formation of blast cell colonies. In the presence
of Ep, rrSCF and IL-3 supported multilineage GEMM
colony formation. However, the number of multilineage
0
0.1
I
I
I
I
1
10
100
1000
Concentration of SCF (ng/ml)
Fig 1. Titration of rrSCF for colony formation from marrow cells of
normal mice.
(GEM, GMM, GEMM, and blast cell) colonies supported
by rrSCF plus Ep was significantly lower that that of the
combination of IL-3 and Ep.
Colony formation from marrow and spleen cells of 5-FUtreated mice. The observation that rrSCF supports formation of multipotential blast cell colonies in cultures of
normal marrow cells was similar to our previous observations with IL-6,4 G-CSF: and IL-11,6 and suggested the
possibility that rrSCF may also act synergisticallywith IL-3
in supporting the proliferation of primitive progenitors. To
test this possibility, we examined colony formation from
marrow and spleen cells harvested 2 or 4 days after
injection of 150 mg/kg 5-FU as we described previo~sly.~.~
Cultures were established in the presence of rrSCF, IL-6,
G-CSF, IL-3, and Ep as single agents and in various
combinations. The results of two experiments with day 2
post-5-FU marrow cells and one experiment with day 4
post-5-FU spleen cells are presented in Table 2. In experiment A, rrSCF, IL-3, or IL-6 in the presence of Ep
supported formation of only a few multilineage colonies. As
we have reported previously: the combination of IL-3 and
IL-6 supported the formation of many multilineage colonies in the presence of Ep. A combination of rrSCF, IL-3,
and Ep also supported formation of many GEMM colonies,
indicating that rrSCF, like IL-6, augments IL-3-dependent
proliferation of multipotential progenitors. Surprisingly, a
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SCF ON BLAST CELL COLONIES
1225
Table 1. Effects of SCF on Colony Formation From Bone Marrow Cells of Normal Mice
No. of Colonied2 x 104Cells
Factors
GM
B
M
EM
Mast
GEM
GMM
GEMM
Blast
None
EP
SCF
SCF,Ep
IL-3
IL-3, Ep
0
121
27 f 2
2823
67 2 5
69 f 3
0
1f1
0
120
0
52 1
0
0
0
0
121
120
0
0
0
0
0
0
0
0
0
121
0
0
0
0
0
0
5 %1
220
0
0
220
0
5 2 1
0
2f1
120
2f 1
121
0
0
4 r 1
321
0
0
0
221
Total
'
0
2f1
29 f 1
32 f 2
79 f 3
90 2 3
Colonies were counted on day 10 of incubation. Data represent mean f SD of quadruplicate cultures. Concentrations of growth factors: Ep, 2
U/mL; SCF, 100 ng/mL; IL-3, 200 U/mL. Colony types are: GM, granulocyte/macrophage colonies; B, erythroid bursts; M, megakaryocyte colonies;
EM, et-ythrocyte/macrophage colonies; Mast, mast cell colonies; GEM, granulocyte/erythrocyte/macrophagecolonies; GMM, granulocyte/macrophage/
megakaryocyte colonies; GEMM, granulocyte/erythrocyte/macrophage/megakaryocytecolonies; Blast, blast cell colonies.
combination of rrSCF, IL-6, and Ep supported formation of
a similar number of multilineage colonies.
In experiment B, we extended the studies to include
other early acting growth factors, G-CSF and IL-4. We have
reported previously that G-CSF also enhances IL-3-
dependent blast cell colony formation? Therefore, we
tested the potential synergism between rrSCF and G-CSF
in the presence of Ep. Similar to the combination of rrSCF,
IL-6, and Ep shown in experiment A, the combination of
rrSCF, G-CSF, and Ep supported multilineage colony
Table 2. Effects of Individual, Early Acting Factors, and Their Combinations on Colony Formation From Postd-FU Marrow and Spleen Cells
No. of Colonies
Factors
M
GM
Experiment A: Day 2 post-5-FU marrow cells
0
None
0
EP
SCF, Ep
421
IL-3, Ep
8f1
IL-6, Ep
5 2 1
SCF, IL-3, Ep
10 r 4
SCF, IL-6, Ep
823
IL-3, IL-6, Ep
1224
SCF, IL-3, IL-6, Ep
12 2 3
Experiment B: Day 2 post4-FU marrow cells
0
None
EP
0
SCF, Ep
4k1
IL-3, Ep
10 f 2
SCF, IL-3, Ep
12 2 2
IL-6, Ep
321
IL-3, IL-6, Ep
14 f 2
SCF, IL-6, Ep
722
G-CSF, Ep
3 f 1
8f 1
SCF, G-SCF, Ep
IL-4, Ep
121
SCF, IL-4, Ep
221
IL-4, IL-6, Ep
92 1
GEM
0
0
0
0
0
0
121
121
121
0
0
0
0
0
0
GEMM
Blast
Total
0
0
0
121
2+1
0
13 2 2
14 f 2
1821
19 f 2
0
0
1 f 1
221
l f 0
121
0
0
0
0
0
6+1
12 f 2
6f1
25 f 6
23 2 3
32 5
33 f 1
0
1%1
120
0
0
2 r 1
0
0
0
0
121
0
0
1 f 1
321
13 r 1
120
13 f 2
1420
0
13 f 2
0
0
14r 1
0
0
2
5
0
2
0
0
2
0
0
0
0
0
5f1
14 2 2
27 f 2
4 f 1
28 f 4
27 f 3
421
26 f 3
l r 1
221
26 2 4
0
0
0
0
2 f 1
3+0
1 f 1
121
0
121
1fO
10 2 2
9+2
321
13r 1
17r 1
21 + 3
20 f 1
0
0
0
0
1%0
0
121
I f 1
0
0
0
0
0
0
0
1k1
0
0
120
0
0
0
0
1fO
0
0
3 r 1
2 f 1
3 r 1
1 f 1
220
5 f 1
4 r 1
0
0
121
121
0
1fO
121
221
1 r o
1r o
1f 1
0
0
GMM
0
0
1
1
1
1
0
0
1
0
0
Experiment C: Day 4 post-5-FU spleen cells
None
EP
SCF, Ep
IL-3, Ep
IL-6, Ep
SCF, IL-3, Ep
SCF, IL-6, Ep
IL-3, IL-6, Ep
SCF, IL-3, IL-6. Ep
0
0
3 r
7 f
2 r
7 f
72
7 f
82
1
1
1
3
0
1
1
0
0
I f 1
220
5 f 2
I f 1
0
0
0
0
0
19 f 2
23 2 4
14f2
23 2 1
27 r 2
31 r 4
34 + 1
5 x l o 4 post-5-FU marrow cells and 1 x lo6 post-5-FU spleen cells were plated per dish. Colonies were scored on day 14 of incubation. Data
represent mean + SD of quadruplicate cultures. Abbreviationsfor colony types are as presented in Table 1. Concentrations of factors: Ep, 2 U/mL;
SCF, 100 ng/mL; IL-3,200 U/mL; IL-6,100 ng/mL; G-CSF, 100 ng/mL; IL-4,lO ng/mL.
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1226
TSUJI, ZSEBO, AND OGAWA
G-CSF, and rrSCF individually supported formation of
both GEMM and blast cell colonies whereas combinations
of these factors supported many GEMM colonies but
almost no blast cell colonies. These observations indicated
that in the presence of combinations of factors, the exit
from Goof dormant stem cells is augmented, allowing more
multipotential progenitors to develop into multilineage
colonies. To test this hypothesis, we performed serial
observations (mapping) of blast cell colony formation from
day 4 post-5-FU spleen cells as we reported previously.'6
Five hundred thousand day 4 post-5-FU spleen cells were
plated per dish in the presence of 2 U/mL E p and either
IL-3, rrSCF, IL-6 alone, or in the presence of rrSCF plus
IL-3, rrSCF plus IL-6, IL-3 plus IL-6, or IL-3 plus IL-6.
Emergence of new blast cell colonies and their proliferation
in two dishes in each group were examined on an inverted
microscope and recorded daily. A portion of the studies of
the growth of the blast cell colonies that later showed
GEMM lineages in cultures is presented in Fig 2. In
cultures containing rrSCF alone, IL-3 alone, rrSCF plus
IL-3, and rrSCF plus IL-6, 14, 12, 14, and 14 blast cell
colonies were identified, respectively. Multipotential blast
cell colonies appeared earlier in cultures containing combinations of synergistic factors such as rrSCF plus IL-3 (C)
and rrSCF plus IL-6 (D) than in cultures containing IL-3
(A) or rrSCF (B) alone. The average time at which
individual blast cell colonies reached 100 cells was calculated to be 10.5 f 2.5, 10.5 2 3.8, 5.4 f 1.0, and 5.5 2 1.2
days for cultures containing IL-3 (A), rrSCF (B), rrSCF
plus IL-3 (C), and rrSCF plus IL-6 (D), respectively. We
then calculated the doubling times of the individual blast
cell colonies based on the most linear portion of the
formation. We have also reported previously that IL-4,
similar to IL-3, is able to support blast cell colony formation
from post-5-FU marrow and spleen cells and that IL-6
significantly enhances IL-Mependent murine blast cell
colony formation." IL-4, rrSCF, and E p failed to support
GEMM colony formation while IL-4, IL-6, and E p acted
synergistically in support of formation of GEMM colonies.
Studies of the synergistic interactions between rrSCF,
IL-3, and IL-6 using day 4 post-5-FU spleen cells are
presented in experiment C of Table 2. The combination of
rrSCF and Ep, similar to the combination of IL-3 and Ep,
supported the formation of many GEMM colonies and a
few blast cell colonies. The combination of IL-6 and Ep
supported the formation of both GEMM colonies and blast
cell colonies. The differences between experiment A and C
with regard to multilineage colony formation supported by
individual synergistic factors may reflect intrinsic differences in the progenitors in the marrow and spleen or they
may be due to differences in state of cell cycling of the
progenitors because day 4 post-5-FU spleen cells may be
recovering from 5-FU toxicity. Alternatively, they may be
caused by differences in the coexisting stromal cells, which
are known to produce factors. Regardless, the combinations of two out of three early acting factors, namely rrSCF,
IL-3, and IL-6, showed enhancement of GEMM colony
formation. Again, the combination of rrSCF and IL-6
supported the-formation of many GEMM colonies similar
to the data in experiment A. Addition of rrSCF to the
combination of IL-6 and IL-3 did not enhance GEMM
colony formation further.
Serial observations of blast cell colony formation. It is of
interest that in experiments A and C of Table 3, IL-3, IL-6,
Table 3. Replating of Blast Cell Colonies Derived From Post-5-FU Spleen Cells Cultured With SCF or IL-3
No. of Secondary Colonies
Primary Culture
SCF
Mean 2 SD
IL-3
Mean f SD
Cell No. Per
Primary Colony
35
41
63
81
88
Replating Efficiency
GM
2
2
3
4
7
2
3
4
E
M
0
0
0
0
0
0
1
0
0
3
0
138
142
208
254
312
136 2 89
7 7
54
53
6
202
0
7
0
8
0
0
0
3
0
10
0
42
47
79
91
96
118
125
131
145
289
116 f 66
8
9
2
2
31
2
8
6
5
124
0
0
0
0
0
0
7
1
8
2
4
9
0
3
5
0
0
0
0
EM
5
0
0
0
0
0
4
GEM
GEMM
Mast
Blast
Total
7
11
8
21
3
4
21
40
147
1
0
0
0
0
0
0
0
1
1
1
0
0
0
0
0
0
0
0
0
0
30
35
43
62
77
81
98
138
205
204
86
85
68
77
88
59
69
66
81
65
7 4 * 10
17
5
41
42
17
33
18
26
15
52
0
0
0
0
0
5
0
1
0
31
40
71
68
75
69
105
92
76
181
74
85
90
75
78
58
84
70
52
63
7' 2 f 12
0
0
0
0
0
0
0
4
5
0
0
11
38
25
0
1
0
1
0
0
0
2
2
3
0
0
0
0
0
1
2
0
0
0
0
0
26
0
0
28
1
5
0
2
0
1
0
2
0
0
0
0
0
0
0
0
1
0
0
1
1%)
GMM
Abbreviations for colony types are as presented in Table 1. Colonies were scored on day 10.
0
0
2
0
1
0
0
0
0
0
0
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1227
SCF ON BLAST CELL COLONIES
B
loo -
8k
SCF
/J
m
0
5
I5
10
M
O
5
10
I5
20
Days in Culture
Fig 2. Growth kinetics of individual blast cell colonies that later
showed GEMM expression. The data represent colonies identified in
two plates, each seeded with 5 x 106 day 4 post-5-FU spleen cells.
Cultures were established in the presence of IL-3 (A), rrSCF (B), IL-3
plus rrSCF (C), and rrSCF plus IL-6 (Dl.
respective curves. The doubling times did not differ significantly and were estimated to be 14.7 f 4.2, 14.8 f 4.1,
12.3 +- 1.8, and 12.7 f 3.0 hours in cultures supported by
rrSCF alone, IL-3 alone, rrSCF plus IL-3, and rrSCF plus
IL-6, respectively. Emergence of multipotential blast cell
colonies was also hastened by combinations of factors such
as IL-3 plus IL-6 or IL-3 plus IL-6 plus rrSCF. The average
times at which individual blast cell colonies reached 100
cells under these conditions were 5.5 2 0.8 and 4.4 f 0.7
days, respectively. These results indicated that the combination of two of these factors significantly shortens the period
of the dormancy state in which the stem cells reside.
Replating studies of individual blast cell colonies. To
define more precisely the differentiation and proliferative
potentials of the blast cell colonies supported by rrSCF, we
performed replating studies of individualblast cell colonies.
Blast cell colonies identified in cultures containing 100
ng/mL rrSCF or 200 U/mL IL-3 were individually picked
and transferred to secondary cultures containing 200 U/mL
IL-3 and 2 U/mL Ep by using an Eppendorf micropipette.
The secondary colonies were counted on day 10. Analysis of
the types and number of secondary colonies derived from
10 representative multipotential blast cell colonies in each
group is shown in Table 3. As we reported previously,' very
heterogeneous distribution of secondary colonies, including
the incidence of GEMM colonies, as well as GM colonies
was seen. The replating efficiencies of the individual blast
cell colonies supported by the two factors were comparable.
Replating studies ofpooled blast cells. As shown in Table
3, the blast cell colonies are highly enriched for progenitors.
Also, they are devoid of stromal cells. Pooled blast cells
constitute unique progenitor cell populations for studies of
growth factor effects. Accordingly, we pooled blast cell
colonies consisting of 50 to 500 blast cells (mean f SD,
159 +- 131) that were identified in cultures of post-5-FW
spleen cells supported by 200 U/mL of IL-3 ahd plated 50
blast cells per plate in the presence of Ep and varying
combinations of early acting factors. The results are presented in Table 4. In the presence of a combination of
rrSCF, IL-3, IL-6, and Ep, 38 of 50 blast cells formed
colonies indicating 76% or higher progenitor incidence.
Similar to the culture studies of normal bone marrow cells
shown in Table 1, IL-3 and Ep supported formation of GM,
GEM, and GEMM colonies. A combination of rrSCF and
Ep and a combination of IL-6 and Ep supported only a few
GEMM colony formations. A combination of rrSCF, IL-3,
and Ep and a combination of IL-3, IL-6, and Ep supported
formation of 12 & 2 and 13 f 3 GEMM colonies, respectively. These numbers were not significantly different from
10 r 2 GEMM colonies supported by IL-3 plus Ep.
Because the majority of progenitors in the blast cell
colonies are in the cell cycle, these observations are
consistent with the notion that rrSCF and IG6 are synergistic factors for the entry of dormant stem cells into cell cycle.
A combination of rrSCF, IL-6, and Ep did not enhance
GEMM colony formation above the levels supported by
IL-3 plus Ep or IL-6 plus Ep in contrast to the observations
Table 4. Effects of Combinations of Growth Factors on Colony Formation From Pooled Blast Cells
No. of Coloniesl50 Cells
Stimuli
GM
E
GEM
GMM
GEMM
Total
0
0
0
0
EP
SCF, Ep
IL-3, Ep
IL-6, Ep
SCF, IL-3, Ep
SCF, IL-6, Ep
IL-3, IL-6, Ep
SCF, IL-3, IL-6, EP
0
0
122 1
23 f 5
1222
26 2 2
17? 1
21 f 1
21 f 3
0
0
0
2?1
0
1 f 1
0
1 f 1
0
0
0
2 f 1
10 f 2*
2 f 1
12 f 2'
2 f 1
13 2 3'
152 1
0
0
1421
35 f 2
1321
39 2 2
19 f 2
35 f 4
38 f 3
None
0
121
0
0
1f 1
0
0
0
0
0
1 f 1
0
0
0
Colonies were scored on day 10 of incubation. Data represent mean f SD of quadruplicate cultures. Abbreviations for colony types are as
presented in Table 1. Concentrationsof factors: Ep, 2 U h L ; SCF, 100 ng/mL; IL-3.200 U/mL; IL-6, 100 ng/mL.
*There were no statistical differences among these numbers.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1228
TSUJI, ZSEBO, AND OGAWA
presented in Table 2, experiment A. This observation also
supports the notion that the primary targets of the rrSCFs
synergistic effects are dormant stem cells.
rrSCF plus E p supported formation of GM colonies from
pooled blast cells. The types of GM colonies supported by
rrSCF are compared with IL-6 and IL-3 by cytologic
analysis in Table 5. IL-3 and Ep supported formation of a
variety of types of colonies. As we have shown previou~ly,'~
IL-6 mostly supported two types of colonies, macrophage
colonies as well as neutrophiVmacrophage colonies. In
contrast, rrSCF supported formation of neutrophil colonies
as well as neutrophil/macrophage colonies. In a separate
report: we have shown that IL-11 supports formation of
only macrophage colonies. All of these synergistic factors
appear to overlap in the effects on dormant stem cells as
well as on later stages of neutrophil/macrophage development.
DISCUSSION
The fact that mice with W and S1 mutations present with
variable multilineage hematopoietic defects"-** indicates
that normal c-kit and the ligand for c-kit are important in
the development of the hematopoietic system. In this
report, we attempted to characterize the effects of rrSCF
(c-kit ligand) on primitive hematopoietic progenitors, including blast cell colony-forming cells using marrow and spleen
cells harvested from mice that had been treated with 150
mgkg 5-FU.We observed that rrSCF, similar to previously
identified syngeneic factors such as IL-6, G-CSF, and IL-11,
augments IL-3-dependent proliferation of multipotential
blast cell colony formation by shortening the dormant
period in which these cells reside; namely, rrSCF in
combination with IL-3 hastened the development of multipotential blast cell and multilineage colonies.
Unexpectedly, the combination of rrSCF and IL-6 and
the combination of rrSCF and G-CSF showed synergism in
the proliferation of the dormant multipotential progenitors.
Because the combination of rrSCF, IL-6, and E p failed to
enhance GEMM colony formation from pooled blast cells
beyond the level supported by the combination of IL-6 and
Ep, it was unlikely that rrSCF, like IL-3, supports continued
proliferation of multipotential progenitors after the cells
exit from Go. It was also possible that rrSCF, IL-6, and E p
interacted with endogenously available growth factors such
as IL-3 when these factors enhanced GEMM colony formation from day 2 post-5-FU marrow cells. Together these
studies indicate that SCF plays a role in the cell cycling of
dormant hematopoietic stem cells.
Previously we have reported that other synergistic factors
possess direct colony-stimulating activities in neutrophil/
macrophage lineages. By using replating of pooled blast
cells, we have shown that IL-6 supports formation of
macrophage colonies as well as neutrophiVmacrophage
colonie~,'~
while G-CSF supports formation of neutrophil
colonies as well as neutrophil/macrophage colonies. Recently, we demonstrated that IL-11 enhances the development of macrophage colonies as well as neutrophil/
macrophage colonies.6 Now we show that rrSCF also acts
directly on the progenitors for neutrophil and neutrophil/
macrophage colonies. Ruscetti et alB reported that neutrophils and their progenitors are reduced in number in the
blood and marrow of Sl/Sld mice. Sonoda et a P reported
that W W mice show poor neutrophil response to CSFproducing tumor. Our observations in culture corroborate
these in vivo observations and indicate the physiologic
importance of SCF during adult life.
In mice with W mutation varying degrees of stem cell
defects have been observed. The homozygous state of W
mutation is not compatible with an adult's life, indicating
the importance of SCF during ontogeny of hematopoiesis.
The culture studies described in this report indicated the
role of rrSCF in the entry into cell cycle of dormant stem
cells and the direct effects of rrSCF on the neutrophil/
macrophage proliferation of adult mice. Further studies are
necessary for defining the precise roles of SCF in adult
hematopoiesis in vivo.
ACKNOWLEDGMENT
The authors thank Geneva Winkler for her excellent technical
assistance and Dr Pamela N. Pharr, Anne G . Leary, and Linda S.
Vann for their assistance in the preparation of this manuscript.
Table 5. Cytologic Analysis of Types of Secondary Colonies Derived From Pooled Blast Cells
Factor in Secondary
Culture
Lineage Expressed in
Secondary Colonies
n
+
+
+
+
+
+
+
+
+
m
+
+
+
+
+
+
+
Mast
+
+
+
e
M
SCF
IL-3
IL-6
5
6
10
1
4
6
5
0
0
0
0
1
5
1
1
t
0
0
2
3
1
1
1
+
+
+
+
0
2
0
1
1
1
4
14
38
+
+
t
+
+
E
t
+
0
+
+
+
+
0
Totals
Cultures contained 2 U/mL Ep.
Abbreviations: n, neutrophil; m, monocyte/macrophage; Mast, mast cell; e, eosinophil; E, erythrocyte; M, megakaryocyte.
0
0
0
0
1
11
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SCF ON BLAST CELL COLONIES
1229
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1991 78: 1223-1229
Enhancement of murine blast cell colony formation in culture by
recombinant rat stem cell factor, ligand for c-kit
K Tsuji, KM Zsebo and M Ogawa
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