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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1097-1105
Delayed Engraftment of Nonobese Diabetic/Severe Combined
Immunodeficient Mice Transplanted With Ex Vivo-Expanded Human
CD34+ Cord Blood Cells
By
G. Güenechea,
J.C. Segovia,
B. Albella,
M. Lamana,
M. Ramírez,
C. Regidor,
M.N. Fernández, and
J.A. Bueren
From the U. Biología Molecular y Celular, CIEMAT and Servicio
de Hematología, H. Puerta de Hierro, Madrid, Spain.
 |
ABSTRACT |
The ex vivo expansion of hematopoietic progenitors is a promising
approach for accelerating the engraftment of recipients, particularly
when cord blood (CB) is used as a source of hematopoietic graft. With
the aim of defining the in vivo repopulating properties of ex
vivo-expanded CB cells, purified CD34+ cells were
subjected to ex vivo expansion, and equivalent proportions of fresh and
ex vivo-expanded samples were transplanted into irradiated nonobese
diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic
intervals after transplantation, femoral bone marrow (BM) samples were
obtained from NOD/SCID recipients and the kinetics of engraftment
evaluated individually. The transplantation of fresh
CD34+ cells generated a dose-dependent engraftment of
recipients, which was evident in all of the posttransplantation times
analyzed (15 to 120 days). When compared with fresh CB, samples
stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor
(SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of
recipients was associated with the transplantation of the ex vivo-expanded versus the fresh CB cells (CD45+
repopulation in NOD/SCIDs BM: 3.7% ± 1.2% v 26.2% ± 5.9%, respectively, at 20 days posttransplantation; P < .005). An impaired short-term engraftment was also observed in mice
transplanted with CB cells incubated with IL-11/SCF/FLT-3
ligand (3.5% ± 1.7% of CD45+ cells in
femoral BM at 20 days posttransplantation). In contrast to these data,
a similar repopulation with the fresh and the ex vivo-expanded cells
was observed at later stages posttransplantation. At 120 days, the
repopulation of CD45+ and
CD45+/CD34+ cells in the femoral BM of
recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%,
respectively, and no significant differences of engraftment between
recipients transplanted with fresh and the ex vivo-expanded samples
were found. The analysis of the engrafted CD45+ cells
showed that both the fresh and the in vitro-incubated samples were
capable of lymphomyeloid reconstitution. Our results suggest that
although the ex vivo expansion of CB cells preserves the long-term
repopulating ability of the sample, an unexpected delay of engraftment
is associated with the transplantation of these manipulated cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE BASIC RESEARCH on the biology of ex
vivo expansion is now offering new advances in the field of
hematopoietic cell transplantation. Data obtained in mouse models have
shown an improved hematologic recovery in recipients transplanted with
ex vivo-expanded versus fresh hematopoietic samples.1-5 In
these models, the advantages and limitations derived from the
transplantation of ex vivo-expanded samples are being progressively
defined6 and data from the first clinical protocols already
reported.7-11 In oncohematology, the conditions required
for the purging of tumor-contaminated grafts during the in vitro
incubation process are being dissected, and new promising results have
been recently reported.12-15 Finally, in the field of gene
therapy, the in vitro incubation with adequate combinations of growth
factors is frequently used to facilitate the transduction of the
primitive repopulating cells or the selection of the transduced
population.16-20
Recent results from clinical cord blood (CB) transplantation have shown
that the number of nucleated cells per kilogram is a major factor in
the recovery of neutrophil and platelet counts.21 This
study, together with others previously reported,22-24 has shown the efficacy of CB grafts for the transplantation of children, although suggested limitations for the transplantation of adults. The
possibility of ex vivo expanding the progenitors present in CB grafts
is, therefore, an attractive approach that may facilitate the
engraftment of adult patients with these samples. To what extent the ex
vivo expansion of CB cells will be clinically useful for this purpose
and whether this will be a safe procedure for preserving the longevity
of the self-renewing hematopoietic stem cells, is still unclear. In
this respect some restrictions,25 and even marked failures
in the repopulating function of in vitro-incubated long-term
repopulating cells (LTRCs) have been reported in mouse hematopoietic
grafts.26-28 However, other studies have shown that primitive repopulating cells from mouse bone marrow (BM) can be maintained29,30 and even modestly expanded in
culture.31 Moreover, in human ex vivo-expanded CB samples,
a modest amplification in the progenitors capable of repopulating
nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice has
been achieved,32,33 something that is consistent with the
idea that ex vivo expansion protocols could at least preserve the human
LTRCs during the amplification of the more mature progenitors.
Regarding the short-term repopulating ability of ex vivo-expanded
grafts, our results with mouse BM indicated that the transplantation of
these samples mediates an accelerated engraftment of recipients. Nevertheless, we showed that the speed of the engraftment was significantly below predictions made on the basis of the large granulocyte-macrophage colony-forming unit (CFU-GM) content, which characterizes the incubated grafts.6 Whether a similar
conclusion is applicable to human ex vivo-expanded samples is unknown
and, therefore, a risk in overestimating the functionality of human ex
vivo-expanded grafts was deduced from that study.
The use of the human-NOD/SCID mouse model20 now offers the
possibility of analyzing the short-term, as well as the long-term, repopulating ability of human hematopoietic samples in an in vivo experimental system. Therefore, with the aim of comparing the in vivo
repopulating properties of fresh and ex vivo-expanded CB cells, we
have followed the kinetics and stability of engraftment of NOD/SCID
mice transplanted with both types of samples. Data presented in this
work are consistent with the idea that the ex vivo expansion of CB is
compatible with an overall preservation of their long-term repopulating
ability. However, an unexpected delay of engraftment was associated
with the transplantation of these ex vivo-expanded samples.
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MATERIALS AND METHODS |
Human samples and isolation of CD34+ cells.
Cells were obtained from umbilical CB after a normal full-term delivery
and the informed consent of the mother. Samples were collected at dawn
and processed during the next 12 hours postpartum. Mononuclear (MN)
cells were obtained by layering the blood onto Ficoll-Hypaque (1.077 g/mL; Pharmacia Biotech, Uppsala, Sweden), and
centrifuging at 400g for 30 minutes. The interface layer was then collected, washed three times, and resuspended in Iscove's Modified Dulbecco's Medium (IMDM; GIBCO-BRL, Grand Island, NY). To
purify the CD34+ cells, the MN fraction was subjected to
immunomagnetic separation using the VarioMACS CD34 progenitor cell
isolation kit (Myltenyi Biotech, Auburn, CA), following the
manufacturer's recommendations. Briefly, MN were washed and
resuspended in phosphate-buffered saline (PBS) containing 0.5% bovine
serum albumin (BSA) (Fraction V; Sigma, St Louis, MO) and
5 mmol/L EDTA. Cells were incubated first with QBEND-10 antibody (mouse
anti-human CD34) (Miltenyi Biotech) in the presence of human IgG as a
blocking reagent and then with an anti-mouse antibody coupled with MACS
microbeads. Labeled cells were filtered through a 30-µm nylon mesh
before loading onto a column installed in the magnetic field. Trapped cells were eluted after the column was removed from the magnet and
further depleted of contaminant CD34 cells by a
second MACS column passage. The purity of the CD34+
population ranged from 95% to 98% as evaluated by flow cytometry using an EPICS Elite ESP analyzer (Coulter, Hialeah, FL).
CD34+ cells were cryopreserved in liquid nitrogen, with
10% dimethyl sulfoxide (DMSO) and 20% fetal bovine serum (FBS). After
thawing, CD34+ cells were diluted 1:1 with human albumin
2.5% (Behring, Hoecht Iberica, Spain) and dextran 40, 5%
(Rheomacrodex 10%; Pharmacia Biotech), and maintained at room
temperature for 10 minutes. Cells were then diluted with medium and
centrifuged at 800g for 15 minutes.34 Freshly
isolated CD34+ cells, as well as cells that had been
cultured for various time intervals, were phenotyped by flow cytometry
as previously described.35 Antibodies used for the
cytometric analysis included anti-CD34 labeled with phycoerythrin (PE)
and anti-CD38 labeled with fluorescein isothiocyanate (FITC) (Becton
Dickinson, San Jose, CA).
Animals.
NOD/LtSz-scid/scid (NOD/SCID) mice (deficient in Fc receptors,
complement function, natural killer, B-, and T-cell function) were used
as recipients of the human hematopoietic cells. Mice were purchased
from The Jackson Laboratory (Bar Harbor, ME). All animals were handled
under sterile conditions and maintained under microisolators. Before
transplantation, 6- to 8-week-old mice were total body irradiated with
2.5 to 3.0 Gy of x-rays (300 kV, 10 mA; Philips MG-324, Hamburg, Germany).
Ex vivo expansion of CD34+ cells.
Purified CD34+ cells were incubated in 25-cm2
tissue culture flasks (Nunc, Roskilde, Denmark) at 2.5 × 104 cells/mL in IMDM containing 20% FBS (GIBCO
Laboratories), recombinant human interleukin-3 (IL-3), IL-6, and stem
cell factor (SCF) at a final concentration of 100 ng/mL (all of them
kindly provided by Immunex, Seattle, WA). At weekly intervals, cell
cultures were diluted with fresh complete medium to reach the initial
cell concentration. In some experiments cells were incubated under the
same experimental conditions using SCF, FLT-3 Ligand (FL; kindly
provided by Immunex), and IL-11 (a kind gift from Genetics Institute,
Cambridge, MA). At the indicated time points, cells were collected for
performing cytologic and cytometric studies, CFU-GM cultures, and
transplantation into NOD/SCID mice.
Cytological analyses and CFU-GM assays.
Fresh CD34+ CB cells and 6-day cultured cells were
cytocentrifuged onto slides, fixed in 100% methanol for 10 minutes,
dried at room temperature, and stained with May-Grünwald-Giemsa
staining solution. CFU-GM colonies were grown in enriched semisolid
culture media from StemBio Research SG*1d (a kind gift of StemBio
Research, Villejuif, France). The appropriate number of cells was
seeded and cultured for 14 days at 37°C in a humidified atmosphere
at 5% of CO2 in air.
Analysis of human cell engraftment.
At periodic intervals after transplantation, BM samples were aspirated
from one femur by puncture through the knee joint, according to a
previously described procedure.36 At the end of the
experiments, mice were killed and peripheral blood and spleen were also
analyzed by flow cytometry for the presence of human cells. Aliquots of
1 to 5 × 105 cells/tube were stained for 25 minutes
at 4°C with anti-human-CD45-FITC (clone HI30,
Pharmingen, San Diego, CA) or -PECy5 (Clone J33, Immunotech, Marseille, France) in combination with anti-human-CD34-PE (Anti-HPCA-2; Becton Dickinson Immunocytometry, San Jose, CA), anti-human-CD33-PE (Anti-Leu-M9; Becton Dickinson), or
anti-human-CD19-PE (Anti-Leu-12; Becton Dickinson). Thereafter, red
blood cells were lysed by adding 2.5 mL of lysis solution (0.155 mol/L
NH4Cl + 0.01 mol/L KHCO3 + 104
mol/L EDTA) and incubation at room temperature for 10 minutes. Cells
were then washed in PBA (phosphate-buffered salt solution with 0.1%
BSA and 0.01% sodium azide), resuspended in PBA + 2 µg/mL propidium
iodide (PI), and analyzed by flow cytometry. For each analysis, a total
of 10,000 viable (PI) cells with appropriate forward
scatter (FS)/side scatter (SSC) were collected. As
controls of nonspecific staining, cells were labeled with
conjugated-nonspecific isotype antibodies. Additionally, cells from
nontransplanted NOD/SCID mice were stained with anti-CD45, anti-CD34,
anti-CD33, and anti-CD19 antibodies.
Statistics.
Data are presented as the mean ± SEM. The significance of
differences between groups was determined by using the two-tailed Student's t-test. The processing and statistical analysis of
the data were performed by using the software SPSS V6.1.2. (SPSS, Inc,
Chicago, IL).
| |
RESULTS |
Ex vivo expansion of CD34+ CB cells incubated with
IL-3/IL-6/SCF.
Data in Fig 1 show the kinetics of
CD34+ CB cells incubated with or without IL-3/IL-6/SCF as
the stimulatory source. As shown in Fig 1A, a progressive amplification
in the cellularity of stimulated cultures was observed during the study
period. At the end of the incubation, a 17,000-fold increase over input
values was observed. The growth of the CFU-GM population reached a
plateau on day 19 of incubation, when the number of
CFU-GMs generally exceeded 45-fold the input value (Fig 1B). When the
proportion of CFU-GMs was evaluated, similar values of
CFU-GM/105 cells were observed during the first 6 days in
culture (Fig 1C), suggesting a modest differentiation pressure at this
time of incubation. A more detailed analysis of the composition of
fresh CD34+ cells and 6-day incubated cells is shown in Fig
1D, which shows an evident increase in the more differentiated cells
(CD34/CD38+ and morphologicaly
differentiated forms) at the expense of a reduced proportion in the
primitive precursors (ie, CD34+/CD38 or
blast cells). This time of incubation was selected for all the
subsequent experiments of transplantation into NOD/SCID mice. The
average amplification in cells and CFU-GM at this time was 15.9 ± 3.9-fold and 19.8 ± 7.9-fold, respectively (n = 7).
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| Fig 1.
Ex vivo expansion kinetics of purified
CD34+ cord blood cells. Cells were cultured in IMDM
supplemented with 20% FBS ( ) or FBS plus IL-3, IL-6, and SCF ( ).
The figure shows the evolution of the cellularity (A) and CFU-GM (B) of
cultures established with an initial input of 2.5 × 104
cells; the proportion of CFU-GM along the culture (C), and (D) the
total composition of fresh ( ) and 6-day incubated samples in the
presence of FBS plus IL-3/IL-6/SCF (). At weekly intervals, cell
cultures were diluted with fresh complete medium to reach the initial
cell concentration. The figure represents data obtained from one
representative experiment.
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NOD/SCID repopulating ability of ex vivo-expanded grafts.
With the aim of evaluating the repopulating properties of ex
vivo-expanded versus fresh CB samples, purified CD34+
cells were subjected to a 6-day incubation period under the conditions described above. To confirm a relationship between the engraftment level of recipients versus the transplanted cell dose, aliquots consisting of 105, 104, and 103
fresh CD34+ cells were transplanted into three groups of
NOD/SCID mice. In addition, the populations that corresponded to the
incubation of 105, 104, and 103
fresh CD34+ cells were transplanted into further groups of
irradiated mice. At different times posttransplantation, small samples
of BM were obtained from the femora of NOD/SCID
recipients, and the proportion of CD45+ and
CD45+/CD34+ cells evaluated by
fluorescence-activated cell sorting (FACS) analysis.
As shown in Fig 2, when fresh samples are
considered, a good relationship between the size of the transplanted
sample and the engraftment of the animals is deduced for all of the
posttransplantation periods analyzed, both for CD45+ and
CD45+/CD34+ determinations. When ex
vivo-expanded samples were transplanted, a dose-dependent engraftment
was seen from day 40 to day 120 posttransplantation. At day 15 posttransplantation, however, increases of 10-fold and even 100-fold in
the graft size did not significantly modify the modest repopulation
associated with the transplantation of 103
CD34+ expanded cells.
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| Fig 2.
Cell dose-dependent engraftment of NOD/SCID mice
transplanted with fresh or ex vivo-expanded human
CD34+ CB cells. The figure represents the proportion of
CD45+ and double-positive
CD45+/CD34+ cells in the femoral BM of
NOD/SCID mice transplanted with 105, 104, and
103 fresh CD34+ CB cells ( ) or with the
IL-3/IL-6/SCF ex vivo-expanded populations, which corresponded to the
incubation of 105, 104, and 103
fresh CD34+ cells ( ). BM was periodically sampled from
day 15 to day 120 posttransplantation. At day 120 posttransplantation,
data from spleen (squares), and PB (triangles) are also represented.
Samples were ex vivo-expanded with FBS plus IL-3/IL-6/SCF, as
indicated in Fig 1.
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To compare more directly the kinetics of repopulation of fresh and ex
vivo-expanded samples, the proportion of CD45+ and
CD45+/CD34+ cells corresponding to the
individually analyzed mice was represented (Fig 3). The analysis of animals
corresponding to the 105 CD34+ cell group shows
a gradual increase in CD45+ cells that reached very high
and stable values by day 40 posttransplantation. The rate of increase
was most significant in mice transplanted with the ex vivo-expanded
samples, as the initial values of CD45+ cells in this group
were well below the values obtained in the control group. At later
stages posttransplantation, high and stable engraftments were also
observed in NOD/SCIDs transplanted with the cultured cells. Similar
conclusions were deduced from the analysis of the double-positive
CD45+/CD34+ cells, although in this case,
differences between both groups of recipients were only apparent in the
first BM sampling (15 days posttransplantation). When mice were
transplanted with smaller grafts, 104 and 103
fresh CD34+ cells or the equivalent ex vivo-expanded
populations, repopulation differences between the fresh and the ex
vivo-expanded grafts could not be established because very
heterogeneous and very poor engraftments were observed, respectively.
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| Fig 3.
Kinetics of engraftment of individual NOD/SCID mice
transplanted with fresh or ex vivo-expanded
CD34+ cells. The figure represents the individual
kinetics of engraftment of NOD/SCID mice shown in Fig 2, which were
transplanted with fresh (closed symbols) or with the equivalent cell
product generated after incubation with IL-3/IL-6/SCF (open symbols).
For further details, see legend to Fig 2.
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With the aim of confirming differences in the engraftment of mice
transplanted with fresh and ex vivo-expanded samples, three further
experiments were conducted, although in this case, purified CD34+ cells were divided in three aliquots. One aliquot was
transplanted before incubation (105 CD34+
cells/mouse), while the second and the third ones were previously incubated for 6 days with IL-3/IL-6/SCF and IL-11/SCF/FL, respectively. In these two cases, each recipient was transplanted with the expanded cell product generated by 105 fresh CD34+
cells. The average number of cells present in IL-3/IL-6/SCF and IL-11/SCF/FL ex vivo-expanded grafts exceeded 18-fold and 6-fold, respectively, the cell numbers present in the fresh samples. Regarding the CFU-GM content of each of the ex vivo-expanded samples, average increases of 13-fold and 3-fold were obtained, respectively, with respect to the fresh samples (not shown).
Data in Fig 4 show the engraftment of
individual NOD/SCIDs transplanted with each of the three populations
considered in this new study. The kinetics of engraftment of mice
transplanted with fresh CD34+ cells were essentially the
same as those described in Fig 3 (see data corresponding to
105 CD34+ cells), although in this case, a
heterogeneous repopulation of CD45+/CD34+ cells
was seen at 20 days posttransplantation. As it happened in the previous
experiment, analyses made soon after the transplantation (day 20 posttransplantation) showed that the CD45+ repopulation of
mice transplanted with the IL-3/IL-6/SCF ex vivo-expanded samples was
clearly below the corresponding values found in the control group.
Significantly, this was also the case in mice transplanted with
samples, which had been incubated with IL-11/SCF/FL. As shown in Fig 4,
recipients transplanted with either the fresh or the ex vivo-expanded
grafts showed a high and stable repopulation of CD45+ cells
in the long-term posttransplantation. Regarding the repopulation of
CD45+/CD34+ cells in mice transplanted with the
ex vivo-expanded populations, a particular delayed engraftment was
apparent in the IL-11/SCF/FL group.
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| Fig 4.
Kinetics of engraftment of individual NOD/SCID mice
transplanted with fresh and two different types of ex vivo- expanded
CD34+ cells. The figure represents the individual
kinetics of engraftment of NOD/SCID mice transplanted with fresh
CD34+ cells (105 cells/mouse; closed symbols)
or with the ex vivo-expanded product generated after a 6-day
incubation of 105 fresh CD34+ cells in the
presence of IL-3/IL-6/SCF () and IL-11/SCF/FLT3L ( ).
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Differences between the engraftment of the three groups of NOD/SCID
mice is best seen in Fig 5, which
represents the mean values of CD45+ and
CD45+/CD34+ cells from day 20 to day 120 posttransplantation. Marked and highly significant reductions
(P < .005) in the repopulation of CD45+ cells
were observed at day 20 posttransplantation in recipients transplanted
with both types of in vitro incubated samples, when compared with
recipients of fresh CB. Differences were maintained when the myeloid
CD45+/CD33+ cells were analyzed (not shown). In
contrast to these data, only minor differences between the fresh and
the ex vivo-expanded groups were seen at later stages
posttransplantation. When CD45+/CD34+ values
were considered, a trend of delayed engraftment of mice transplanted
with the ex vivo-expanded samples was also observed, although
statistical differences with the control group were only obtained in
mice transplanted with the IL-11/SCF/FL-stimulated CB and sampled at 40 days posttransplantation.
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| Fig 5.
Delayed engraftment of NOD/SCID mice transplanted with
fresh and ex vivo-expanded CD34+ cells. The figure
represents the mean values of CD45+ and double-positive
CD45+/CD34+ cells in the femoral BM of
NOD/SCID mice shown in Fig 4, which were transplanted with fresh ()
and with cells incubated in the presence of IL-3/IL-6/SCF () and
IL-11/SCF/FL (). For further details, see legend to Fig 4.
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Long-term differentiation ability of fresh and ex vivo-expanded
CD34+ CB cells.
The multilineage differentiation capacity of fresh CD34+
and 6-day ex vivo-expanded samples was investigated at 120 days
posttransplantation. Thymus was routinely reconstituted by less than
1% of CD45+ cells and therefore it was not considered in
this analysis. Figure 6 shows the
hematopoietic differentiation pattern in the BM, spleen, and peripheral
blood (PB) of representative NOD/SCID mice transplanted with IL-3/IL-6/SCF ex vivo-expanded cells. As shown in Fig 6, in
addition to a high expression of the CD34 marker in the BM, an evident
lymphomyeloid engraftment was observed in the three tissues analyzed.
Similar histograms were also obtained in all recipients transplanted
with fresh and IL-11/SCF/FL cultured cells (not shown).
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| Fig 6.
Multilineage repopulation ability of IL-3/IL-6/SCF ex
vivo-expanded samples. BM, spleen, and PB of NOD/SCID mice were
examined at 120 days posttransplantation. Histograms represent cells
labeled with anti-CD45-PECy5 and anti-CD34-PE, anti-CD19-PE, or
anti-CD33-PE antibodies. Quadrants were set according to
isotype-matched negative control stainings.
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A detailed analysis of the lymphohematopoiesis of NOD/SCIDs
transplanted with fresh and ex vivo-expanded samples is shown in
Table 1. These data confirm the capacity of
either of the ex vivo-expanded samples to repopulate NOD/SCIDs with
lymphomyeloid cells. As shown, T-cell reconstitution was significantly
below B-cell and myeloid repopulation in all analyzed tissues,
regardless of the manipulation of the graft. It is worth mentioning
that a trend of increased B-cell over myeloid reconstitution was found in the BM and spleen, but not in PB, of mice transplanted with the ex
vivo-expanded samples. Also, a trend of reduced T-cell repopulation
was associated with the in vitro incubation process, although no
significant data were obtained in this respect.
| |
DISCUSSION |
Two main aspects in the biology of ex vivo-expanded CB cells will
ultimately define the applicability of these grafts in human hematopoietic transplantation. The first one relates to the actual efficacy of the ex vivo-expanded samples for improving the short-term engraftment of recipients. The second point relates to the possibility of preserving the longevity and the multipotentiality of CB cells subjected to ex vivo expansion.
Data obtained with mouse BM cells have shown an accelerated engraftment
of recipients transplanted with ex vivo-expanded
samples,1-6 while also showing some limitations in the
short-term functionality of these grafts.6 The experiments
presented in this study aimed to confirm these observations using human
CB cells. Nowadays, the human-NOD/SCID mouse model20 offers
one of the best approaches for investigating the in vivo repopulating
ability of human hematopoietic samples.20 In addition, the
possibility of monitoring the engraftment of these animals after the
transplantation of human hematopoietic samples36 allowed us
to investigate for the first time the kinetics of engraftment
associated with the transplantation of CB cells before and after ex
vivo expansion.
In our initial experiments, CB samples were stimulated with
IL-3/IL-6/SCF, a growth factor combination widely used in ex vivo expansion and retroviral-mediated gene transfer
protocols.19,37-40 Consistent with earlier
studies,36,41 maximum values of human repopulation were
reached in about 40 days after the transplantation of fresh CB. In
comparison to the kinetics of engraftment observed in these recipients,
the human repopulation of mice transplanted with
IL-3/IL-6/SCF-incubated CB was markedly delayed (see Figs 3 through
5). Significantly, this was produced even though the cellularity and
CFU-GM content of the incubated samples was highly increased (16-fold
and 20-fold, respectively) with respect to the fresh samples. To
determine whether the delayed repopulation was a direct consequence of
the IL-3/IL-6/SCF stimulation, a different combination of growth
factors was also used. However, as observed after the transplantation
of IL-3/IL-6/SCF-stimulated CB, a significant delay of engraftment was
also observed in recipients transplanted with IL-11/SCF/FL-incubated
cells (Fig 5).
Although a relative engrafting defect, as a per CFU-GM basis, was
observed in ex vivo-expanded mouse BM,6 an overall
improved engraftment was associated with the transplantation of these
samples. In the current experiments with human CB, the engrafting
defect of the cultured cells was apparent not only in a per CFU-GM
basis, but also in absolute terms when comparing the engrafting ability of the samples before and after the ex vivo expansion.
Whether the engraftment delay associated with the incubation process is
related to the xenogenic transplantation model used in these assays or
on the contrary, whether our results are actually predicting the
behavior of the ex vivo-expanded samples in human recipients is still
unclear. However, consistent with our results, recent data in
myeloablated baboons have shown that the transplantation of autologous
ex vivo-expanded BM also results in a delayed engraftment of
recipients.42 The possibility of impairing rather than
improving the engraftment of patients transplanted with in
vitro-incubated samples is clearly deduced from these findings and
would be considered in the design of new clinical trials related to ex
vivo expansion. These experimental data also suggest that other
biological properties apart from the content in hematopoietic
progenitors are modulating the grafting ability of the sample. This
hypothesis is consistent with the current clinical results, which have
not demonstrated a significant shortening in the engraftment of
patients transplanted with ex vivo-expanded grafts.8-11
The extent to which the proliferative activation of the
progenitors26,28 or changes in the expression of adhesion
molecules,43 chemokine receptors,44 or other
unknown molecules are critical for the correct seeding of the
hematopoietic grafts in the recipient BM is currently unknown.
Although the experiments conducted in this study cannot discriminate
the generation of small changes in the content of LTRCs, our data show
that no changes in the long-term engraftment of the animals are
observed as a result of the transplantation of in vitro-incubated
samples (Fig 5). This observation, together with the dose-dependent
engraftment shown in Fig 2, indicates that no overall changes in the
long-term repopulating ability of the grafts are produced during the
incubation process. The generation of relative alterations in the
homing and in the size and self-renewal ability of the LTRC compartment
cannot be deduced, however, from our study.
Consistent with earlier studies, a lymphomyeloid engraftment of
recipients was observed both when fresh41,45 and ex
vivo-expanded CB cells were
transplanted.32,33 The quantitative analysis of data obtained at 120 days postengraftment showed, however, a trend
of reduced myeloid and T-cell reconstitution at the expense of an
increased B-cell repopulation (see Table 1). Taking into account that
the T-cell repopulation of NOD/SCIDs is most probably the consequence
of the in vivo amplification of inoculated T cells,46 the
reduced T-cell engraftment observed in the ex vivo-expanded groups
could be explained by considering that residual T cells present in the
CD34+ population are being purged during the incubation of
the samples; something, which could be exploited in T-cell depletion
strategies. The reason why recipients of ex vivo-expanded grafts
contained a reduced repopulation of myeloid over B cells in BM and
spleen, but not in PB, is currently unknown and should be subject of
further studies.
Taken together, our data indicate that although no changes in the
long-term repopulation of NOD/SCID mice are produced after the
transplantation of ex vivo-expanded CB cells, a very significant delay
in the engraftment of these recipients is associated with the in vitro
incubation of the samples. Our observations have direct implications in
the design of new clinical protocols of ex vivo expansion and emphasize
the relevance of investigating more deeply the biological insights
involved in the ex vivo expansion of hematopoietic progenitors.
| |
ACKNOWLEDGMENT |
The authors thank I. Ormán for expert assistance with the flow
cytometry, S. García for excellent technical collaboration, and
J. Martínez for careful maintenance of the animals. We also thank Dr C. Garaulet, D. Monteagudo, and N. Somolinos and the staff of midwives from Hospital General de Móstoles for umbilical cord blood collection.
| |
FOOTNOTES |
Submitted August 7, 1998; accepted October 2, 1998.
Supported by Grant No. SAF 98-0008-C04-01 from the Comisión
Interministerial de Ciencia y Tecnología and Fundación
Ramón Areces.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to J.A. Bueren, PhD, Unidad de
Biología Molecular y Celular, CIEMAT, Avenida Complutense 22, 28040 Madrid, Spain; e-mail: bueren{at}ciemat.es.
| |
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Abstract
Introduction