Enhancement of Murine Hematopoiesis by Synergistic Interactions Between Steel
Factor (ligand for c-kit), Interleukin-11, and Other Early Acting Factors
in Culture
By Kohichiro Tsuji, Stewart D. Lyman, Tetsuo Sudo, Steven C. Clark, and Makio Ogawa
Entry into the cell cycle of dormant hematopoietic progenitors appears to be regulated by multiple synergistic factors,
including interleukin-6 (IL-6). granulocytecolony-stimulating
factor (G-CSF), IL-11, and the ligand for c-kit, which is also
known as steel factor (SF). We have tested the effects of
these and other hematopoieticfactors on the proliferation of
partially enriched dormant murine progenitors in the presence and absence of serum. In serum-containing cultures, SF
and IL-11 interacted to support the formation of multilineage
colonies; the level of colony formation was comparablewith
the colony formation supportedby other effective two-factor
combinations. In serum-free cultures, colony formation supported by two factors was significantly less than that in
serum-containing culture and the most effective two-factor
combination in serum-free culture was SF plus IL-3. In
serum-free cultures, three-factor combinations consisting of
SF, IL-3, and one of IL-6, G-CSF, or IL-11 yielded colony
formation that was comparable with that seen in serumcontainingcultures. These studies indicatethat IL-11 belongs
to a group of early-acting hematopoietic synergistic factors
that now includes IL-6, G-CSF, and IL-11. In contrast, SF is
unique among the synergistic factors in that it interacts
either with growth factors such as IL-3 or GM-CSF or with
synergistic factors such as IL-6, IL-11, or G-CSF.
o 1992by The American Society of Hematology.
I
enriched about 150-fold by a combination of density gradient centrifugation and negative immunomagnetic bead
selection using lineage-specificmonoclonal antibodies (MoAbs).15 We have found that IL-1l, like IL-6 and G-CSF,
acts synergistically with SF in support of multilineage
colony formation with enriched bone marrow cells in
serum-containing cultures. In the absence of serum, IL-3
or, less efficiently, GM-CSF, is also required.
N THE STEADY STATE bone marrow, most hematopoietic stem cells are in a quiescent state termed Go. In
this state, they divide infrequently to yield actively proliferating multipotential progenitors. During regeneration from
marrow aplasia, the stem cells are activated to divide more
frequently. Several cytokines appear to be involved in the
process of activation and regulation of the proliferation of
the stem cells and their progeny. Interleukin-3 (IL-3)
supports the proliferation of multipotential progenitors,
but it does not appear to trigger cycling of dormant stem
cells.’ IL-4 also supports proliferation of multipotential
progenitors in much the same fashion as IL-3.2Granulocytemacrophage colony-stimulatingfactor (GM-CSF) was also
shown to support proliferation of a subpopulation of
multipotential progenitors responding to IL-3.3 More recent studies indicate that the entry into cell cycle of the
dormant stem cell is regulated by several synergistic factors.
Studies of murine multipotential blast cell colonies showed
that IL-64 and granulocyte-CSF (G-CSF)5 enhance IL-3dependent proliferation of progenitors, in part by shortening the Go period of the dormant cells. While a similar
effect was observed with IL-1 in cultures of crude post-5fluorouracil (5-FU) marrow cells$ analysis of cultures of
purified human marrow cells indicated that the IL-1 effect
is indirect, probably mediated in part by IL-6 and G-CSF.7
Recently, we identified two additional cytokines that
appear to regulate the kinetics of early hematopoietic
progenitors. IL-11, which was identified in medium conditioned by primate bone marrow stromal cells (PU-34):
works synergistically with IL-39 and IL-4lo in support of
proliferation of early progenitors by shortening their Go
period. More recently, steel factor (SF) (also known as mast
cell growth factor,l1kit ligand,I2and stem cell factor13)was
shown to act synergisticallywith IL-3 in support of progenitor proliferation by shortening the Go period.14 Synergism
in support of proliferation of multipotential progenitors
was also observed between SF and IL-6 or G-CSF, but not
between SF and IL-4.14 In the present study, we have
examined the effects of SF and IL-11 in varying combinations with other cytokines on early hematopoietic progenitors in serum-containing and serum-free cultures. We used
the bone marrow cells of 5-FU-treated mice that were
Blood, Vol79, No 11 (June 11,1992: pp 2855-2860
MATERIALS AND METHODS
Cellpreparation. Female (C57B116 x DBA/Z) F1 mice, 10 to
15weeks old, were obtained from ARS Spraque Dawley (Indianapolis, IN). Cells were obtained from marrows of mice that had been
injected with 150 mglkg 5-FU (Adria Laboratories, Columbia,
OH) through the tail veins 2 days before marrow harvest. Density
separated, lineage-negative cells were prepared from the day 2
post-5-FU marrow cells as described previo~sly.~~
The cell preparation is about 150-fold enriched for the hematopoietic progenitors
that can be assayed with IL-3 and IL-6.
Factors. Recombinant murine SF expressed in yeast was purified as described previously.I6 Recombinant human IL-11 expressed in COS-1 cells was purified as described? Escherichia
coli-derived recombinant human IL-6 had a specific activity of 2 x
lo6 Ulmg. Recombinant murine GM-CSF was purified from
conditioned medium of Chinese hamster ovary (CHO) cells trans-
From the Department of Medicine, Medical University of South
Carolina and Ralph H. Johnson Department of Veterans Affairs
Medical Center, Charleston, SC; Immunpx Corporation, Seattle WA;
Biomaterial Research Institute Co, Lid, Yokohama, Japan; and the
Genetics Institute, Cambridge, MA.
Submitted November 12,1991; accepted January 29,1992.
Supported by National Institutes of Health Grant No. DK32294, the
Research Service of the Ralph H. Johnson Department of Veterans
Affairs Medical Center, and contributions from Erin Brewery Co,
Japan. M. 0.is VA Medical Investigator.
Address reprint requests to Makio Ogawa, MD, PhD, VA Medical
Center, 109 Bee St, Charleston, SC 29403.
The publication costs of this am‘cle were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-49711921 7911-OO18$3.00/0
2855
2856
TSUJI ET AL
fected with cDNA encoding murine GM-CSF. The source of IL-3
was conditioned medium of CHO cells that were engineered to
produce murine IL-3 in high titer (30,000 U/mL). Recombinant
human G-CSF that was expressed in E coli and recombinant
human erythropoietin (Epo) were generously provided by Drs
Lawrence Souza and Fu-kuen Lin of AmGen (Thousand Oaks,
CA). Recombinant murine IL-4, expressed in yeast, was purified as
described" and was provided by Dr Connie Flatynek of Sterling
Drug Inc (Malvern, PA). Concentrations of factors in these
experiments were as follows: SF, 100 ng/mL; IL-11, 100 U/mL;
IL-3, 200 U/mL; GM-CSF, 200 U/mL; IL-6, 100 ng/mL; G-CSF,
100 ng/mL; and IL-4,lO ng/mL.
Clonal cell culture. Methylcellulose cell cultures were established in 35-mm Lux suspension culture dishes (#5221R, Nunc,
Inc, Naperville, IL). In serum-containingculture, 1 mL of culture
mixture contained 500 enriched cells, a-medium (Flow Laboratories, Inc, McLean, VA), 1.2% 1,500 cp methylcellulose (Fisher
Scientific Co, Norcross, GA), 1% deionized, fraction V bovine
serum albumin (BSA; Sigma Chemical Co, St Louis, MO), 1 x
mol/L 2-mercaptoethanol (Eastman Organic Chemicals, Rochester, NY),30% fetal calf serum (FCS; Hyclone Laboratories, Inc,
Logan, UT), 2 U/mL Epo, and designated hematopoietic factors.
Because Epo was added to all cultures, the effect of one-, two-, or
three-factor combinations are due, in actuality, to combinations of
two, three, or four growth factors, respectively. Dishes were
incubated at 37°C in a humidified atmosphere with 5% COz in air.
In serum-free culture, FCS was replaced by a combination of 1%
deionized crystalized BSA (Sigma Chemical Co), 600 kg/mL fully
iron-saturated human transferrin ( > 98% pure) (Sigma Chemical
Co), 10 kg/mL lecithin (Sigma Chemical Co),6 +g/mL cholesterol
(Sigma Chemical Co),and 1 X lo-' mol/L sodium selenite (Sigma
Chemical Co); fraction V BSA was omitted. Dishes were incubated
at 37°C in a humidified atmosphere with a mixture of 5% C02,5%
0 2 , and 90% N2. Except for megakaryocyte colonies, colonies
consisting of 50 or more cells were scored on an inverted microscope on day 14 of incubation. Megakaryocyte colonieswere scored
when they contained four or more megakaryocytes. Abbreviations
for colony types are as follows: GM, granulocyte/macrophage
colonies; M, megakaryocyte colonies; GEM, granulocyte/erythrocyte/macrophage colonies; GMM, granulocyte/macrophage/
megakaryocyte colonies; GEMM, granulocyte/erythrocyte/macrophage/megakaryocyte colonies; and B1, blast cell colonies.
RESULTS
Colony formation from nonenriched day 2 post-5-FU
marrow cells. Results of the analysis of colony formation
from day 2 post-5-FU marrow cells in FCS-containing
culture are presented in Table 1. SF and IL-11 individually
supported formation of a small number of colonies, most of
which contained granulocyte and monocytes (GM colonies), as reported previo~sly?.'~Other factors, including
IL-3, IL-6, G-CSF, IL-4, and GM-CSF, also supported the
growth of G M colonies. In addition, IL-3 and G-CSF
supported formation of a few multilineage and blast cell
colonies. A marked synergy was evident between S F and
IL-11 in support of multilineage colony formation in the
presence of Epo. As presented p r e v i o u ~ l yS, ~F ~in combinations with IL-3, IL-6, o r G-CSF supported formation of
many multilineage colonies. Also noted previously were the
synergies between 1L-11 and IL-3: IL-11 and IL-4,'" and
IL-11 and GM-CSF in support of multilineage colony
formation, although the combination of IL-11 and GM-CSF
produced half of the number of colonies supported by IL-3
plus IL-11. S F and IL-4 did not show synergism, also as
Table 1. Effects of SF, IL-11, and Other Factors on Colony Formation
From Day 2 Post-5-FU Marrow Cells in Serum-Containing Culture
No. of Colonias/5 x lO4Cells
Factors
GM
IL-11
IL-3
IL-6
G-CSF
IL-4
GM-CSF
SF, IL-11
SF, IL-3
SF, IL-6
SF, G-CSF
SF, IL-4
SF, GM-CSF
IL-11, IL-3
IL-11, IL-6
IL-l1.G-CSF
IL-11. IL-4
IL-l1,GM-CSF
1 2 1
221
522
3 2 1
5 2 1
1 2 1
3+0
1022
8+1
1021
6 2 1
3 2 1
6 2 2
9 2 1
3+1
421
9+0
7 2 0
SF
M
0
0
0
0
0
0
0
1 2
l e
1 2
0
0
0
1 2
0
1 2
0
0
GEM
1
1
1
1
1
0
1 2 1
1 2 1
0
0
0
0
1 2 0
1+1
1 2 0
1+1
0
0
1 2 1
0
0
0
121
GEMM
61
0
0
0
0
221
1 2 1
0
1 2 1
1 2 0
1 2 1
0
0
0
0
1 4 ~ 1 0~
0
11+3*
0
14+2*
0
13+2*
0
1 2 1
3 2 1
0
0
1321*
221
1 2 0
4 2 1
0
10+1*
0
721"
0
Total
1 2 1
3 2 1
9 2 2
4 2 1
7 2 2
1 2 1
3 2 0
2622
2125
2622
20+2
4 2 1
9+1
2422
6 2 2
9 2 1
19+1
1520
Culture was performed in the presence of 2 U/mL Epo. Numbers
indicate mean + SD of colony numbers in quadruplicate cultures.
*These numbers are different from those supported by single factors
atP < .01 by Student's t-test.
noted p r e v i ~ u s l y A
. ~ ~few multilineage colonies were observed in cultures containing IL-11 plus IL-6 and IL-11 plus
G-CSF.
Colony formation from enriched day 2 post-5-FU marrow
cells in serum-containing and serum-free culture. W e next
examined colony formation from enriched day 2 post-5-FU
marrow cells and the results are presented in Tables 2 and
3. In serum-containing cultures (Table 2), the overall
pattern of colony formation was very similar to that reported in Table 1 with nonenriched day 2 post-5-FU
marrow. However, S F and IL-11 as single agents failed to
support colony formation from enriched marrow cells. The
most potent two-factor combinations for multilineage colony formation were again S F plus IL-11, S F plus IL-3, SF
plus IL-6, SF plus G-CSF, and 1L-11 plus IL-3. In contrast
to SF, which interacted with other synergistic factors, IL-11
did not show synergism with other synergistic factors,
including IL-6 and G-CSF. The combinations of IL-11 with
IL-4 or GM-CSF supported a few multilineage colonies
from the enriched marrow cells, although less than that
observed with nonenriched cells (Table 1).
In serum-free cultures (Table 3), no colony formation
was observed when individual factors were tested in the
presence of Epo. Formation of significant number of
multilineage colonies by two-factor combinations were
observed only with S F plus IL-3. Other two-factor combinations, including SF plus IL-11, supported no or few multilineage colonies.
Effects of three-factor Combinations containing SF and
IL-I1 on colony formation from enriched day 2 post-5-FU
marrow cells. In serum-free cultures of enriched marrow
cells, combinations of S F and IL-11, S F and IL-6, and S F
and G-CSF did not support multilineage colony formation
2857
SYNERGY AMONG EARLY ACTING FACTORS
Table 2. Effects of SF, IL-11, and Other Factors on Colony Formation
From Enriched Day 2 Post-5-FU Cells in Serum-Containing Culture
No. of Colonies1500Cells
Factors
GM
SF
IL-11
IL-3
IL-6
G-CSF
IL-4
GM-CSF
SF, IL-11
SF, IL-3
SF, IL-6
SF, G-CSF
SF, IL-4
SF, GM-CSF
IL-11, IL-3
IL-11, IL-6
IL-11, G-CSF
IL-11, IL-4
IL-11, GM-CSF
0
0
4 2
2 2
4 2
1 2
12
4 2
4 2
4 2
4 2
2 2
2 2
6 2
7 2
2 2
4 2
4 2
1
1
1
1
1
1
0
2
1
0
1
1
2
1
1
2
M
GEM
GEMM
Bl
Total
0
0
6 2 1
2 2 1
5 2 2
1 2 1
121
16 2 1
12 2 2
142 1
14 2 0
2 2 0
4+ 1
192 1
7 2 2
4 2 0
8 2 2
8 2 1
0
0
0
0
0
0
0
0
0
0
121
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1 2 1
0
0
0
0
0
0
0
0
0
0
0
2 2 1
0
0
0
0
1022'
822"
1021'
1021*
0
2 2 0
13+2*
0
1 2 0
421t
42Ot
0
0
0
121
0
0
0
0
0
0
0
0
0
0
1 2 1
0
0
Culture was performed in the presence of 2 U/mL Epo. Numbers
indicate mean 2 SD of colony numbers in quadruplicate cultures.
*These numbers are different from those supported by single factors
at P < .01 by Student's t-test.
tColony formation supported by these factor combinations is less
than the colony formation supported by the factor combinations
designated with (") at P < .05.
effectively (Table 3), whereas in serum-containing cultures,
these combinations effectively supported multilineage colony formation (Table 2). It was possible that the apparent
synergism between SF and one of the synergistic factors on
multilineage colony formation required additional factors
in serum-free culture. Consequently, we tested three-factor
Table 3. Effects of SF, IL-11, and Other Factors on Colony Formation
From Enriched Post-5-FU Cells in Serum-Free Culture
No. of Colonies/500Cells
Factors
SF
IL-11
IL-3
IL-6
G-CSF
IL-4
GM-CSF
SF, IL-11
SF, IL-3
SF, IL-6
SF, G-CSF
SF, IL-4
SF, GM-CSF
IL-11, IL-3
IL-11, IL-6
IL-11, G-CSF
IL-11, IL-4
IL-11, GM-CSF
GM
GEM
GMM
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2 1
2 2
2 1
2 1
0
2 2 1
2 2 1
0
121
0
0
0
0
121
0
0
0
7 2 2
121
0
0
0
0
0
4
3
3
3
0
0
0
0
0
GEMM
0
0
0
81
0
0
0
0
0
0
0
0
0
0
0
0
121
0
0
1*1
0
0
0
0
0
0
0
5 2 2
1121
5 2 0
4 2 1
0
3 2 1
2 2 1
0
0
0
0
121
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Table 4. Colony Formation From Enriched Post-5-FU Marrow Cells
in Serum-Containing and Serum-Free Cultures
No. of Colonies/500Cells
Total
0
0
0
combinations, including SF and IL-11, in serum-containing
and serum-free cultures of enriched marrow cells. The
results are shown in Table 4. In serum-containing cultures,
addition of IL-3, IL-6, G-CSF, or GM-CSF to the combination of SF and IL-11 failed to enhance multilineage colony
formation over the level supported by SF and IL-11.
Addition of IL-4 to the combination of SF and IL-11
partially inhibited multilineage colony formation, possibly
due to the reported negative effects of IL-4 on hematopoietic proliferation.1s In serum-free cultures, the combination
of SF and IL-11 did not support formation of multilineage
colonies, as already shown in Table 3. Only when IL-3 or
GM-CSF was added to the combination of SF and IL-11
were multilineage colonies obtained. The number of multilineage colonies supported by SF, IL-11, and IL-3 were
comparable with serum-containing cultures. Combinations
of three synergistic factors without IL-3 or GM-CSF did not
effectively support multilineage colony formation. These
results may indicate that FCS contains other growth factors
possessing IL-3- or GM-CSF-like functions.
The observations in the serum-free cultures (Table 4)
indicated that the combinations of SF, IL-11, and GM-CSF
supported significant multilineage colony formation. In the
next experiment, we tested the three-factor combinations,
including SF and IL-3 or SF and GM-CSF. The results of
analysis in serum-containing and serum-free cultures are
presented in Table 5. In serum-containing cultures, the
combination of IL-3 and SF with either IL-11, IL-6, or
G-CSF resulted in the formation of similar numbers of
multilineage and total colonies. Three-factor combinations
including GM-CSF and SF with either IL-11, IL-6, or
G-CSF also supported formation of multilineage and total
colonies. In general, cultures containing IL-3 produced
more colonies than the combinations including GM-CSF,
agreeing with the earlier observations that the targets of
IL-3 may be at earlier stages of development than those of
0
0
0
Culture was performed in the presence of 2 U/mL Epo. Numbers
indicate mean f SD of colony numbers in quadruplicate cultures.
Factors
Serum-containing
culture
SF- IL-11
SF, IL-11, IL-3
SF, IL-11, IL-6
SF,IL-11,G-CSF
SF, IL-11, IL-4
SF, IL-11, GM-CSF
Serum-free culture
SF, IL-11
SF, IL-11, IL-3
SF,IL-11, IL-6
SF,IL-1l.G-CSF
SF, IL-11, IL-4
SF, IL-11, GM-CSF
GM
M
GEM
GEMM
Total
2
2
2
2
2
0
1?1
0
1 2 1
0
0
1 2 1
0
0
1 2 1
2 2 1
0
1421
1524
1422
1523
621*
1422
1723
1924
1622
2022
1321
1722
2 5 1
3 2 1
2 2 0
42 1
1 2 1
221
0
0
0
0
0
1 2 1
1 2 1
221
0
1522
0
0
0
0
0
8 2 2
2*1
1922
3 2 1
72 1
1 2 1
1022
2
3
2
3
5
3
1
1
0
0
1
2 0
0
12 1
Culture was performed in the presence of 2 U/mL Epo. Numbers
indicate mean 2 SD of colony numbers in quadruplicate cultures.
*Significantly (P < .01) different from colonyformation supported by
other factor combinations in serum-containing culture.
2858
TSUJI ET AL
Table 5. Colony Formationby Enriched Day 2 Post-5-FU Marrow
Cells Supportedby Three-Factor Combinations
Including IL-3 Plus SF or OM-CSF
No. of ColoniesI500 Cells
Factors
Serum-containing
culture
IL-3, SF, IL-11
IL-3, SF, IL-6
IL-I,SF,G-CSF
GM-CSF,SF,IL-11
GM-CSF,SF,IL-6
GM-CSF,SF,G-CSF
Serum-freeculture
IL-3, SF
IL-3, IL-I1
IL-3, IL-6
IL-3, G-CSF
IL-3, SF, IL-I 1
IL-3, SF, IL-6
IL-3,SF,G-CSF
GM-CSF, SF
GM-CSF,SF,IL-11
GM-CSF,SF,IL-6
GM-CSF,SF,G-CSF
GM
M
GEM
0
0
622
522 121
0
5 2 1 12 1
0
4 k 1
0
121
522
0
0
6* 3
0
0
321
422
322
522
320
521
622
220
2+1
2k 1
221
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1k
0
12
12
0
0
0
12
1
0
1
1
GMM
GEMM
Total
0
0
0
0
0
0
14?1*
16k2*
152 3t
8 2 2*
1 0 2 1'
9 2 It
2021
2221
21 2 1
13 2 0
1522
15 2 2
0
7 2 2 1021
0
121
521
121 2k1
6k1
0
121
721
0
13+0* 1 6 2 0
0
13+2* 1 9 2 1
0
12 k 3 t 1 9 2 1
0
120
320
0
5 2 I* 7 2 0
0
7 + I* 9 2 0
0
62Ot 9 2 2
Culture was performed in the presence of 2 U/mL Epo. Numbers
indicate mean 2 SD of colony numbers in quadruplicate cultures.
*Colony formation seen in GM-CSF-containing cultures was less
than that in IL-3-containing cultures atP < .01.
tColony formation seen in GM-CSF-containing cultures was less
than than in IL-hontaining cultures at P < .02.
GM-CSF? In serum-free cultures, the order of the effectiveness of factor combinations was similar to that in serumcontaining cultures. Again, the best combinations for total
and multilineage colony formation were SF and IL-3 with
either IL-11, IL-6, or G-CSF. GM-CSF was less effective
than IL-3 in these three-factor combinations.
Cultures in mouseplasma. It was possible that the batch
of FCS we used contained exceptionally high concentrations of IL-3, GM-CSF, or other proteins with similar
hematopoietic functions, thereby significantly affecting our
results from serum-containing cultures. To test the physiologic relevance of our studies in FCS-containing cultures,
we performed the culture studies of day 2 post-5-FU
marrow cells using 5% mouse plasma and compared the
results with those from cultures supplemented with 5%
FCS. As shown in Table 6, mouse plasma supported the
synergistic effects of the hematopoietic growth factors on
multilineage colony formation that were identical to those
observed in 5% FCS culture.
DISCUSSION
Analysis of the hematopoietic blast cell colony culture
system in our laboratory has shown that the entry into cell
cycle of dormant stem cells can be regulated by several
combinations of cytokines, including IL-3, IL-6, G-CSF,
and two more recently identified cytokines, IL-11 and SF.
IL-11 was originally identified in the supernatant of cultures of nonhuman primate stromal cell line PU-34.8 The
reported biologic functions of recombinant IL-11 overlap
significantly with those of IL-6, including enhancement of
IL-3-dependent megakaryopoiesis in culture,19stimulation
of hepatic acute phase protein synthesis,20and enhancement of IL-S9 and IL-4-dependentlO proliferation of
multipotential progenitors in culture. SF, which was identified and its DNA cloned by investigators in several laboratories, is variously called mast cell growth factor," kit ligand,12
and stem cell factor.'* SF is encoded by the SI locus in
mouse and is a ligand for c-kit, a proto-oncogene encoding a
tyrosine kinase receptor.21 Mutations in the SI gene may
cause severe macrocytic anemia, mast cell deficiency, hypopigmentation, and sterility in mice.22These adverse effects
on hematopoiesis caused by the mutations in the SI locus
indicate that SF must play an important role in ontogeny of
the hematopoietic system. Currently, a number of investigators are engaged in the characterization of the effects of SF
on hematopoiesis in adult animals and there have already
been a number of reports on significant effects on hematopoiesis in c u l t ~ r eand
~ - in
~ vivo.%
~
Generally, SF appears to
be a very potent synergistic factor with other cytokines. In
our laboratory, we have observed that SF interacts with
IL-3, IL-6, and G-CSF and enhances blast cell colony and
multilineage colony formation by shortening the dormancy
(Go) period of the early pr0genit0rs.l~
In this study we examined the effects of interactions
between SF and IL-11 in various combinations with other
early acting cytokines on the growth of early hematopoietic
progenitors. We used enriched bone marrow cells from
5-FU-treated mice that had been purified about 150-foldby
a combination of density gradient centrifugation and negative immunomagnetic bead selection using lineage-specific
MoAbs.15 The results indicated that SF and IL-11 interact
synergistically to support proliferation of hematopoietic
stem cells in serum-containing culture and that this combination was equally effective as some of the other two- or
three-factor combinations. In serum-free cultures, only the
Table 6. Comparision Between Mouse Plasma and FCS in Support of
Colony FormationFrom Day 2 Post-5-FU Marrow Cells
No. of Coloniesll x lV Cells
GM
Factors
~
~
~
Mouse plasma (5%)
SF
IL-3
IL-6
SF, IL-3
SF, IL-6
IL-3, IL-6
SF, IL-3, IL-6
FCS (5%)
SF
IL-3
IL-6
SF, IL-3
SF, IL-6
IL-3 ,IL-6
SF, IL-3, IL-6
~
2k1
9+2
421
821
1421
15k1
1520
2+0
5k2
121
722
1521
1621
16+3
M
~
BI
GEMM
~
~~
Total
~
0
1+1
0
620
1123
1322
1522
0
1+1
1k1
120
121
121
0
221
11+3
5 k l
1521
2622
3023
32k1
0
0
121
0
121
121
120
0
1120
1724
1423
1523
0
0
0
221
0
0
0
220
722
121
2122
3324
3024
3351
0
0
0
0
0
1k1
221
0
2 k l
Culture was performed in the presence of 2 U/mL Epo. Numbers
indicate mean 2 SD of colony numbers in quadruplicate cultures.
SYNERGY AMONG EARLY ACTING FACTORS
2859
combinations containing SF and IL-3 were effective in
support of GEMM colony formation, in agreement with the
recent observation by Migliaccio et
IL-11 appears to
belong to the class of hematopoietic factors that includes
IL-6 and G-CSF that can function to enhance the colonysupporting abilities of combinations of IL-3 and SF or
GM-CSF and SF.
These observations confirmed and extended the previous
model of early h e m a t o p ~ i e s i sIn
. ~this
~ model, we proposed
that IL-3 and GM-CSF are intermediate-acting growth
factors regulating the survival and proliferation of actively
cycling multipotential progenitors and that IL-6 and G-CSF
individually can act to shorten the dormancy (Go) period of
the stem cells. There is a significant overlap in the functions
of these two factors and there appears to be no interaction
between IL-6 and G-CSF in support of stem cell prolifera-
tion. Now we add IL-11 to this group because IL-11
interacts neither with IL-6 nor G-CSF but can interact with
IL-4.l0 Although SF participates in stimulation of the
cycling of dormant progenitors, several features render this
factor unique; it interacts with IL-3, IL-11, IL-6, and
G-CSF, but not IL-4,I4 in support of proliferation of early
progenitors. It remains to be clarified how these factors
interact with each other at the cellular and molecular level.
Recent efforts have focused on in vivo and ex vivo manipulation of dormant hematopoietic stem cells because of the
potential application in bone marrow transplantation and
in treatment of therapy-induced bone marrow failure. Our
results in culture indicate that useful combinations of
factors for stimulation of stem cells might include SF and
IL-3 along with one member of the group of synergistic
factors IL-6, G-CSF, or IL-11.
REFERENCES
1. Suda T, Suda J, Ogawa M, Ihle J N Permissive role of
interleukin 3 (IL-3) in proliferation and differentiation of multipotential hemopoietic progenitors in culture. J Cell Physiol 124:182,
1985
2. Kishi K, Ihle JN, Urdal DL, Ogawa M: Murine B-cell
stimulatory factor-1 (BSF-1)linterleukin-4 (IL-4) is a multi-CSF
which acts directly on primitive hemopoietic progenitors. J Cell
Physioll39463,1989
3. Koike K, Ogawa M, Ihle JN, Miyake T, Shimizu T, Miyajima
A, Yokota T, Arai K: Recombinant murine granulocyte-macrophage (GM) colony-stimulatingfactor supports formation of GM
and multipotential blast cell colonies in culture: Comparison with
the effects of interleukin-3.J Cell Physiol131:458,1987
4. Ikebuchi K, Wong GG, Clark SC, Ihle JN, Hirai Y, Ogawa M
Interleukin-6 enhancement of interleukin-3-dependent proliferation of multipotential hemopoieticprogenitors. Proc Natl Acad Sci
USA 84:9035,1987
5. Ikebuchi K, Clark SC, Ihle JN, Souza LM, Ogawa M:
Granulocyte colony-stimulating factor enhances interleukin-3dependent proliferation of multipotential hemopoietic progenitors. Proc Natl Acad Sci USA 85:3445,1988
6. Mochizuki DY, Eisenman JA, Conlon PJ, Larsen AD, Tushinski RJ: Interleukin 1 regulates hematopoietic activity, a role
previously ascribed to hemopoietin 1. Proc Natl Acad Sci USA
845267,1987
7. Leary AG, Ikebuchi K, Hirai Y, Wong GG, Yang Y-C, Clark
SC, Ogawa M: Synergism between interleukin-6 and interleukin-3
in supporting proliferation of human hemopoietic stem cells:
Comparison with interleukin-la. Blood 71:1759,1988
8. Paul SR, Bennett F, Calvetti JA, Kelleher K, Wood CR,
OHara RM Jr, Leary AC, Sibley B, Clark SC, Williams DA, Yang
Y-C Molecular cloning of a cDNA encoding interleukin 11, a
stromal cell-derived lymphopoietic and hematopoietic cytokine.
Proc Natl Acad Sci USA 87:7512,1990
9. Musashi M, Yang Y-C, Paul SR, Clark SC, Sudo T, Ogawa M
Direct and synergisticeffects of interleukin-11on murine hemopoiesis in culture. Proc Natl Acad Sci USA 88:765,1991
10. Musashi M, Clark SC, Sudo T, Urdal DL, Ogawa M:
Synergistic interactions between interleukin-11 and interleukin-4
in support of proliferation of primitive hematopoietic progenitors
of mice. Blood 78:1148,1991
11. Williams DE, Eisenman J, Baird A, Rauch C, Van Ness K,
March CJ, Park LS, Martin U, Mochizuki DY, Boswell HS,
Burgess GS, Cosman D, Lyman SD: Identification of a ligand for
the c-kit proto-oncogene. Cell 63:167,1990
12. Huang E, Nocka K, Bieier DR, Chu T-Y, Buck J, Lahm
H-W, Wellner D, Leder P, Besmer P: The hematopoietic growth
factor KL is encoded by the SZ locus and is the ligand of the c-kit
receptor, the gene produce of the W locus. Cell 63:225,1990
13. Zsebo KM, Wypych J, McNiece IK, Lu HS, Smith KA,
Karkare SB, Sachdev RK, Yuschenkoff VN,Birkett NC, Williams
LR, Satyagal VN, Tung W, Bosselman RA, Mendiaz EA, Langley
KE: Identification, purification, and biological characterization of
hematopoietic stem cell factor from buffalo rat liver conditioned
medium. Cell 63:195, 1990
14. Tsuji K, Zsebo KM, Ogawa M: Enhancement of murine
blast cell colony formation in culture by recombinant rat stem cell
factor (rrSCF), ligand for c-kit. Blood 78:1223, 1991
15. Shih JP, Zeng HQ, Ogawa M: Enrichment of murine
marrow cells for progenitors of multilineage hemopoietic colonies.
Leukemia (in press)
16. Anderson DM, Lyman SD, Baird A, Wignall JM, Eismann J,
Rauch C, March CY, Boswell HS, Gimpel SD, Cosman CJ,
Williams DE: Molecular cloning of mast cell growth factor, a
hematopoietin that is active in both membrane bound and soluble
forms. Cell 63:235, 1990
17. Park LS,Friend D, Grabstein K, Urdal D L Characterization of the high affinity cell-surface receptor for murine B-cellstimulating factor 1. Proc Natl Acad Sci USA 84:1669, 1987
18. Rennick D, Yang G, Muller-Sieburg C, Smith C, Arai N,
Takabe Y, Gemmell L: Interleukin 4 (B-cell stimulatory factor 1)
can enhance or antagonize the factor-dependent growth of hemopoietic progenitor cells. Proc Natl Acad Sci USA 84:6889, 1987
19. Bruno E, Briddell RA, Cooper RJ,Hoffman R: Effects of
recombinant interleukin 11 on human megakaryocyte progenitor
cells. Exp Hematol 19:378, 1991
20. Baumann H, Schendel P: Interleukin-11 regulates the hepatic expression of the same plasma protein genes as interleukin-6.
J Biol Chem 266:20121,1991
21. Chabot B, Stephenson DA, Chapman VM, Besmer P,
Bernstein A The proto-oncogene c-kit encoding a transmembrane
tyrosine kinase receptor maps to the mouse W locus. Nature
335:88,1988
22. Russell ES, Bernstein SE: Blood and blood formation, in
Green EL (ed): Biology of the Laboratory Mouse. New York, NY,
McGraw-Hill, 1966,p 351
23. McNiece IK, Langley KE, Zsebo KM: Recombinant human
stem cell factor synergiseswith GM-CSF, G-CSF, IL-3 and Epo to
stimulate human progenitor cells of the myeloid and erythroid
lineages. Exp Hematol19:226,1991
2860
24. Broxmeyer HE, Hangoc G, Cooper S, Anderson D, Cosman
D, Lyman SD, Williams D E Influence of murine mast cell growth
factor (c-kit ligand) on colony formation by mouse marrow hematopoietic progenitor cells. Exp Hematol19:143,1991
25. De Vries P, Base1 KA, Eiseman JR, Alpert AR, Williams
D E The effect of recombinant mast cell growth factor on purified
murine hematopoietic stem cells. J Exp Med 173:1205,1991
26. Migliaccio G, Migliaccio AR, Valinsky J, Langley K, Zsebo
K, Visser JWM, Adamson JW: Stem cell factor induces proliferation and differentiation of highly enriched murine hematopoietic
cells. Proc Natl Acad Sci USA 88:7420,1991
TSUJl ET AL
27. Metcalf D, Nicola N A Direct proliferative actions of stem
cell factor on murine bone marrow cells in vitro: Effects of
combination with colony-stimulating factors. Proc Natl Acad Sci
USA 88:6239,1991
28. Molineaux G, Migdalska A, Szmitkowski M, Zsebo K,
Dexter TM: The effects on hematopoiesis of recombinant stem cell
factor (ligand for c-kit) administered in vivo to mice either alone or
in combination with granulocyte colony-stimulating factor. Blood
78:961,1991
29. Ogawa M: Effects of hemopoietic growth factors on stem
cells in vitro. Hematol Oncol Clin North Am 3:453,1989