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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 353-359
In Vitro and In Vivo Evidence That Ex Vivo Cytokine Priming of Donor
Marrow Cells May Ameliorate Posttransplant Thrombocytopenia
By
Mariusz Z. Ratajczak,
Janina Ratajczak,
Boguslaw Machalinski,
Rosemarie Mick, and
Alan M. Gewirtz
From the Departments of Pathology and Laboratory Medicine,
Biostatistics and Epidemiology, and Internal Medicine, University of
Pennsylvania School of Medicine, Philadelphia, PA.
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ABSTRACT |
Thrombocytopenia is typically observed in patients undergoing
hematopoietic stem cell transplantation. We hypothesized that delayed
platelet count recovery might be ameliorated by increasing the number
of megakaryocyte colony- forming units (CFU-Meg) in the hematopoietic
cell graft. To test this hypothesis, we evaluated cytokine combinations
and culture medium potentially useful for expanding CFU-Meg in vitro.
We then examined the ability of expanded cells to accelerate platelet
recovery in an animal transplant model. Depending on the cytokine
combination used, we found that culturing marrow CD34+
cells for 7 to 10 days in serum-free cultures was able to expand
CFU-Meg ~40 to 80 times over input number. Shorter incubation periods
were also found to be effective and when CD34+ cells were
exposed to thrombopoietin (TPO), kit ligand (KL), interleukin-1
(IL-1), and IL-3 in serum-free cultures for as few as 48 hours, the
number of assayable CFU-Meg was still increased ~threefold over input
number. Of interest, cytokine primed marrow cells were also found to
form colonies in vitro more quickly than unprimed cells. The potential
clinical utility of this short-term expansion strategy was subsequently
tested in an in vivo animal model. Lethally irradiated Balb-C mice were
transplanted with previously frozen syngeneic marrow mononuclear cells
(106/mouse), one tenth of which (105) had been
primed with [TPO, KL, IL-1a, and IL-3] under serum-free conditions
for 36 hours before cryopreservation. Mice receiving the primed frozen
marrow cells recovered their platelet and neutrophil counts 3 to 5 days
earlier than mice transplanted with unprimed cells. Mice which received
marrow cells that had been primed after thawing but before
transplantation had similar recovery kinetics. We conclude that
pretransplant priming of hematopoietic cells leads to faster recovery
of all hematopoietic lineages. Equally important, donor cell priming
before transplant may represent a highly cost-effective alternative to
constant administration of cytokines during the posttransplant recovery
period.
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INTRODUCTION |
THROMBOCYTOPENIA remains a significant
cause of morbidity in patients undergoing high-dose chemotherapy and
hematopoietic stem cell transplantation. The platelet transfusions used
to treat this problem are a potential source of blood borne pathogens,
not infrequently lead to alloimmunization of the
recipient,1 and consume millions of scarce health care
dollars each year.2 For all these reasons, it is
straightforward that an effective measure for ameliorating this problem
would make an important contribution to the care of patients undergoing
hematopoietic cell transplantation.
Although many factors could be hypothesized to be of etiologic
importance in the pathogenesis of posttransplant thrombocytopenia,
three that may be of particular importance are inadequate production of
megakaryocyte growth factors, release of inhibitory cytokines after
marrow damage, and inadequate numbers of megakaryocyte progenitor cells
in the donor graft.3 The first two considerations have
received the most attention to date. However, transplantation of
peripheral blood stem cells harvested after cytokine stimulation has
not led to consistent shortening of the thrombocytopenic
period,4 and attempts to stimulate platelet recovery in the
posttransplant period with cytokines such as interleukin-3 (IL-3), and
IL-6 have been associated with unacceptable toxicity.5-7
IL-11 and thrombopoietin (TPO) may prove more useful in this
regard8,9 but this has not been formally proven.
Regardless, administration of additional cytokines to transplant
patients is likely to significantly increase the cost of an already
expensive procedure. Avoiding platelet transfusions might decrease a
patient's exposure to megakaryocyte colony-forming unit (CFU-Meg)
inhibitory cytokines, such as PF4, which are released from platelet
alpha granules,10-13 but platelets cannot be withheld when
clinically indicated since the immediate consequences may be far more
severe.
Our group has been interested in the technical feasibility of expanding
CFU-Meg in the cells to be transplanted as a practical way of enhancing
platelet production in the recipient until more primitive progenitor
cells have had time to engraft and expand. We report herein in vitro
and in vivo studies which suggest that this approach is feasible,
economical, and likely to be successful in the population of interest.
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MATERIALS AND METHODS |
Human cells.
Light-density marrow mononuclear cells (MNC) were obtained from seven
consenting normal human donors and depleted of adherent cells and T
lymphocytes (A TMNC) as
described.14-17 CD34+ cells were enriched from
the ATMNC population by incubation with
anti-HPC1 murine monoclonal antibody (Becton Dickinson, Mountain View,
CA) and subsequent immunoselection of antibody-labeled cells with
magnetic beads according to the manufacturer's protocol (Dynal, Oslo,
Norway). Briefly, 2 × 107
ATMNC were incubated (1 hour at 4°C on
a rotating rack) in 1 mL of Iscove DMEM (GIBCO, Grand Island,
NY) containing 5% (vol/vol) bovine calf serum (BCS;
Hyclone, Logan, UT) and 50 µL of anti-CD34 antibodies (anti-HPC1).
After incubation, the cells were washed 3× in Iscove DMEM + 5% BCS,
then resuspended in 1 mL of Iscove DMEM + 5% BCS with 150 µL of
sheep antimouse coated paramagnetic beads (Dynal). Cells were incubated
again (45 minutes at 4°C on a rotating rack) and then transferred to
5-mL polystyrene plastic tubes for magnetic collection of tagged cells
as directed by the manufacturer (Dynal). The CD34 enrichment procedure
was performed a total of 3×. After the final magnetic separation, the
remaining cells were resuspended in Iscove DMEM + 10% BCS. Cell
viability and purity was >98%.
Murine cells.
Light density MNC were enriched from marrow flushed from the femurs of
pathogen free, 4- to 6-week old female Balb-C mice (Taconic Labs,
Germantown, NY) by Ficoll-Paque density gradient centrifugation. The
light density MNC were further depleted of monocytes by adherence to
plastic dishes for 2 hours.
Human CFU-Meg assay.
CFU-Meg were assayed in plasma clot cultures as previously
described.14,17 CFU-Meg colony growth was stimulated with
recombinant human TPO (50 ng/mL) and IL-3 (20 U/mL).
Colonies were enumerated after 10 to 11 days of culture by
immunofluorescence with an antiplatelet glycoprotein IIb/IIIa
monoclonal antibody (Immunotech, Marseille, France) also as previously
reported.14,17 Data points represent mean ± SD of
quadruplicate culture dishes.
Megakaryocyte phenotyping of expanded cells.
Cells from expansion cultures were washed twice with Ca++
and Mg++ free phosphate buffered saline (GIBCO-BRL, Grand
Island, NY), stained with the Immunotech murine monoclonal antihuman
IIb/IIIa antibody (1:100) for 30 minutes. The cells were then washed
and binding of the primary antibody was detected with a
phycoerythrin-conjugated sheep antimouse Ab (1:100) (Sigma, St Louis,
MO). Expression of IIb/IIIa was evaluated by fluorescence-activated
cell sorter (FACS).
Cryopreservation of MNC.
Cells (2 × 104 human CD34+ MNC/mL, or
5 × 106 murine A-MNC/mL) were suspended in freezing
medium (Iscove DMEM with 10% dimethyl sulfoxide [Sigma] + 20% BCS
[Hyclone] placed in cold 2-mL cryotubes (Corning, Corning, NY) and
then transferred immediately to a  80°C freezer. Twenty-four hours
later the vials were placed into liquid nitrogen (196°C) for
long-term storage. Four weeks later, samples were thawed and assayed
for CFU-Meg as described above.
Cell thawing procedure.
Cryotubes were placed in a 37°C water bath for 2 minutes. The cell
suspension was then transferred to 50-mL polypropylene tubes (Corning)
containing 30 mL of ice-cold Iscove DMEM + 20% BCS. After 10 minutes
of incubation on ice, cells were centrifuged (1,000 rpm, 10 minutes)
and the pellet resuspended in fresh culture medium. Cell viability was
assessed by trypan blue (0.5%) exclusion test and expressed as a
percentage of 100 cells counted.
Serum free, ex vivo expansion of human CD34+ MNC.
CD34+ AT MNC were suspended
in Iscove DMEM (5 × 103 cells/mL) supplemented with
25% (vol/vol) serum substitute or 25% animal sera (12.5% horse serum
[HS] + 12.5% BCS). Cells were seeded into 24-well
plates (1 mL/well) and incubated for 7 to 21 days (37°C, 95%
humidity, 5% CO2). Serum substitute consisted of 1%
delipidated, deionized, charcoal treated BSA, 270 µg/mL iron
saturated transferrin, insulin (20 µg/mL), cholesterol (5.6 µg/mL)
and 2 mmol/L L-glutamine (all from components from Sigma). Cytokines (R
& D, Minneapolis, MN; unless otherwise indicated) added to the wells
consisted of recombinant human (rH) TPO (50 ng/mL), rH kit ligand (KL)
(10 ng/mL), rH IL-3 (20 U/mL, Genetics Institute, Cambridge, MA), rH
interleukin-1 (100 pg/mL, Genzyme, Cambridge, MA), IL-6 (40
U/mL, Robert Wood Johnson Pharmaceutical Institute,
Raritan, NJ), IL-11 (100 ng/mL, Genetics Institute).
Short-term cytokine priming of human MNC.
CD34+ cells (4 × 104/mL) were suspended for
varying periods of time in 0.5-mL aliquots of expansion medium (Iscove
DMEM [GIBCO; 25% (vol/vol) serum substitute; TPO [50 ng/mL], KL
[10 ng/mL], IL-3 [20 U/mL], and IL-1 [100 pg/mL]) for 48 hours
in 4-mL polypropylene tubes. Thereafter, an aliquot of cells
(2 × 104/0.5 mL) was removed for CFU-Meg assay, while
the remainder of the cells were stored in liquid nitrogen as described.
On occasion, freshly isolated CD34+ cells were
cryopreserved as described above, and upon thawing were suspended in
expansion medium for 48 hours after which time cells were employed for
evaluation of CFU-Meg numbers. Control cells were cultured briefly in
expansion medium, washed, and then plated immediately in plasma clot
containing rH TPO (50 ng/mL), and IL-3 (20 U/mL).
Short-term cytokine priming of murine MNC.
Murine A MNC (fresh or newly thawed) were primed for 36
hours under serum-free conditions with the mixture of rH TPO (50
ng/mL), rH IL-1 (100 pg/mL), recombinant murine (rM) IL-3 (10 ng/mL,
Genzyme), and rM KL (10 ng/mL, Genzyme). After priming, fresh cells
were washed and then cryopreserved, while thawed cells after priming
were mixed with newly thawed, unprimed cells in a ratio of 1:9 and then
used for transplantation.
Marrow transplantation in lethally irradiated mice.
Balb-C mice were lethally irradiated from  -irradiation source (900
cGy). Twenty-four hours later, mice were transplanted by tail vein
injection with unprimed MNC (106 cells/mouse), or
106 total cells consisting of a mixture of cytokine primed
and unprimed cells in a ratio of 1:9. Transplanted mice were bled at
intervals from the retro-orbital plexus for leukocyte, platelet, and
hematocrit determinations using unopette microcollection (Becton
Dickinson, Rutherford, NJ) and heparinized microhematocrit capillary
tubes (Oxford Labware, St Louis, MO), as described.18
Statistical analysis.
Arithmetic means and standard deviations of numbers of colonies, or
cultured cells, were calculated using Instat 1.14 statistical software
package (GraphPad 1993, San Diego, CA). Differences
between means were judged for statistical significance
(P < .01) using Student's t-test for unpaired
samples. To analyze platelet recovery kinetics in the murine
transplantation experiments mean nadir platelet values between the
control and treatment groups were compared using an independent samples
t-test. We also examined and compared the rate of platelet
count recovery from day 9 (nadir) through day 16 using a random effects
linear model that compares the fitted slopes of the linear increase
between the groups. The linear models for each group were constructed
based on individual animal data and were fit by maximum likelihood
methods using a linear random effects model that compares the slopes of
the line of increase between the groups. The lines for each group were
constructed from the individual animal's data and were fit by maximum
likelihood methods using the XTREG command in STATA version 5.0 m
(STATA Corp, College Station, TX).
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RESULTS |
Serum free, ex vivo expansion of human CFU-Meg.
Our group,10,19 and others,11,12 have reported
that megakaryocytes and platelets contain a variety of negative
regulatory proteins in their alpha granules that are released upon
activation of these cells. For this reason, and because serum is known
to contain other hematopoietic inhibitory substances, we hypothesized
that a serum-free culture system might be more conducive to
megakaryocyte progenitor cell expansion than one containing serum.
Proceeding from this premise, we isolated CD34+
AT MNC and cultured the cells serum-free
in Iscove DMEM containing different cytokine cocktails designed to
stimulate human megakaryocyte growth. The effects of these cytokines on
total megakaryocytes and CFU-Meg produced by the serum-free system was
assessed after varying times in culture using a combination of
immunocytochemistry, FACS analysis and colony forming assays.
Results of initial culture experiments are shown in Tables 1 and
2. We first
examined CFU-Meg expansion as a function of cytokine exposure kinetics.
CD34+ cells were exposed to a maximally stimulating
cytokine cocktail for 7, 10, 14, and 21 days. The number of assayable
CFU-Meg, in comparison to colonies obtained after immediately plating
cells in cultures containing the same cytokine cocktail is shown in
Table 1. These experiments indicated that optimal CFU-Meg expansion was
obtained by expanding cells for ~10 days, which resulted in an
~50-fold expansion of megakaryocyte progenitor cells. Of interest,
the number of megakaryocytes/colony paralleled the kinetics of
progenitor cell expansion in a similar but somewhat delayed fashion
(Table 1). After a 7-day exposure to cytokines, when the number of
assayable CFU-Meg had increased ~15-fold, the number of cells/colony
was not different from control colonies. However, after 10 and 14 days
of cytokine stimulation, when expansion of colony-forming cells had
peaked, the number of megakaryocytes/colony was ~doubled. After 21
days, the number of CFU-Meg declined to ~day 7 value, while the
number of cells/colony declined to control values.
We then examined a number of different cytokine combinations for their
ability to expand CFU-Meg after 10 days of culture (Table 2). Depending
on the combination of cytokines employed, the number of megakaryocytic
progenitors increased from 40 to 80 times over input number. Of
interest, medium supplemented with animal serum was much less effective
for CFU-Meg expansion purposes. In cultures supplemented with [12.5%
HS + 12.5% BCS] the number of CFU-Meg increased only ~2 to 3
times over input number. In addition, we also found that cells expanded
in serum-free medium appeared to express IIb/IIIa at higher
concentrations on their surface than cells that had been expanded in
serum containing medium (Fig 1A through D).
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| Fig 1.
Expression of glycoprotein IIb/IIIa by
CD34+ MNC after 10 day exposure to TPO + KL + IL-3
(A); TPO + KL + IL-3 + IL-11 + IL-6 (B); TPO + IL-3
+ IL-11 + IL-6 (C); and TPO + KL + IL-3 + IL-11 + IL-6 +
IL-1 (D). Cells displayed in upper panels of A, B, C, and D were
cultured in Iscove DMEM supplemented with 25% animal serum (12.5%
BCS + 12.5% HS), whereas cells displayed in lower panels of A, B,
C, and D were cultured in Iscove DMEM supplemented with 25% serum
substitute. Representative data from three independent studies are
shown.
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Short-term cytokine priming of CD34+
MNC effect on human CFU-Meg.
Incubation of CD34+ MNC for 7 to 14 days in serum-free
culture appeared to be an effective strategy for expanding
megakaryocyte progenitors and accelerating their maturation.
Nevertheless, since the goal of our studies was to develop an efficient
and practical approach for CFU-Meg expansion we began to investigate
the utility of shorter cytokine preincubation periods. Accordingly, in
the next series of experiments, we cultured cells serum-free for 48
hours in a cytokine cocktail consisting of
(TPO + KL + IL-3 + IL-1a). As noted previously, this combination
of cytokines was found to expand CFU-Meg >50-fold over the input
number of CFU-Meg (Tables 1 and 2). Using these conditions, we found
that CD34+ derived CFU-Meg were increased ~threefold in
comparison to untreated controls (Fig 2B).Further, when cytokine primed CFU-Meg were cryopreserved, subsequently
thawed, and then plated in plasma clot cultures, the CFU-Meg not only
remained clonogenic (Fig 2C), they appeared to form colonies faster
than CFU-Meg present in an unmanipulated population of progenitor
cells. Of interest, it was also possible to expand CFU-Meg in
progenitor cells that had been previously frozen but unexposed to
cytokines. The degree of expansion, ~twofold to threefold over
noncytokine exposed cells, was similar to that observed in cells
exposed to cytokines ex vivo before cryopreservation (Fig 2D).
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| Fig 2.
Effect of prefreezing or postthawing cytokine stimulation
on CFU-Meg expansion from 104 CD34+ MNC. (A)
CFU-Meg from cells cultured immediately. (B) CFU-Meg from cells
cultured serum-free for 48 hours in the presence of TPO + KL +
IL-1a + IL-3. (C) CFU-Meg from cytokine stimulated MNC before
cryopreservation. (D) CFU-Meg from cytokine stimulated MNC after
thawing. Data are pooled from four independent experiments, each
performed in quadruplicate.
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Developing a murine model for ex vivo cytokine priming of
transplanted progenitor cells.
The above experiments suggested that exposure of hematopoietic
progenitor cells to an appropriate cytokine cocktail before
transplantation might lead to accelerated platelet count recovery in
animals transplanted with these cells. To test this hypothesis we
exposed 104 adherent cell depleted murine marrow
mononuclear cells to a cytokine cocktail consisting of (IL-3 [10
ng/mL] + TPO [50 ng/mL] + IL-1 [100 pg/mL] + KL [10
ng/mL]) to determine if murine CFU-Meg would expand in a manner
similar to human progenitor cells. We also assessed the effect of
cytokine priming on expansion of CFU-GM. After cytokine exposure, the
murine CFU-Meg were manipulated in the same manner as human CFU-Meg,
ie, they were either cultured immediately following cytokine
stimulation; frozen after stimulation and then thawed and cultured; or
frozen, thawed, and then stimulated for 36 hours before immediate
plating in plasma clots containing IL-3 + TPO. CFU-GM were
plated immediately after cytokine exposure. We found that short-term
cytokine exposure of murine progenitor cells expanded CFU-Meg
~threefold, regardless of when in relation to freezing cells were
treated (Fig 3A). CFU-GM were expanded ~twofold (Fig
3B).
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| Fig 3.
Effect of short-term cytokine stimulation on expansion of
murine CFU-Meg (Panel A) and CFU-GM (Panel B). Adherent cell-depleted
murine marrow mononuclear cells were exposed to cytokines for 36 hours
as detailed in the text. Panel A: (A) CFU-Meg colonies (mean ± SD)
derived from cells (104) cultured immediately; (B) CFU-Meg
colonies (mean ± SD) derived from cells (104) cultured
serum-free for 36 hours in presence of
(TPO + KL + IL-1 + IL-3); (C) CFU-Meg colonies
(mean ± SD) derived from cells (104) cytokine
stimulated before cryopreservation; (D) CFU-Meg colonies
(mean ± SD) derived from cells (104) cytokine
stimulated after thawing. Data are pooled from three independent
experiments, each performed in quadruplicate. Panel B: (A) CFU-GM
colonies (mean ± SD) derived from cells (104) cultured
immediately; (B) CFU-GM colonies (mean ± SD) derived from cells
(104) cultured serum-free for 36 hours in presence of
(TPO + KL + IL-1 + IL-3). Data are pooled from three
independent experiments, each performed in triplicate.
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Because murine and human marrow cells appeared to expand in an
essentially identical manner, we employed a murine transplant model to
test the ability of ex vivo stimulated marrow cells to accelerate
platelet recovery. Lethally irradiated mice were transplanted with
hematopoietic progenitor cells that were preincubated for only 36 hours
with a cytokine mixture that consisted of (rHTPO + rH IL-1 + rM
KL + rM IL-3). In addition, because we were aware of reports which
suggested that exposure of progenitor cells to so-called "early
acting" cytokines might impair long-term engraftment of transplanted
cells,20-24 we primed only a small part of the marrow graft
(~10% of transplanted cells) to avoid this potential complication.
Accordingly, marrow harvested from donor mice was divided into two
aliquots. One, consisting of the majority of cells (~90%) was
cryopreserved after processing without exposure to cytokines. The other
aliquot (~10%) of cells was primed by 36 hours exposure to the
cytokine combination just described. Priming was performed either
before cryopreservation or immediately after thawing of cells to be
transplanted. At the time of transplantation primed cells were mixed
with unexposed marrow cells in a ratio of 1:10.
Mice transplanted with cytokine primed cells showed slightly improved
rates of recovery of hematocrit (Fig 4A),and enhanced reconstitution of leukocyte counts (Fig 4B). Most germane
to this report, however, were the effects of cytokine priming on the
animals' platelet count nadirs and recovery kinetics (Fig 4C). As
shown in Fig 4C, the nadir platelet count in the animals transplanted
with unprimed donor cells occurred at day 9. While the mean day 9
platelet counts were not significantly different between the control
group (56,500/µL) and the animals that were transplanted with cells
that had been frozen/thawed and then cytokine stimulated (F/T/S)
immediately before transplantation (48,200/µL), the difference
between day 9 platelet counts in the control group and animals which
had been transplanted with cells that were cytokine stimulated before
being frozen, thawed, and then transplanted (S/F/T) was highly
significant (125,300/µL; P < .001). In addition, while
the platelet count nadirs occurred at day 9 for both the control
animals and those which received F/T/S marrow cells, the platelet nadir
count for those animals which received S/F/T marrow cells occurred on
day 7 (P < .001).
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| Fig 4.
Peripheral blood hematocrit (%) (A), leukocyte (B), and
platelet (C) counts in lethally irradiated mice transplanted with
unprimed (control) donor cells ( ), donor cells that were cytokine
stimulated, frozen, and then thawed (S/F/T) ( ), or cells that were
frozen, thawed, and then cytokine stimulated (F/T/S) ( ) before
transplantation. Leukocyte counts are absolute per µL, platelet
counts are (×106/µL). Data are pooled from two
independent experiments each with 5 to 10 mice/group.
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We also examined the rate of platelet count recovery using a random
effects linear model. Analysis of platelet count recovery from day 9
(nadir) through day 16 showed an average increase in platelet count of
28,300/µL/d in the control group of animals. In the F/T/S group the
rate of increase was estimated to be 83,250/µL/d (z = 8.37,
P < .001) while in the S/F/T group it was 103,150/µL/d
(z = 11.23, P < .001). It is important to note that
lethally irradiated control animals died by day 18 without signs of
engraftment.
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DISCUSSION |
Depending on the source of hematopoietic stem cells, and the patients'
underlying disease, restoration of hematopoiesis in transplant
recipients can take as few as 7 days or up to a month or
longer.4,25 During the period of marrow aplasia/hypoplasia,
the patient is at considerable risk for infectious complications.
Anemia and thrombocytopenia are more amenable to intervention, but
transfusion therapy is not completely benign. Further, if a patient
becomes alloimmunized, platelet transfusions are largely ineffective at
controlling thrombocytopenic bleeding. To surmount these problems,
patients are often infused with hematopoietic cytokines in an attempt
to accelerate marrow growth.5-9 There is little doubt that
presently available cytokines, in particular G-CSF and GM-CSF,
accelerate neutrophil recovery by several days in comparison to
patients who are not treated with hematopoietic growth
factors.26,27 This strategy for accelerating myelopoietic
recovery has been reported to be clinically useful by some
investigators,28 but not by others.29 In no
case have consistent effects on platelet count recovery, in the absence
of clinically significant toxicity, been reported. Quite recently,
IL-11 and TPO have entered into clinical trial but the utility of these
growth factors remains unproved. However, even if effective it would
not be unreasonable to expect that routine use of these, and other
cytokines, would add significantly to the cost of transplantation and
might also increase the morbidity of the procedure.
With the above considerations in mind, we began to explore alternative
strategies for approaching the problem of posttransplant
thrombocytopenia. We hypothesized that by expanding the number of
megakaryocyte progenitors in the hematopoietic cell graft before
transplantation, such cells might be primed to proliferate relatively
rapidly upon homing to the host microenvironment. Proliferation in turn
would be accompanied by maturation of platelet shedding megakaryocytes
and consequent alleviation of thrombocytopenia. To test this
hypothesis, we needed to design a viable strategy for CFU-Meg
expansion. Our approach was based on the premise that use of serum-free
culture medium, and the appropriate cytokines, would synergistically
promote megakaryocyte progenitor cell expansion by stimulating cell
proliferation in the absence of known inhibitors of megakaryocyte
growth such as transforming growth factor- and PF4.10-12
There were reported already attempts to stimulate bone marrow cells
before transplantation with cytokines yielding different
results.28,29 We were also cognizant of reports that
cytokines useful for stem/progenitor cell expansion, such as IL-1,
IL-3, IL-6 and KL, while immediately useful for progenitor cell
expansion, might prove detrimental for long-term engraftment by
downregulating cytoadhesion molecules necessary for stem cell
homing.20-30 To obviate this problem we split the cells we
wished to transplant into two separate aliquots. One of the aliquots
consisted of the majority of cells (~90%) and was transplanted
unmanipulated. The other aliquot was exposed to a cytokine cocktail
designed to promote the expansion of CFU-Meg.
In support of our hypothesis, and in accord with previously reported
serum-free expansion systems designed to expand long-term culture
initiating cells (LTCICs), colony-forming unit-granulocyte-macrophage
(CFU-GM), and burst-forming unit erythroid,31-33 we found
that megakaryocyte progenitor cells present
in CD34+ enriched, as well as adherent cell depleted,
marrow mononuclear cells were greatly expanded when cultured under
serum-free conditions. We also found that with an appropriate cytokine
cocktail, (TPO + KL + IL-1 + IL-3), the period of exposure
required to obtain up to a threefold expansion of these cells was quite
brief, between 36 and 48 hours. Cytokine exposed marrow cells also
appeared to acquire an increased proliferative capacity as reflected by
an increased number of megakaryocytes comprising colonies arising after
10 and 14 days of prestimulation (Table 1). Of interest, it also
appeared to us that cytokine primed progenitor cells formed colonies in
vitro at a faster rate (~2 days) than cells which were not so
treated. These results supported the idea that transplantation of
primed cells in vivo might lead to early enhancement of platelet
production and this in fact was shown in an in vivo model employing
murine cells (Fig 4). To provide data that our approach might be useful
in patients, we used a murine transplant model to test our hypothesis.
This enabled us to show that transplantation of ex vivo expanded
megakaryocyte progenitor cells resulted in platelet recovery 3 to 5
days faster than syngeneic animals who were transplanted with nonprimed
cells. Interestingly, it appeared that these animals also had
accelerated engraftment of CFU-GM as evidenced by the similar
acceleration of neutrophil recovery in these animals (Fig 4B).
Presently, we cannot be certain of the mechanism(s) responsible for the
observed acceleration of platelet engraftment. Since enhanced platelet
recovery was observed in mice that received marrow cells primed for as
little as 36 hours, it seems unlikely that enhanced proliferative
capacity of progenitors is responsible for the effects we have observed
(Table 1). Nevertheless, it was our impression that morphologically
identifiable megakaryocyte colonies appeared earlier in cultures
initiated with cytokine primed cells. We hypothesize then that enhanced
platelet recovery is likely the result of transplanting an increased
number of progenitors with an accelerated ability to complete their
maturation program. Finally, while any regimen of cytokine priming
appeared to accelerate platelet count recovery, statistical
analysis of the data suggested that cells which were cytokine primed
before being frozen engrafted and developed earlier as reflected by
a higher nadir platelet count, and an earlier and more rapid recovery
in animals transplanted with such cells (Fig 4C). Based on these
results, we suggest that short-term stimulation of hematopoietic cells
in serum-free cultures before cryopreservation will enhance platelet,
and perhaps neutrophil, recovery in transplant patients in a timely and
cost-efficient manner. Clinical trials designed to test this hypothesis
appear warranted and we hope to begin such trials in the very near
future.
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FOOTNOTES |
Submitted March 10, 1997;
accepted September 3, 1997.
Presented at the XXVIII Annual Meeting of the American Society of
Hematology, held in Orlando, FL, December 6-10, 1996.
Address reprint requests to Alan M. Gewirtz and Mariusz Z. Ratajczak,
Room 514 Stellar-Chance Laboratories, University of Pennsylvania School
of Medicine, 422 Curie Blvd, Philadelphia, PA 19104.
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Abstract
Introduction
Abstract