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ЗмістImdm+ fcs 18.19
1999; 84:675-682 original paper
Short-term, serum-free, static culture of cord blood-derived CD34+ cells:
effects of FLT3-L and MIP-1a on in vitro expansion of hematopoietic
GEMMA CAPMANY, SERGI QUEROL, JOSE ANTONIO CANCELAS, JOAN GARCIA
Cryobiology and Cell Therapy Department and Barcelona Cord Blood Bank, Institut de Recerca Oncologica, Barcelona,
Background and Objective. The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The aim of this study was to fix the optimal condition for the generation of committed progenitors without affecting the stem cell compartment.
Design and Methods. Analysis of the effects of FLT3L and MIP-1a when combined with SCF, IL-3 and IL6, in short-term (6 days), serum-free expansion cultures of CB-selected CD34+ cells.
Results. An important expansion was obtained that ranged between 8-15 times for CFU-GM, 21-51 times for the BFU-E/CFU-Mix population and 11 to 30 times for CD34+ cells assessed by flow cytometry. From the combinations tested, those in which FLT3-L was present had a significant increase in the expansion of committed progenitors, while the presence of MIP-1a had a detrimental effect on the generation of more differentiated cells. However, stem cell candidates assessed by week 5 CAFC assay could be maintained in culture when both MIP-1a and FLT3-L were present (up to 91% recovery). This culture system was also able to expand megakaryocytic precursors as determined by the co-expression of CD34 and CD61 antigens (45-70 times), in spite of the use of cytokines non-specific for the megakaryocytic lineage.
Interpretation and Conclusions. The results obtained point to the combination of SCF, IL-3, IL-6, FLT3-L and MIP-1a as the best suited for a pre-clinical short-term serum-free static ex vivo expansion protocol of CB CD34+ cells, since it can generate large numbers of committed progenitor cells as well as maintaining week 5 CAFC.
©1999, Ferrata Storti Foundation
Key words: cord blood, CD34+ cells, FLT3-L, MIP-1a,
Correspondence: Sergio Querol, M.D., Institut de Recerca Oncologica,
Autovia de Castelldefels km 2,7, 08907 L’Hospitalet de Llobregat,
Spain. Phone: international +34-93-2607826 – Fax: international +3493-2607776 – E-mail: firstname.lastname@example.org
The transplantation of cord-blood (CB) derived hematopoietic progenitors has become an alternative to other hematopoietic progenitor sources (bone marrow and mobilized peripheral blood) in those cases in which no suitable donor is available.1-5 In two reports that analyzed engraftment following CB transplantation, median time to neutrophil recovery and platelet transfusion independence was 23 and 75 days respectively.3,4 This delay, particularly in platelet engraftment, has conditioned a high rate of transplant related mortality. In order to overcome this problem, the use of ex vivo expanded cells has been suggested as a possible means of accelerating the speed of engraftment.6-8 Previous reports have shown that it is possible to generate considerable numbers of committed hematopoietic progenitors in vitro using cytokine combinations that include early acting (SCF, IL-6) and proliferative cytokines (IL-3).7,9-12 However, the ex vivo expansion of more immature progenitors in stroma-free cultures has yielded more controversial results.7,8,13,14 It has recently been suggested that fetal liver tyrosine kinase ligand (FLT3-L) and macrophage inhibitory protein 1a (MIP-1a) play roles in the maintenance of stem cells in stroma-free cultures. FLT3 (also known as FLK2 and CD135) is expressed in 88% to 95% of CB-derived CD34+ cells,15 and its ligand (FLT3-L) has been found to induce proliferation of CD34+ cells, in synergy with other cytokines such as IL-3, IL-6, G-CSF and GM-CSF.7-9,16,17 MIP1a is a chemokine that inhibits the proliferation of immature subsets of progenitors in clonogenic cultures, while it stimulates the proliferation of more mature progenitors such as CFU-E, CFU-G and CFUM.18,19 The use of MIP-1a in liquid cultures has shown that its effects are not only dependent on the phenotype of the cultured cells, but also on the cytokines present in the culture.20 Taken together, the results obtained have led to the suggestion that MIP-1a might be involved in preventing the terminal differentiation of immature progenitors.21
In view of these initial reports and in order to define a clinically focused procedure, we sought to test the effects of FLT3-L and an MIP-1a analog (BB10010) on the short term ex vivo expansion of CB-derived
vol. 84(8):August 1999
G. Capmany et al.
CD34+ cells. These cytokines were added to SCF, IL-3
and IL-6, which in our hands22 have been able to
expand CB-derived CD34+ cells efficiently in a serum-
dependent medium. The first part of the report
describes the results of the ex vivo expansion of CD34+
cells in the presence of a serum-free medium (Stem-
Pro 34; Life Technologies). In the second part of the
study we compare several combinations of cytokines
containing early acting cytokines, such as SCF and
FLT3-L, and an inhibitory cytokine MIP-1a, and analyze the effect that the presence of these cytokines during short ex vivo expansion cultures has on immature
and committed progenitors, including megakaryocytic
progenitors, as assessed by flow cytometry.
Design and Methods
Cord blood samples (n=16) were obtained from
term deliveries, informed consent having previously
been obtained from the mothers. Briefly, immediately after delivery and while the placenta was still in
utero, the umbilical cord was clamped and sectioned
and the umbilical vein was punctured. Blood was
allowed to drain by gravity into a blood collection
bag containing CPD-A as anticoagulant, and was
processed within 24 hours of collection.
The mononuclear cell fraction (MNC) present in
CB was isolated by density gradient separation (Ficoll-
Hypaque, density 1.077 g/mL; Pharmacia, Uppsala,
Sweden). CD34+ cells were purified from the MNC by
positive selection of CD34+ cells, using an immunomagnetic system for peripheral blood (Isolex-50, Baxter, Deerfield, IL, USA)23 as previously described . The
CD34-enriched cell fraction was washed and re-suspended in serum-free culture medium (StemPro-34,
Life Technologies, Grand Island, NY, USA).
Recombinant human IL-3 and IL-6 were obtained
from Sandoz (Basel, Switzerland); recombinant
human SCF and FLT3-L were a kind gift from Amgen
(Thousand Oaks, California, USA); the MIP-1a analog BB-10010 was a kind gift from British Biotech
SCF, IL-3 and IL-6 were used at a concentration of
50 ng/mL each; FLT3-L was used at 100 ng/mL and
the MIP-1a analog was used at 20 ng/mL. To supplement the cultures, 4 different cytokine combinations were used: to a basic combination containing
SCF, IL-3 and IL-6 (s,3,6), either FLT3-L (s,3,6,f),
MIP-1a (s,3,6,m) or both (s,3,6,f,m) were added.
A static, short term, stroma-free culture system was
used to expand CD34+ cells. Fifty thousand cells of
the enriched fraction were seeded in 1 mL of culture
medium in 4-well dishes (Nunc, Roskilde, Denmark),
and cultured at 37.C and 5% CO2 for 6 days.
Cytokines were added to the culture on days 0 and 3.
Two different culture media were used; a serum-
dependent medium consisting of IMDM (Life Technologies) supplemented with 25% fetal calf serum
(FCS; Life Technologies); and a serum free medium
(StemPro-34, Life Technologies; SP34) supplemented
with 2% human albumin (Instituto Grifols, Parets del
Valles, Spain). Nine initial experiments were done to
evaluate the performance of a serum-free media compared to a serum-dependent condition using SCF, IL3 and IL-6 for expansion. Following this, seven experiments were performed in order to evaluate the different cytokine combinations proposed using a
Expansion cultures were monitored on days 0 and
6. At both these times, CFU-Mix assay, flow cytometry and cobblestone-area-forming cell (CAFC) assays
Cells for the assay were plated in methylcellulose
containing 30% fetal bovine serum, SCF (50 ng/mL),
recombinant human GM-colony-stimulating factor
(rh-GM-CSF; 10 ng/mL), rhIL-3 (10 ng/mL) and rherythropoietin (3 U/mL; Methocult GF H4434; Stem
Cell Technologies, Vancouver, Canada). Cells of the
enriched fraction were plated at 750 cells/mL and
cells obtained after 6 days of ex vivo expansion were
plated at a concentration of 1,200 cells/mL. Cultures
were kept at 37.C and 5% CO2 for 2 weeks, and were
then scored for the presence of CFU-GM, BFU-E and
CFU-Mix. For analysis BFU-E and CFU-Mix were considered together. Only immature erythroid colonies
(BFU-E) were scored; CFU-E were not included in the
BFU-E/CFU-Mix group. Colonies that contained cells
from erythroid and myeloid lineages were scored as
CFU-Mix. When used, colony-forming cells (CFC)
were related to the total amount of colonies including all the lineages scored.
Flow cytometry analysis
The following monoclonal antibodies were used
for flow cytometry: phycoerythrin-labeled anti CD34,
clone 8G12 (HPCA-2, Becton-Dickinson, San Jose,
CA, USA); tri-color labeled anti-CD45, clone HI30
(Caltag Laboratories, Burlingame, CA, USA); fluorescein-labeled anti-CD61, clone RUU-PL 7F12 (Becton-Dickinson). Isotypic controls (phycoerythrinIgG1 and fluorescein-IgG1) were obtained from Becton-Dickinson and Coulter (Miami, Florida). Samples were processed as described elsewhere,23 and
analyzed using an Epics XL-MCL flow cytometer and
Epics-XL software (version 1.5, Izasa-Coulter, Barcelona, Spain). CD34+ cells that showed a level of fluorescence superior to that of the isotypic control were
termed CD34+, or total CD34+ cells. This population
includes cells with low intensity of fluorescence for
CD34 antigen that could not be clonogenic. Cells,
that after culture showed a level of fluorescence
equivalent to that shown by the cells of the enriched
fraction on day 0 were termed CD34bright cells. In Fig-
vol. 84(8):August 1999
Effect of FLT3-L and MIP-1a on ex vivo expansion of CB CD34+ cells
Figure 1. Strategies for defining the CD34bright and CD34+
regions. The histogram shows the definition of the region for
analysis after positive selection. Two regions were created
using the same instrument settings at day 0 and after 6
days of expansion. R1 is a fixed region created after selection (day 0) and shows the cluster population positive for
CD34 antigen. The events in this region assessed after 6day expansion were termed CD34bright. R2 is the region that
includes the events with a level of fluorescence superior to
that of the isotypic control. These cells were termed overall CD34+ cells in each point of analysis.
ure 1, the gating strategies used to monitor the follow-up of this population are presented. In order that
the analyses at day 0 and day 6 were comparable,
the same antibody concentration, labeling time and
instrument settings were used. Previous reports24,25
have shown an enrichment of immature progenitors
in CB-CD34+ cells with a high intensity fluoresence
Cobblestone-area-forming cell (CAFC) assay was
performed as described previously,26 except that irradiated human stroma was used as a feeder layer
instead of a stromal cell line. Briefly, 203106 MNC
obtained from human bone marrow were seeded in
25 cm2 flasks and cultured at 33.C and 5% CO2 in
long-term culture medium. This medium consists of
McCoy’s medium (Life Technologies) supplemented
with 12.5% fetal calf serum (Life Technologies) and
12.5% horse serum (Biological Industries, Kibbutz
Beit Haemek, Israel), 1 .M hydrocortisone (Biological Industries), 30 mM HEPES (Biological Industries),
2 mM L-glutamine (Biological Industries) and vitamins and amino acids (Biological Industries) at
appropriate concentrations. When the cultures had
reached 75% confluence (3-4 weeks), cells were
trypsinized, irradiated with 15 Gy and seeded onto 96
well plates at a concentration of 30,000 cells/well.
Plates thus prepared were overlaid in a limiting dilution set-up with cells from the CD34+ cell-enriched
fraction or with cells obtained after 6 days of ex vivo
expansion; 4 dilutions (range between 5000 and 500
cells per well) and 15 replicates per dilution were
seeded for each sample. Cells were cultured for 5
weeks at 33.C in long-term culture medium, with
weekly medium feedings. Plates were scored for the
presence of cobblestone areas (CA) in week 5 of culture. The frequency of CAFC was calculated using
Poisson statistics as described elsewhere.27 Stroma
from the same donor were used for all the CAFC
assays, in order to ensure that the results obtained
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, release 6.1).
The level of significance was set at p<0.05. The Student’s t-test was used. Unless otherwise stated, data
are expressed as mean and standard deviation.
A total of 16 cord blood units were used in these
experiments. The median percentage of CD34+ cells
present in whole cord blood was 0.31% (0.13-0.48).
After Ficoll separation, the median percentage of
CD34+ cells was 0.74% (0.38-1.76), with a median
recovery of CD34+ cells of 83% (52-117). In the
enriched fraction, the median purity of CD34+ cells
was 86% (72-93), with a median yield of 44% (38-57)
of the CD34+ cells present before selection (after density gradient separation).
Performance of a serum-free medium vs.
a serum-dependent medium
In order to evaluate the effect of a serum-free medium on the ex vivo expansion of cord-blood derived
hematopoietic progenitors, cells from the enriched
fraction (n=9) were expanded in both serum-free and
serum-dependent media, in the presence of SCF, IL3, and IL-6. The results are summarized in Table 1.
No significant differences were observed between the
Table 1. Fold expansion of nucleated cells (NC), CD34+ cells,
CD34bright cells and colony-forming cells (CFC) in the presence of serum (IMDM + FCS) or in its absence (SP34), after
6 days of ex vivo expansion of CB-derived CD34+ cells in the
presence of SCF, IL-3 and IL-6 (50 ng/mL each). Mean and
standard deviation of 9 experiments.
Total CD34+ CD34bright
vol. 84(8):August 1999
G. Capmany et al.
Figure 2. Expansion of nucleated cells. Fold expansion of
nucleated cells in the presence of 4 cytokine combinations
after 6 days of ex vivo expansion culture of CB-derived
CD34+ cells. Data shown are the mean and standard deviation of 7 experiments. Abbreviations: s = SCF (50 ng/mL);
3 = IL-3 (50 ng/mL); 6 = IL-6 (50 ng/mL); f = FLT3-L (100
ng/mL); m = MIP-1a (20 ng/mL); *= fold expansion in
groups containing FLT3-L was significantly higher than in
groups without FLT3-L (p . 0.05); § = the rate of expansion
in the s,3,6,f group was significantly higher than in the
s,3,6,f,m (p . 0.05).
fold-increase of nucleated cells (NC), total CD34+
cells, CD34bright cells and clonogenic precursors
(Table 1). Moreover, serum-free expansion cultures
showed a lower variability between samples than
when the expansion was carried out in the presence
of serum. The variation coefficients between experiments were 42% vs 62% for nucleated cells, 51% vs
96% for CD34+ cells, 57% vs 115% for CD34bright cells
and 58% vs 91% for colony-forming cells, in serum-
free and fetal calf serum-containing medium, respectively. These results suggest that the use of a serum-
free media would allow greater reproducibility of
results between samples, as well as permitting a better analysis of the effect of cytokines by eliminating
possible interactions with unknown factors present in
Ex vivo expansion of CD34+ cord-blood derived
cells in a serum-free medium
In a second series of experiments (n=7), the effects
of the above described 4 different cytokine combinations (s,3,6; s,3,6,f; s,3,6,m; s,3,6,f,m) in ex vivo
expansion cultures carried out in SP34 were tested.
Nucleated cell expansion
The results of the expansion of NC are summarized
in Figure 2. The greatest increase in nucleated cells
was obtained with the s,3,6,f combination (50 fold ±
Figure 3. Expansion of CD34+ and CD34bright cells. Fold
expansion of CD34+ and CD34bright cells in the presence of
several cytokine combinations after 6 days of ex vivo expansion culture of CB-derived CD34+ cells. Data are expressed
as mean and standard deviation (n=7). Abbreviations: s =
SCF (50 ng/mL); 3 = IL-3 (50 ng/mL); 6 = IL-6 (50 ng/mL);
f = FLT3-L (100 ng/mL); m = MIP-1a (20 ng/mL). *=fold
expansion in groups containing FLT3-L was significantly
higher than in groups without FLT3-L (p<0.05); § = the rate
of expansion in the s,3,6,f group was significantly higher
than in the s,3,6,f,m group (p<0.05).
9), while the cytokine combinations least efficient at
achieving NC expansion were those that did not contain FLT3-L (s,3,6: 18 fold ± 6 and s,3,6,m: 23 fold ±
5). Analysis of the fold-increase obtained with each
cytokine combination showed that the groups containing FLT3-L caused significantly greater increase
than those groups in which this cytokine was not present. Furthermore, the fold-increase achieved in the
s,3,6,f group was significantly higher than in the other group containing FLT3-L (s,3,6,f,m: 42 fold ± 10),
suggesting that the presence of MIP-1a has an
inhibitory effect on the total number of cells generated when FLT3-ligand is also present.
Expansion of CD34+ cells
Analysis of the generation of CD34+ and CD34bright
cells revealed a similar pattern to that obtained for
NC (Figure 3). Thus, the greatest expansion of CD34+
and CD34bright cells was achieved with the s,3,6,f combination (30 fold ± 5 and 13 fold ± 3, respectively).
In the case of CD34+ cells, as with nucleated cells, the
expansion was significantly higher in the 2 groups
containing FLT3-L (s,3,6,f: 30 fold ± 5; s,3,6,f,m: 24
fold ± 4) than in the 2 groups in which this cytokine
was not present (s,3,6: 11 fold ± 4; s,3,6,m: 14 fold
± 2). The fold expansion in the group supplemented
with s,3,6,f was also significantly higher than the
expansion with the s,3,6,f,m combination.
vol. 84(8):August 1999
Effect of FLT3-L and MIP-1a on ex vivo expansion of CB CD34+ cells
For CD34bright cells, the expansion achieved in the
2 groups containing FLT3-L (s,3,6,f: 13 fold ± 3;
s,3,6,f,m: 11 fold ± 3) was again significantly higher
than in the 2 groups that did not contain it (s,3,6: 4
± 2; s,3,6,m: 6 ± 2); however, no significant differences resulted from the addition of MIP-1a in the
presence of FLT3-L. This suggests that MIP-1a affects
mainly the generation of mature cells rather than a
more immature population (represented by the
Expansion of CD34+ CD61+ cells
The mean percentage of cells co-expressing CD34
and CD61 antigens in the enriched fractions used for
this study was 1.43% (± 1.00). All the combinations
of cytokines analyzed were capable of expanding this
subpopulation of cells after 6 days of in vitro culture,
although a great deal of variation between samples
was observed. Fold-increases obtained were 45±18,
70±35, 57±32, and 61±36 for the combinations
s,3,6; s,3,6,f; s,3,6,m; and s,3,6,f,m respectively. The
increase observed was as high as the myeloid/erythroid expansion as assessed by CFU-Mix assay, suggesting a non-lineage restricted (multilineage) expansion using these combinations of cytokines, all of
which include early-acting cytokines. No significant
differences exist between the different groups analyzed.
Expansion of clonogenic precursors (CFC)
The results of the expansion of progenitor cells
(CFU-GM and BFU-E/CFU-Mix) are illustrated in Figure 4. As is the case with CD34bright cells, the fold-
increases of CFU-GM and BFU-E/CFU-Mix were significantly higher in the groups supplemented with
FLT3-L than in the groups that did not include FLT3
L. No differences were observed between the s,3,6,f
group and the group containing FLT3-L and MIP-1a.
The MIP-1a analog did not affect the generation of
CFC, in contrast with the results obtained for NC and
CD34+ cells. In all the combinations tested the expansion of BFU-E/CFU-Mix population was higher than
the CFU-GM. This was mainly due to a considerable
generation of mature erythroid colonies with burst-
forming unit characteristics. Cloning efficiency (CE)
of CD34+ cells was lower after six days of expansion,
suggesting a generation of CD34+ cells, expressing
low antigen intensity, that is not clonogenic. After
positive selection CE was 42±30% (including CFUGM, and BFU-E/CFU-Mix group). In contrast, the
mean CE ranged from 25-37% after six days of expansion culture, depending on the combination used.
Evolution of CAFC during ex vivo expansion
Week 5 CAFC were assessed in order to evaluate
the functional behavior of the stem cell compartment
in the short-term cultures (n=4). In the conditions
used, week 5 CAFC failed to expand, even though
input levels could be maintained when FLT3-L or
Figure 4. Expansion of clonogenic precursors. Fold expansion of CFU-GM and BFU-E/CFU-Mix in the presence of several cytokine combinations after 6 days of ex vivo expansion
culture of CB-derived CD34+ cells. Data are expressed as
mean and standard deviation of 7 different experiments.
Abbreviations: s = SCF (50 ng/mL); 3 = IL-3 (50 ng/mL); 6
= IL-6 (50 ng/mL); f = FLT3-L (100 ng/mL); m = MIP-1a (20
ng/ml). *= fold expansion in groups containing FLT3-L was
significantly higher than in groups without FLT3-L (p<0.05);
§ = the rate of expansion in the s,3,6,f group was significantly higher than in the s,3,6,f,m group (p<0.05).
MIP-1a were added to the cultures. Thus, while the
combination s,3,6 only maintained 63% (± 26) of
input CAFCs after 6 days of ex vivo expansion, addition of FLT3-L or MIP-1a alone increased the percentage to 79% (±57) and 84% (±44) respectively,
and addition of both FLT3-L and MIP-1a allowed
the maintenance of 91% (±49) of the input week 5
CAFC. Figure 5 shows the mean recovery after 6-day
expansion of 5th week CAFC depending on the combination of cytokines used for proliferation.
This report describes the results obtained after ex
vivo expansion of CB-derived CD34+ cells in a short-
term, stroma-free, static culture which, once scaled
up, could be used in a clinical setting. In the first part
of the study, the performance of a serum-free medium (StemPro-34) in the ex vivo expansion of cord-
blood derived CD34+ cells was analyzed; a serum-free
medium would avoid the risk of immunologic reactions and transmissible diseases linked to the use of
FCS, thus making it better suited for clinical applications. In addition, a serum-free medium allows better assessment of the effects of individual cytokines
on the culture. The results obtained using 2 different
combinations of cytokines (s,3,6 and s,3,6,f) show
vol. 84(8):August 1999
G. Capmany et al.
Figure 5. Recovery of week 5
CAFC in 6-day cultures. Evolu-
tion of week 5 CAFC after 6 days
of ex vivo expansion cultures in
the presence of 5 different cyto-
kine combinations. The data
shown are the mean of 4 inde-
Abbreviations: s = SCF (50
ng/mL); 3 = IL-3 (50 ng/mL); 6
= IL-6 (50 ng/mL); f = FLT3-L
(100 ng/mL); m = MIP-1a (20
Day 0 Day +6
that the presence of serum is not necessary for efficient expansion of progenitor cells, and suggest that
no additional factors contained in FCS appear to be
necessary for an optimal expansion in culture. Furthermore, better reproducibility was observed using
serum-free media than when serum was present in
the cultures; this is probably due to the lack of effects
from factors contained in the serum.28
The second part of the study was aimed at determining the effect of several cytokine combinations
containing the early acting cytokine FLT3-L and the
inhibitory chemokine MIP-1a on the expansion of
CB-derived CD34+ cells. To evaluate progenitor
expansion, the fold increases of NC, CFC, CD34+ cells
were assessed, while in order to determine the expansion of the immature compartment, the week 5 CAFC
assay was used. This assay was chosen since it has
been reported to be a good predictor of engraftment
in the human model.14
Our results show that addition of FLT3-L (100
ng/mL) to expansion cultures supplemented with
SCF, IL-3 and IL-6 results in significantly greater
expansion of committed progenitor cells, both in the
presence and absence of MIP-1a. An increase in the
proliferation rate and a higher recruitment of immature cells into the cell-cycle was suggested and could
explain the results observed in our assays. These
results are consistent with other observations regarding a potent effect of FLT3-L on the activation of
primitive progenitor cells.29-33
Addition of MIP-1a in the presence of SCF, IL-3
and IL-6 did not have a significant effect on the expansion of either the more mature (NC and CD34+ cells)
or the more immature compartments (CD34bright cells
and colony-forming-unit population). When a stimulatory cytokine (FLT3-L) was also present in the culture medium, the presence of MIP-1a resulted in significantly lower expansion in the more committed
compartment (compared to the group with SCF, IL3, IL-6 and FLT3-L) while no differences existed in the
expansion of more immature populations. These
results agree with the hypothesis suggesting that MIP1a acts by preventing the differentiation of immature
progenitors, leading them to accumulate, rather than
affecting their proliferation. Thus, the presence of
MIP-1a would result in slightly lower amounts of
mature and precursor cells than when the chemokine
is not present, while leaving the size of the immature
compartment unaffected or increased.21,34 Although
it is still unclear how the effect of MIP-1a is achieved,
it is believed that it prevents immature progenitors
from going into the cell cycle.35,36
The results obtained from the week 5 CAFC assay
show that both FLT3-L and MIP-1a have a positive
effect on CAFC maintenance, an effect that is most
beneficial when both cytokines are present, when up
to 91% input CAFC can still be detected after 6 days
culture. Moreover, CAFC maintenance in the presence of either FLT3-L or MIP-1a is higher than with
SCF, IL-3 or IL-6 alone. Nevertheless, none of the
combinations of cytokines analyzed was capable of
expanding these primitive progenitors. Even though
both Kogler et al.7 and Moore et al.37 demonstrated
LTC-IC expansion in the presence of IL-3, it is possible that the presence of this cytokine in our cultures
can explain, at least partly, our failure to expand week
5 CAFC. Piacibello et al.8 obtained LTC-IC expansion
in cultures supplemented with thrombopoietin and
FLT3-L, but could only maintain input LTC-IC levels
after 2 weeks of ex vivo culture when IL-3 was added
to the cultures. Zandstra et al.38 showed that ex vivo
expansion of bone marrow LTC-IC required the use
of high concentrations of FLT3-L (300 ng/mL); furthermore, they also showed that the presence of relatively high levels of IL-3 (60 ng/mL) when the concentration of FLT3-L in the medium was low (10
ng/mL) was detrimental for the maintenance of LTCIC. Similar results on the negative effect of IL-3 over
the expansion of immature progenitors were reported by Verfaillie et al.21 using stroma-dependent cul-
vol. 84(8):August 1999
Effect of FLT3-L and MIP-1a on ex vivo expansion of CB CD34+ cells
tures. Thus, it is likely that failure of the stem cell
compartment to expand in our culture system was
due to the presence of IL-3 together with relatively
low concentrations of FLT3-L.
Delayed engraftment of the megakaryocytic lineage
is a common feature of CB transplants reported to
date.3,4 We sought to make a preliminary assessment
of the megakaryocytic lineage analyzing cells coexpressing CD34 and CD61 antigens, a population
which is very low in the initial CB CD34+ cell fraction.
Interestingly, the combinations of cytokines tested
show that it is possible to expand the megakaryocytic
precursors present in CB ex vivo in a serum-free medium, in the absence of specific cytokines for this lineage (MGDF and IL-11). This finding suggests that
combinations including early acting proliferative
cytokines promote the expansion of progenitor cells
representing all hematopoietic lineages (erythrocytic,
myelo-monocytic and megakaryocytic) and can therefore be useful for clinically oriented ex vivo expansion
protocols.39,40 There are not, however, any data supporting a direct involvement of this population in the
short-term repopulating potential.
Taken together these results suggest that, from
those tested, the most appropriate cytokine combination for clinically oriented serum-free ex vivo expansion of cord blood-derived progenitor cells would be
that containing SCF, IL-3, IL-6, FLT3-L and MIP-1a,
since this is capable of generating considerable
expansion in the mature compartment and can maintain the levels of stem cell candidates. It would be of
interest to evaluate the progenitors generated in vivo,
in order to define the role of cells thus obtained in
short-term repopulating ability.
Contributions and Acknowledgments
GC carried out the laboratory work and did the data analysis. SQ participated in the design, analysis and discussion.
JAC carried out the flow cytometry determination and participated in the discussion of the draft. JG was responsible for
the establishment of the study and critical evaluation.
The authors wish to thank Drs Lluis Amat, Santi Gonzalez
and Dolors Gomez from the Hopital Universitari Sant Joan
de Deu for collecting the cord blood, and Dr Lourdes Petriz
for her help with the stroma irradiation.
This work was supported by grants from the FIS 97/2120
and 98/0753, DGICYT PB95-0904 and from the Fundacion Jose Carreras para la Lucha contra la Leucemia.
Conflict of interest: none.
Redundant publications: < 50%; in BMT 1998; 21 suppl. 3, S77-S80 in which we published the preliminary data.
This previous paper appeared in a non-peer reviewed symposium supplement.
Manuscript received December 22, 1998; accepted April
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