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The Action of Bryostatin on Normal Human Hematopoietic Progenitors Is
Mediated by Accessory Cell Release of Growth Factors
By Saul J. Sharkis, Richard J. Jones, Mary L. Bellis, George D. Demetri, James D. Griffin,
Curt Civin, and W. Stratford May
Since enrichment of human bone-marrow hematopoietic
progenitors is becoming more feasible and since purified
growth factors are now available, w e sought t o study the
action of growth factors on CD34-positive enriched cultures of human bone-marrow cells. We tested the effect of
recombinant human (rh) granulocyte-macrophage colonystimulating factor (GM-CSF), rh interleukin-3 (IL-3). or a
unique biologic response modifier, bryostatin 1, on the
growth of purified CD34 cells obtained by limiting dilution
in single-cell cultures. We have shown previously that
bryostatin 1 stimulates both myeloid and erythroid progenitors of human origin in vitro. In this study both IL-3 and
GM-CSF supported colony formation from 500. 100. or
single-cell cultures at equivalent plating efficiencies, suggesting a direct action of these factors on hematopoietic
cell growth. Conversely, bryostatin 1 did not support the
growth of CD34 cells in single-cell cultures, and the cloning
efficiency increased with increasing the number of cells in
the culture. To test whether the indirect action of bryostatin 1 might be mediated through the production of growth
factors by accessory cells, studies were performed using
antibodies directed against human IL-3 and OM-CSF in
culture with bryostatin 1 and normal human bone-marrow
cells. Results are consistent with the hypothesis that
bryostatin 1 could have a stimulatory effect on the accessory cell populations t o produce either IL-3 or GM-CSF.
Further support for this notion was obtained by demonstrating that T cells, which are cells known t o be able t o produce
IL-3 and GM-CSF, are stimulated by bryostatin 1 t o express
messenger RNA (mRNA) for specific growth factors, including GM-CSF. These results provide further support that
bryostatin 1 may be a useful clinical agent t o stimulate
hematopoiesis in vivo.
0 1990 by The American Society of Hematology.
T
similar/identical plating efficiency observed at all levels of
the limiting dilution analysis. If accessory cells, either
directly or indirectly through release of any growth factors,
were required for proliferation, then a t cell numbers approaching single cells in the culture (less possibility for cellkcell
interaction), the plating efficiency would decrease. This
system allowed us to examine the growth potential of the
recombinant purified growth factors as well as bryostatin 1
on individual progenitor cells. In addition, neutralization of
bryostatin 1-stimulated growth, by antibodies to known
growth factors, would further suggest an indirect mechanism
of action for bryostatin 1. Finally, we examined the possibility that bryostatin would activate messenger R N A (mRNA)
for known hematopoietic growth factors if its mechanism of
action was via the release of these factors from accessory-cell
populations.
H E INTERACTION between hematopoietic progenitors and accessory-cell types (which either produce
humoral agents or interact directly with progenitors) has
recently become an important research study area due to
advances made in the technologies available to isolate these
progenitors. Direct interaction between purified growth factors and isolated enriched populations of human progenitors
has been established.’,* In addition to the role of growth
factors, alteration of culture conditions will allow modified
proliferation and/or differentiation of progenitor^.^ We have
reported that bryostatin 1, a macrocyclic lactone purified
from a marine invertebrate, Bugula neritina, can support the
growth of both human4 and murine’ progenitor cells in vitro.
To determine whether both growth factors and bryostatin 1
could directly stimulate human bone marrow progenitor
cells, enriched human CD34-positive cells6 were used for
limiting dilution analysis. Single cells could then be evaluated for their growth in recombinant human (rh) granulocytemacrophage colony-stimulating factor (GM-CSF), rh interleukin-3 (IL-3), or bryostatin 1 at
or lo-’’ mol/L
concentrations in a methylcellulose clonogenic assay for
hematopoietic progenitors. A direct action would result in
From The Johns Hopkins Oncology Center, Baltimore. MD; and
the Dana-Farber Cancer Institute. Boston. MA.
Submitted January 31,1990; accepted April 24,1990.
Supported in part by National Institutes of Health Grants No.
R01 CA47993-02. CA32318. and CA06973, and a grant from
Gemini Science, Inc.
W.S.M.
is a Scholar of the Leukemia Society of America.
Address reprint requests to Saul J. Sharkis. PhD. 2-1 27 Oncology, Johns Hopkins Oncology Center, 600 N Wove St, Baltimore,
M D 21 205.
The publication costs of this article 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 1990 by The American Society of Hematology.
0006-4971/90/7604-0008$3.00/0
716
METHODS
Collection of bone marrow cells. Bone marrow aspirates from
normal volunteers were harvested from their posterior iliac crest.
Informed consent was obtained, and the study participation design
was approved by The Johns Hopkins Institutional Review Board.
Mononuclear bone marrow cells (density less than 1.078 g/mL)
were recovered by Ficoll-Hypaque density centrifugation as
described:
Preparation of CD34-positive cells. Nonadherent cells (adherent cells were removed by overnight incubation at 37OC in tissue
flasks with McCoy’s 5 A medium in 10% fetal bovine serum [FBS])
were labeled with 4 to 8 mL of anti-CD34 antibody and were
incubated in the cold (4OC) for 45 minutes. These incubation
conditions are in antibody excess. Cells were then washed and
exposed to sheep or goat antimouse (FAB,) fluorescein-conjugated
IgG. Cells were placed through the fluorescence-activated cell sorter
(EPIC 752 Coulter Electronics, Hialea, FL) and sorted aseptically
into bright (top 1%) and dull (lowest 1%) populations. We had
shown previously6that this isolation procedure resulted in significant
enrichment of hematopoietic progenitors (greater than 35-fold).
This allowed us to segregate progenitors to the bright fraction and
relative depletion of the progenitors to the dull fraction. Flow
cytometry reanalysis after sorting indicated that CD34 bright cells
Blood, Vol 76, No 4 (August 15). 1990: pp 7 16-720
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717
BRYOSTATIN AND RHCSFSON SINGLE PROGENITORS
were greater than 95% positive and that CD34 dull cells were greater
than 95% negative for the CD34 antigen.
Colony-stimulatingfactors and clonogenic assay. Cells, including those labeled but not sorted (unseparated control cells), were
plated in 35-mm plates in methylcellulose (1.2%). For CD34positive cells, 5 x 10’ cells were plated. For CD34-negative cells, 2.5
x IO5 cells were plated. Enriched CD34-positive cells were also
plated in microtiter wells at 100 or 1 cell/well in 60 wells of the
96-well plate (remaining wells contained water for hydration). Cells
were plated with 1.0 U of rh erythropoietin and either 100 U/mL of
GM-CSF or 20 U/mL of IL-3 (Amgen, Thousand Oaks, CA).
These concentrations routinely produced optimum colony formation
in our laboratory. Controls contained either no growth factor or 10%
vol/vol leukocyte-conditioned media (LCM). In the absence of
growth factor, no colonies were formed (data not shown). As
demonstrated previously4
mol/L bryostatin 1 (known to stimulate primarily granulocyte-macrophage colony growth) or lo-’’
mol/L bryostatin 1 (known to stimulate primarily erythroid colony
growth) was added to various cell concentrations to determine if
these concentrations of this biologic response modifier would produce
colonies. All cultures contained 30% FBS (Hyclone, Logan, UT).
Colonies of at least 40 cells (myeloid, erythroid, or mixed) were
counted after 13 to 14 days after plating and incubation at 5% CO,
in air at 37OC.
Antibody studies. As a further attempt to determine if bryostatin 1 allowed myeloid and erythroid colony formation, we plated
mol/L or lo-’’
normal bone-marrow cells ( 5 x lo3) with either
mol/L bryostatin 1 with or without the addition of anti-GM-CSF or
anti-IL-3. Titer of these neutralizing antibodies used were as
recommended by the manufacturer (anti-IL-3, a gift from Amgen,
was used at a dilution of ‘/‘a, which neutralized 20 U of IL-3;
anti4M-CSF, purchased from Genzyme (Boston, MA), was used
at a dilution of %a, which reportedly neutralized 100 U of GM-CSF).
Ten individual microtiter wells were scored for colonies of both
erythroid (BFU-E) and myeloid (CFU-GM) phenotype in these
methylcellulose cultures 12 to 14 days following plating. These
experiments were repeated five times, and the results are expressed
as the mean + 1 SEM for the total of 50 wells per group counted.
Microtiter culture was used to conserve the total amount of antibody
used.
Northern blot analysis of cells exposed to bryostatin. To
determine if mRNA for known growth factors was expressed upon
exposure of cells to bryostatin, normal human peripheral-blood
mononuclear cells were incubated with bryostatin 1 in vitro for 2, 16,
and 40 hours. The cells were lysed and RNA prepared for Northern
blot analysis. The lysate was ultracentrifuged through a cushion of
5.7 mol/L cesium chloride at 36,000 RPM in an AH650 rotor for 18
hours. The RNA pellet was harvested and ethanol precipitated twice
and dissolved in diethylpyrocarbonate-treated water. Total cellular
RNA (10 fig/lane) was separated by size via electrophoresis through
a denaturing gel (1.2% agarose with 2.2 mol/L formaldehyde), then
blotted to nylon transfer membranes (Gene Screen Plus, New
England Nuclear, Boston, MA). Plasmids containing cDNAs encoding the human CSF were generously supplied by Drs Steven Clark
and Gordon Wong (Genetics Institute, Cambridge, MA). The
4.0-kb M-CSF transcript was detected using an EcoRl fragment
from the human M-CSF cDNA. The 0.8-kb GM-CSF mRNA was
detected using an EcoRl/NCOl fragment from the human GMCSF cDNA. The 1.8-kb G-CSF and 1.0-kb IL-3 mRNA transcripts
were searched for using near full-length cDNA clones inserted in the
Xhol site of the pXMT2 vector. After restriction endonuclease
release, the cDNA probes were ’,P labeled by random oligonucleotide primer technique. After prehybridization, blots were incubated
for 18 hours at 65°C in hybridization buffer with labeled probes
present at a final concentration of 2.5 x IO5cpm/mL. The blots were
then washed, and autoradiograms were made by exposure at -7OOC
using an intensifying screen.
RESULTS
Colony formation in 35-mm plates. As can be seen in
Table 1, the frequency of colony formation is dramatically
increased in the CD34-positive population of cells and is
markedly reduced in the CD34-negative fraction for all the
growth promoters. The best overall growth promoter was our
leukocyte-conditioned media, and the least active materials
were the two concentrations of bryostatin 1. In these cultures
at least 500 cells (CD34 positive) and as many as 2.5 x lo5
cells (CD34 negative) were plated. It is apparent that
cell-ceil interactions were possible for these cultures.
Colonyformation in microtiterplates. Preliminary studies indicated that plating various numbers (100, 50, 25, etc)
of cells by limiting dilution in microtiter plates resulted in
similar frequencies of colony formation for LCM, GM-CSF,
and IL-3 (data not shown). In Table 2 the plating efficiency
was compared for 5 x lo4 unseparated cells and CD34positive cells plated at single cell/culture. The plating
efficiency is not statistically different for the two populations,
suggesting a direct effect of these factors on progenitor cells
(see below). However, if plating efficiency for bryostatin 1
exposures are compared for unseparated cells, 2 x lo4CD34positive cells (35-mm plates) to 1 CD34-positive cell in
microtiter plates, the efficiency increases significantly with
increased density of cells plated (Table 3), suggesting a
requirement for accessory cells to interact with bryostatin 1
to stimulate colony formation.
Types of colonies formed in response to various growth
promoters. Table 4 shows the total number of colonies
observed from CD34-positive cells in single-cell cultures as a
function of the total number of microtiter wells examined
and the percent colony type (differential) with the various
growth factors. The highest plating efficiency observed, once
again, was LCM, followed by GM-CSF and IL-3. Both
LCM and GM-CSF produce similar types of colonies, but
IL-3 produces relatively more erythroid and less myeloidtype colonies. This suggests not only a direct action but also a
selective and directed proliferation pattern. For the bryostatin 1 cultures a total of 5000 cultures were examined, and
only 5 colonies were observed. Four of the five colonies were
erythroid grown in lo-’’ bryostatin 1, a concentration that
favors erythroid colony g r ~ w t h . ~
Table 1. Growth of Unseparated, CDWNegative, and
CD34-Positive Cells With Various Growth Stimuli
Mean No. of Colonies/106
Bone Marrow Cells Plated
Growth Factor
LCM
GM-CSF
IL-3
Bryostatin ( 10-smol/L)
Bryostatin (lO-”mol/L)
Unseparated CD34-Negative.
104
59
36
18
24
3.5
0.5
1.5
0.75
0.125
CD34-Positiva.
7,868
5,733
4,250
830
680
“Both the CD34-positive and the CD34-negative populations represent 1% of the cells.
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718
SHARKIS ET AL
Table 2. Plating Efficiency for Unseparated Compared W i t h
CD34-Positive Cells in Single-Cell Culture Exposed t o LCM.
GM-CSF, or IL-3
% Plating Efficiencv
Growth Factor
LCM
GM-CSF
IL-3
Table 4. Stimulation of Enriched Progenitors W i t h Hematopoietic
Growth Factors in Single-Cell Culture
Growth Stimulus
Unseparated.
1 CD34-Positive
P Valuet
5.8
5.9
3.6
5.4
3.9
3.5
.8
.7
.9
~
*Plating efficiency is normalizedfor unseparated cells by multiplying by
100, since the CD34-positive population represents 1% of the total. A
total of 5 x 1O4 unseparated cells were plated.
tChi-square analysis revealed no significant difference in plating
efficiency between unseparated and single cells.
Studies with neutralizing antibodies to growth factors,
Table 5 shows the results of five experiments in which
bryostatin at
mol/L or lo-’’ mol/L were incubated
with bone marrow mononuclear cells simultaneously with or
without neutralizing antibody to either GM-CSF or IL-3.
The results demonstrate that a significant reduction in colony
formation attributable to the effect of bryostatin 1 occurs in
the presence of either anti-GM-CSF or anti-IL-3. Furthermore, in combination these two antibodies reduce colony
formation even further. These studies support the likely
possibility that IL-3 and/or GM-CSF can be released by
cells exposed to bryostatin 1 .
Northern blot studies, In Figure 1 it can be seen that the
expression of mRNA for human GM-CSF, G-CSF, and
M-CSF from blood mononuclear cells is increased after
exposure to bryostatin 1. We are unable to detect mRNA for
IL-3 by Northern analysis either at 16 or 40 hours after
exposure to bryostatin 1 (data not shown). The ethidium
bromide staining pattern correlated well with hybridization
of a labeled cDNA probe for actin in these cells (data not
shown). Visualization of the 28s and 18s ribosomal R N A
bands by ethidium bromide staining indicated that the
16-hour control lane was somewhat underloaded compared
with the other lanes.
% Colony Type
No. of Colonies/
Total Cultures Counted.
E
GM
Mixed
28/520
52/1,320
46/1,320
17.9
16.1
37.0
82.1
80.4
58.7
0
3.4
4.3
0/2,500
-
-
-
5/2.500
80
20
0
LCM
rh GM-CSF
rh IL-3
Bryostatin 1
(io-’ mol/L)
Bryostatin 1
( l o - ” mol/L)
*No more than a single colony was observed per culture.
reported.’-2Using a liquid culture system, rh GM-CSF can
stimulate the growth of CD34-positive c e k 3We have tested
this hypothesis using microtiter-plate methylcellulose cultures of single cells. When sufficient single-cell cultures are
performed (1 320 cultures), it is clear that both GM-CSF and
IL-3 can act directly on cells to initiate colony formation.
While we never observed more than one colony in a microtiter well, it remains possible that cells produced by the single
cell could subsequently produce growth factors in an autocrine fashion, and our data cannot exclude this possibility.
Using umbilical-cord blood progenitors, Kannourakis et
al’ showed that cell sorting, limiting dilution, and micromanipulating single cells resulted in comparable results, suggesting that limiting dilution following isolation by cell sorting
truly does result in single-cell cultures and not simply cluster
(10 to 20 cells) formation. Furthermore, by Poisson distribution analysis, only 24% of wells should contain more than one
cell; therefore, the similar cloning efficiency for single-cell
and unseparated cell cultures for LCM, IL-3, and GM-CSF
are consistent with single cells being plated in our studies.
Absence of colony formation with bryostatin 1 in single-cell
cultures, but the presence at the higher cell concentrations,
Table 5. Effect of Antibodies t o IL-3 and GM-CSF on
Bryostatin-Stimulated Human Progenitors
Colonies/5 x
DISCUSSION
Treatment
A direct action on human bone marrow cells for purified
recombinant growth factors such as rh GM-CSF has been
Table 3. Plating Efficiency for Unseparated Compared With
CDBCPositive Cells at Either 2 x lo4 or Single Cells
Exposed t o Bryostatin
% Platina Efficiencv
Unseparated.
Bryostatin
(io-’ mol/L)
Bryostatin
(lo-’’ mol/L)
1.8
2.1
2 x io4
CD34-
1 x ioo
CD34-
Positive
Positive
0.83
0.68
0.0
0.20
Bryostatin (lo-’ mol/L)
Bryostatin ( l o - ” mol/L)
Bryostatin (lo-’ mol/L)
Bryostatin (lo-’’ mol/L)
CSF
Bryostatin (lo-’ mol/L)
Bryostatin ( l o - ” mol/L)
Bryostatin (lo-’ mol/L)
CY IL-3
Bryostatin (10-l1mol/L)
a IL-3
CSF
IL-3
GM-CSF
GM-CSF
GM-CSF
IL-3
CY IL-3
No growth factor*
+ a GM-CSF
+ a GM+ a IL-3
+ a IL-3
+ a GM-CSF
+
P Valuet
<1 x
<l
x
‘Plating efficiency is multiplied by 100 to normalize, since CD34positive cells represent 1% of the cells collected. A total of 5 x 1O4
unseparated cells were plated.
t P values are significantly different by chi-square analysis comparing
unseparated with either concentration of CD34-positive cells.
+
+
+
+ a GM-
Cells Platedt
Erythroid
Myeloid
3.0 f 1.3
3.2
1.0
0.9 2 0.6
2.4 f 0.9
2.9 f 0.9
1.5 f 0.5
1.6 f 0.6
0.7 f 0.4
0.7 f 0.1
1.9 f 0.6
1.4 f 0.9
1.8 f 0.7
0.7 f 0.3
1.0 f 0.2
0.5
2.16
3.36
3.1
0.4
0.0
f 0.3
f 0.2
1.4
.15
f 0.01
f 0.0
2
2
0.6 f 0.2
2.9 f 0.4
4.7 2 1.0
3.4 f 1.0
2.7 f 0.8
0.0 f 0.0
*Erythropoietin and 30% FBS are present in all cultures except this
group, which only contains the 30% FBS.
tEach group represents the mean 2 SEM of five experiments in which
10 cultures per experiment were done.
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BRYOSTATlN AND nnCSFs ON SINGLE PROGENITORS
A
I
719
B
2
1
3 4
2
3
4
-it M-CSF
.
)
G-CSF
CI)
GM-CSF
m
-283-
18s-
+ *
Fig 1. (A) Exprodon of CSF mRNA by normal human peripheral-blood " c l e a r
cells purified far T cells by E resetting's
and adherence depletion. Cells were incubeted for 16 hours with
media (lane 1) or 2 x lo-' mol/L bryostatin 1 (lane 2) or 40 houra
(media, lane 3 and b r y o m i n 1. lane 41. ( 8 )Ethidium bromide stain
of RNA gel demonstratingthe 28s and 18s bands. Lanes 1 and 3 arb
mediaonly incubation for 16 hours and 40 hours, respectively.
Lanes 2 and 4 represent incubation with bryostatin for 16 and 4 0
hours, respectively.
indicates indirect action of bryostatin 1. Our early studies
with bryostatin 1 demonstrated that this agent could support
the growth of both myeloid and erythroid colonies from
whole human bone marrow? We suggested that the mechanism of action on normal bone-marrow progenitors might be
a direct one?This was implied by results obtained when eight
cell clusters were removed and transferred to separate dishes.
Additional colony formation in the secondary cultures containing bryostatin 1 was observed. Using single cells, we
observed only five colonies. The Poisson distribution analysis,
consistent with those five colonies being the consequence of
more than 1 cell per well, leads us to conclude that the effect
of bryostatin 1 is an indirect one. To explore the mechanism
of bryostatin stimulation, we turned to an animal model.' In
that study, murine anti-113 antibody blocked the erythroidpotentiating ability of bryostatin 1. In the present study both
human anti-GM-CSF and anti-IL-3, alone and in combination, also blocked the growth of bryostatin I-induced colonies.
The accumulation of mRNA transcripts encoding GMCSF, G-CSF, and M-CSF by peripheral-blood mononuclear
cells exposed to bryostatin I indicated that this molecule
stimulates hematopoietic accessory-cell populations to produce cytokines. The inability of this technique to document
the increased expression of mRNA for IL-3 is consistent with
the recent finding that this transcript is quite difficult to
detect by Northern analysis.' The antibody-blocking experiments and the mRNA transcript analysis strongly suggest
that bryostatin 1 might stimulate growth-factor production.
Taken together with our single-cell culture data, which
eliminates the possibility of an accessory cell interacting with
bryostatin 1 (and consequential failure to produce growth of
purified progenitors), these findings indicate a role for
accessory-cell stimulation of progenitors by bryostatin. The
neutralization of cytokines in cultures by GM-CSF and IL-3
antibodies was not complete. These are very low-titer antibodies without total neutralizing ability but have little or no cross
reactivity. At present they are the best available. The
expression of G-CSF message by bryostatin 1 suggests that
release of this cytokine by accessory cells might play a role in
growth stimulation. G-CSF has been shown to have multiple
targets for increased growth of progenitors: Recently Sieff et
a13 have suggested that IL-3 or GM-CSF in cultures lacking
FBS are inadequate to support the full growth potential of
progenitor cells. Their findings thus indicate that FBS may
contain additional growth promoters or cofactors. Since the
cell culture conditions employed in our study include 30%
FBS. it is possible that other factors could likewise influence
the formation of clones. These cultures are complex, and
cofactors or bryostatin can induce several growth potentiators from accessory cells.
The mechanism by which bryostatin 1 stimulates growth
factor production is unknown. At the molecular level, bryostatin 1 has been shown to activate protein kinase C.9'" This
enzyme, which has growth-related signal transduction
properties," may be important in hematopoietic progenitorcell proliferation. Accessory cells producing growth factors
(ie, G, M, GM-CSF, and IL-3) may do so in response to the
activation of enzymes, such as protein-kinase C.
Plating-enriched progenitors in a single-cell culture system should prove useful for further delineating the interaction between colony-forming cells, cofactors, and growth
promoters.
We have recently shownt3that bryostatin 1 can inhibit the
growth of leukemic cell lines, fresh-explanted acutemyelogenous leukemia cells, and, most interesting!y, CFUG M from myelodysplastic patients. The hematopoietic stimulatory activity of bryostatin 1 coupled to the antileukemic
activity may make this agent a valuable antineoplastic agent
in the clinic.
REFERENCES
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vitro of individual murine and human hematopoietic cells after
fluorescence-activated cell sorting. Exp Hematol 16:367.1988
2. Mayani H, Baines P. Jones A, Hoy T, Jacobs A: Effects of
rccombinant human granulocyte-macrophage colony stimulating
factor (rh GM-CSF) on single CD-34 positive hemopoietic progenitors from human bone marrow. Int J Cell Cloning 7:30,1989
3. Sieff CA. Ekern SC. Nathan DG,Anderson JW: Combination
of recombinant colony-stimulating factors are required for optimal
hematopoietic differentiation in serum-deprived culture. Blood 73:
688,1989
4. May WS. Sharkis SJ. Esa AH, Gebbia V, Kraft AS. Pettit
GR, Sensenbrenner L L Antineoplastic bryostatins are multipotential stimulators of human hematopoietic progenitor cells. Proc Natl
Acad Sci USA 848483,1987
5. Leonard JP, May WS. lhle JN. Pettit GR. Sharkis SJ:
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SHARKIS ET AL
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1990 76: 716-720
The action of bryostatin on normal human hematopoietic progenitors
is mediated by accessory cell release of growth factors
SJ Sharkis, RJ Jones, ML Bellis, GD Demetri, JD Griffin, C Civin and WS May
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