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 icon

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




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Haematologica

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

progenitor cells


GEMMA CAPMANY, SERGI QUEROL, JOSE ANTONIO CANCELAS, JOAN GARCIA


Cryobiology and Cell Therapy Department and Barcelona Cord Blood Bank, Institut de Recerca Oncologica, Barcelona,


Spain


ABSTRACT


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,

expansion


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: squerol@iro.es


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

Haematologica

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


Progenitor cells


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).


Cytokines


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

(Oxford, UK).


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.


Expansion cultures


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

serum-free media.


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

were performed.

CFU assay


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-


Haematologica

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

level (CD34+++).


CAFC assay


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

were comparable.


Statistical analysis


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.


Results


Positive selection


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.


Culture medium

NC

Fold expansion

Total CD34+ CD34bright

cells cells

CFC

^ IMDM+ FCS 18.19

(± 10.05)

9.16

(± 7.06)

4.26

(± 3.94)

12.50

(± 14.51)

SP34 16.71

(± 7.23)

10.97

(± 5.86)

3.53

(± 2.11)

13.60

(± 8.77)


Haematologica

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

serum.


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.


Haematologica

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

CD34bright cells).


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

cultures


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.


Discussion


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


Haematologica

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-

pendent 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).

Day 0 Day +6

Expansion rate

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-


Haematologica

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.


Funding


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.


Disclosures


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 processing


Manuscript received December 22, 1998; accepted April

26, 1999.


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