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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4509-4515
A Potential Molecular Approach to Ex Vivo Hematopoietic Expansion
With Recombinant Epidermal Growth Factor Receptor-Expressing Adenovirus
Vector
By
Tokiharu Takahashi,
Kaoru Yamada,
Tomoyuki Tanaka,
Keiki Kumano,
Mineo Kurokawa,
Tsuyoshi Takahashi,
Naoto Hirano,
Hiroaki Honda,
Shigeru Chiba,
Kohichiro Tsuji,
Yoshio Yazaki,
Tatsutoshi Nakahata, and
Hisamaru Hirai
From The Third Department of Internal Medicine, the Department of
Transfusion and Immunohematology, and the Department of Cell Therapy & Transplantation Medicine, Faculty of Medicine, University of Tokyo,
Bunkyo-ku, Tokyo, Japan; and the Department of Clinical Oncology,
Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo,
Japan.
 |
ABSTRACT |
Ex vivo expansion of hematopoietic stem cell (HSC) is an attractive
technology for its potency of a variety of clinical applications. Such
a technology has been achieved to some extent with combinations of
various cytokines or continuous perfusion cultures. However, much more
improvement is required especially for expansion of primitive
hematopoietic progenitors. We propose here a novel molecular approach
that might have the potential to compensate the current expansion. We designed an adenovirus vector to transiently express human epidermal growth factor receptor (EGFR), which is known to
transduce only a mitogenic, but not a differentiation signal to mouse
bone marrow cells on human purified CD34+ peripheral
blood (PB) cells, and tried to expand these cells with EGF ex vivo.
Because we found that exposure of CD34+ PB cells to
cytokines induced surface expression of adenovirus-internalization receptor and rendered these cells permissive to adenovirus infection, we infected these cells with the adenovirus vector carrying EGFR gene
in the presence of cytokines. Two-color flow cytometric analysis demonstrated that 60.3% ± 22.4% of CD34+ cells
expressed the adenovirus-mediated EGFR. Moreover, long-term culture-initiating cell assay showed that adenovirus
vector could transduce more primitive progenitors. Subsequently, we
tried to expand these cells in suspension culture with EGF for 5 days. Methylcellulose clonal assay showed that EGF induced 5.0- ± 2.4-fold proliferation of the colony-forming unit pool during 5 days of expansion. The simple procedure of efficient adenovirus gene delivery to immature hematopoietic cells proved promising, and this technique was potentially applicable for a novel strategy aiming at ex vivo expansion of hematopoietic progenitors.
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INTRODUCTION |
A GREAT INTEREST has been focused on ex
vivo expansion of hematopoietic stem cells (HSCs) in clinical
hematology for a variety of clinical applications.1,2 It
could complement current bone marrow (BM) or peripheral blood stem cell
transplantation (PBSCT) technology. We would be able to use cord blood
stem cells for allogeneic transplantation to adults, or much fewer
PBSCs would be sufficient for PBSCT. Finally, as a target of
hematological gene therapy, ex vivo expansion of HSCs is an
indispensable technology. To date, many previous studies reported
amplification of hematopoietic progenitors, and most of them used
various combinations of cytokines3 or stroma
cells.4 In particular, incubation of purified
CD34+ cells with combinations of interleukin-3 (IL-3) and
other cytokines has been the most commonly studied technique, and some
groups reported successful clinical application of this
technique.5-7 However, IL-3 acts on relatively late-stage
progenitors and would differentiate these cells rather than
proliferate.1 Therefore, a technique to expand primitive
progenitors with keeping their immaturity is required.
To address these issues, we propose here a novel molecular approach
that might have the potential to enhance the current
expansion. We tried to express epidermal growth factor receptor (EGFR)
transiently on human hematopoietic progenitors ex vivo and expand them
with EGF as a self-renewal factor, because ectopic EGFR expressed on mouse BM cells was known to transduce only a mitogenic, but not a
differentiation signal.8 Thus, it could be expected that hematopoietic immature cells expressing ectopic EGFR could be expanded
by EGF without differentiation.
For this purpose, we used an adenovirus vector for gene delivery,
because adenoviral DNA rarely integrate into host cell genome, which is
favorable for our purpose in that no adverse effects would occur in
patients after reinfusion of expanded progenitors. Furthermore, an
adenovirus vector could transduce nonreplicating cells; thus, it has an
advantage for transduction of quiescent HSCs.
Recently, gene transfer technique with an adenovirus vector has greatly
advanced, and replication-deficient adenovirus vectors were used in
some clinical trials for cystic fibrosis.9,10 Karlsson et
al11 first succeeded in transducing an exogenous gene to
hematopoietic cells using a recombinant adenovirus and suggested its
potential use. However, only a few studies reported the application of
this vector for hematopoietic systems.12,13
In the present study, we first examined the adenoviral gene
transduction of hematopoietic progenitors. In particular, we analyzed the expression of adenovirus-internalization receptor on the surface of
CD34+ cells. Second, we transduced exogenous EGFR to
hematopoietic progenitors with a recombinant adenovirus vector and
tried to expand them ex vivo to examine the feasibility of this novel
molecular strategy.
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MATERIALS AND METHODS |
Cells and cell separation procedures.
Human peripheral blood (PB) mononuclear cells (MNCs) mobilized with
cyclophosphamide and filgrastim were collected by apheresis from 7 patients undergoing therapy for nonhematologic malignancies after
informed consent was obtained.
CD34+ cells were enriched using Isolex 50 Stem Cell
Research Reagent Kit (Baxter Healthcare Corp, Deerfield, IL) according to the manufacturer's instructions. Final CD34+ cell
fraction was stained with fluorescein isothiocyanate (FITC)-conjugated 8G12 monoclonal antibody (MoAb) to CD34 (anti-HPCA-2; Becton
Dickinson, San Jose, CA) and fixed with paraformaldehyde, and the
purity was quantitated with a FACSort flow cytometer (Becton
Dickinson). More than 85% of the purified cells were verified to be
CD34+ by flow cytometric analysis.
For the experiments of Table 1, Fig 3, and Fig 5, CD34+
cells, CD34+ EGFR+ cells, and CD34+
EGFR cells were sorted with a flow cytometer (FACS
Vantage; Becton Dickinson), respectively. The purity of each sorted
population as verified by reanalysis was greater than 95%.
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| Fig 3.
Quantitation of LTC-IC by limiting dilution analysis.
Four dilutions of CD34+ EGFR+ cells ( )
and CD34+ EGFR cells ( ) were cultured
over irradiated stromal layers, and the number of clonogenic cells
detectable after 7 weeks was determined. In this experiment, the
frequency of LTC-IC was 1:768 cells in CD34+
EGFR+ cells ( ) and 1:1,206 in CD34+
EGFR cells (---).
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| Fig 5.
Generation of total progenitors after infection and
liquid culture of sorted 200 CD34+ cells. One-milliliter
cultures were initiated with 1.0 × 104 sorted
CD34+ cells in the StemPro-34 SFM Complete Medium
containing IL-6 and SCF in the presence or absence of EGF. These cells
were incubated with the Ax1w or Ax/hEGFR virus, and control cells were
cultured without adenovirus vectors. After 5 days of incubation, 1/50
of the cells of each fraction were subjected to methylcellulose clonal culture with SCF, IL-6, IL-3, and Epo in triplicate. The data presented
are from a single patient, and the mean ± SD of results from three
dishes is shown.
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Determination of expression of integrin
 v 3 on CD34+ cell
surface.
PB MNCs mobilized and collected by apheresis were separated by
Lymphoprep (1.077 g/mL; Nycomed Pharma AS, Oslo, Norway). Cells were
washed with phosphate-buffered saline (PBS) containing 1% nonimmune
rabbit serum and incubated in the presence of 25 µg of anti-integrin
v3 MoAb, LM609 (Biogenesis, Poole,
UK) per milliliter for 30 minutes at 4°C. Cells were
washed twice and then incubated with rabbit antimouse Ig antibody
conjugated to phycoerythrin (PE; DAKO AS, Glostrup, Denmark) for 30 minutes at 4°C. Control cell samples were incubated with the
secondary antibody alone. Cells were washed twice and then incubated
with FITC-8G12 MoAb to CD34 or isotype FITC control (Becton Dickinson). After additional washes, cells were analyzed by a FACSort flow cytometer with the Cell Quest program (Becton Dickinson).
For cell activation studies, CD34+ cells were purified as
described above and cultured with IL-3 (5 ng/ml; Kirin Brewery Inc, Takasaki, Japan), IL-6 (20 ng/mL; Kirin Brewery Inc), or
stem cell factor (SCF; 100 ng/mL; Kirin Brewery Inc) in various
combinations in RPMI 1640 medium containing 10% fetal calf serum
(FCS). After 48 hours of incubation, the cells were double-stained and
analyzed as described above. CD34+ cells cultured in medium
containing 10% FCS without cytokines were stained in the same way and
served as a control. To set the CD34+ fraction, cells that
were incubated with FITC-labeled isotype control (mouse Ig G1; Becton
Dickinson) instead of antihuman CD34 MoAb were used as a negative
control (data not shown), and gated cells were analyzed for the
expression of integrin v3.
Recombinant adenovirus vectors.
The recombinant, replication-deficient adenovirus vectors encoding
human EGFR were generated as previously described.14 Briefly, the human EGFR coding sequence was placed under the control of
CAG promoter in the pAx1CAwt expression plasmid15
containing almost whole genome of adenovirus without E1A, E1B, and E3
regions (pAxCAT-hEGFR). The replication-deficient recombinant
adenovirus AxCAT-hEGFR (Ax/hEGFR) was obtained by in vivo homologous
recombination after cotransfection into the 293 human embryonic kidney
cell line with pAxCAT-hEGFR, and adenovirus DNA-terminal protein
complex was digested with EcoT22I.14 Viruses were purified
by ultracentrifugation through two cesium chloride gradients and stored
in Ca2+-Mg2+-free PBS with 10% glycerol at
 80°C.16 The titer of produced adenovirus vectors was
determined by a limiting dilution assay using the 293 cells.16 The mock replication-deficient vector Ax1w14 was generously provided by Dr I. Saito (University
of Tokyo, Tokyo, Japan) and used as a control.
Determination of surface expression of EGFR on
CD34+ cells.
For determination of adenovirus-mediated transduction efficiency,
enriched CD34+ cells were cultured in 2 mL of RPMI 1640 medium containing 1% human serum albumin (HSA) at 1 × 105 cells per well. The cells were incubated at the
indicated MOI of the Ax1w or Ax/hEGFR virus with 5 ng of IL-3, 20 ng of
IL-6, and 100 ng of SCF per milliliter.
After 60 hours of infection, cells were incubated with antihuman EGFR
MoAb (Ab-1; Oncogene Science Inc, Cambridge, MA) at a concentration of
1 µg/mL. Control cell samples were incubated with the isotype control
antibody (anti-keyhole limpet hemocyanin, IgG2a; Becton Dickinson) in
the same way. After washed with PBS twice, the cells were incubated
with rabbit antimouse Ig antibody conjugated to PE (DAKO AS) for 30 minutes at 4°C. The cells were washed twice and then incubated with
FITC-8G12 MoAb to CD34 or FITC-labeled isotype control (mouse Ig G1).
After additional washes, cells were analyzed by a FACSort flow
cytometer with the Cell Quest program (Becton Dickinson). The cells
incubated with the FITC-labeled isotype control were used to set the
upper boundary of the negative cell autofluorescence and
CD34+ fraction (data not shown).
Clonal culture of sorted CD34+
EGFR+ and CD34+
EGFR cells.
CD34+ PB cells were enriched by immune beads and infected
with Ax/hEGFR in the same way. After 60 hours of infection, the cells were sorted into CD34+ EGFR+ or
CD34+ EGFR fractions and were subsequently
incubated in triplicate at concentrations of 200 cells/mL in
methylcellulose culture, as previously reported.17,18 One
milliliter of culture mixture containing cells, 0.9% methylcellulose (Shinetsu Chemical, Tokyo, Japan) - minimum essential medium (MEM), 30% fetal bovine serum (FBS; Hyclone
Laboratories Inc, Logan, UT), 1% deionized fraction V bovine serum
albumin (BSA; Sigma Chemical Co, St Louis, MO), 0.05 mmol/L
2-mercaptoethanol, 20 ng/mL IL-3, 100 ng/mL IL-6, 100 ng/mL SCF, 10 ng/mL granulocyte colony-stimulating factor (G-CSF; Kirin Brewery Inc),
10 ng/mL thrombopoietin (Tpo; Kirin Brewery Inc), and 2 U/mL
erythropoietin (Epo; Kirin Brewery Inc) was plated into
35-mm Lux standard nontissue culture dishes (Nunc, Roskilde, Denmark)
and incubated at 37°C in a humidified atmosphere flushed with 5%
CO2 in air. All cultures were scored at day 14 according to
criteria reported previously.17-19
A limiting dilution analysis of long-term
culture-initiating cells (LTC-IC).
To prepare feeders, MNCs obtained from the adult BM donor were used as
described.20-22 The MNCs were first cultured at a
concentration of 2 × 106 cells/mL in LTC medium
(MyeloCult H5100; Stem Cell Technologies Inc, Vancouver, British
Columbia, Canada) supplemented with 106 mol/L
hydrocortisone. After culturing for 3 weeks, greater than 80% of the
confluent stromal layers were irradiated (15 Gy of 250-kV peak x-rays)
and trypsinized. The cells were resuspended in LTC medium and seeded in
96-well flat-bottomed microwell plates at 3 × 104 cells
per well for reestablishing the stromal feeder layer. The following
day, the sorted CD34+ EGFR+ cells and
CD34+ EGFR cells were plated into each of
the 96 wells at four different dilutions (range, 100 to 800 cells/well), with a total volume of 200 µL/well. At weekly intervals,
half of the nonadherent cells were removed; at the same time, half of
the medium was replaced. After 7 weeks, the nonadherent cells and the
adherent cells suspended by repetitive pipetting were plated in clonal
methylcellulose culture supplemented with SCF, IL-6, IL-3, G-CSF, and
Epo to determine the total clonogenic cell content of each LTC.
Ex vivo expansion and colony-forming assay.
For expansion in suspension culture, purified 1.0 × 104
CD34+ cells were cultured in 1 mL of RPMI 1640 medium
containing 1% HSA, 20 ng of IL-6, and 100 ng of SCF per milliliter in
the presence or absence of 2 ng of EGF per milliliter. The cells were
incubated with the Ax1w or Ax/hEGFR virus at a multiplicity of
infection (MOI) of 1,000 plaque-forming units (pfu)/cell and control
cells were cultured without adenovirus vectors. After 5 days of incubation at 37°C in 5% CO2 and 5%
O2, cells were washed with PBS and 1/50 cells were
subjected to semisolid cultures in 1 mL of 1.2% methylcellulose-MEM containing 30% FBS, 1% BSA, 0.05 mmol/L 2-mercaptoethanol, 5 ng/mL IL-3, 20 ng/mL IL-6, 100 ng/mL SCF, and 2 U/mL Epo in duplicate after
removal of viral particles. After 14 days of incubation at 37°C in
5% CO2 and 5% O2, colony-forming
unit-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid
(BFU-E) were scored using a dissecting microscope and standard criteria
for their identification.17 The experiments were repeated
five times using CD34+ cells from 5 patients, respectively.
For the experiment of Fig 5, sorted CD34+ cells from 1 patient and specific serum-free medium (StemPro-34 SFM Complete Medium;
Life Technologies, Rockville, MD) were used, and other procedures were
performed in the same way.
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RESULTS |
Cytokine-induced expression of integrin
v3 on CD34+ cell
surface.
To investigate the feasibility of adenoviral transduction to normal
hematopoietic progenitor cells, we analyzed surface expression of
integrin v3, which is known to be one of
the internalization receptors for adenovirus, on human
CD34+ PB cells (Fig 1). A
previous work demonstrated that the adenovirus attachment to the cell
surface and the subsequent step of internalization occur through
distinct receptors23; they showed that the
vitronectin-binding integrins v3 and v5 promote internalization of adenovirus.
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| Fig 1.
Flow cytometric analysis of integrin
v3 expression on human
CD34+ PB cells. For cell activation study, purified human
CD34+ PB cells were incubated in the absence or presence
of IL-3 (5 ng/mL), IL-6 (20 ng/mL), or SCF (100 ng/mL) for 48 hours.
Flow cytometric analysis was performed by staining for both CD34 and integrin v3 simultaneously as described
in the Materials and Methods. First, CD34+ PB cells were
gated, and the expression of integrin v3
on gated CD34+ PB cells was shown.
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We found that a very small population (1.76% ± 1.12%) of
CD34+ PB cells expressed integrin
v3 and that this was unchanged after
these cells were cultured in the absence of cytokines for 48 hours.
However, exposure of these cells to various cytokines induced cell
surface expression of integrin v3 on
CD34+ PB cells (Fig 1). Among the cytokines examined, IL-3
raised the surface expression of integrin
v3 most strongly. Moreover, the various
combinations of cytokines induced the expression more than a single
cytokine. In particular, exposure of CD34+ cells to the
combination of IL-3, IL-6, and SCF for 48 hours induced cell surface
expression of integrin v3 on 33.3% ± 26.5% of these cells (Fig 1). These results suggested that exposure of
CD34+ PB cells to cytokines could confer susceptibility to
adenovirus infection and increase adenoviral transduction efficiency.
Adenovirus transduction of human CD34+ PB cells.
Whereas adenoviral gene transfer into the adherent cells is achieved
with quite a high efficiency, that into hematopoietic cells was
reported to be lower in the previous study.24 Next, we
evaluated efficiency of adenoviral gene transfer into human hematopoietic progenitors. We constructed a recombinant
replication-deficient adenovirus vector expressing human EGFR
(Ax/hEGFR) and used them to monitor viral transduction. The gene
product is transmembrane protein; thus, we could detect expression of
this gene in viable cells by the standard flow cytometric analysis.
We infected CD34+ cells purified from human PB MNCs (purity
was >85% in each experiment) with the Ax/hEGFR virus at an MOI of
250 or 1,000 pfu/cell and analyzed surface expression of EGFR after 60 hours of incubation in the serum-free medium containing IL-3, IL-6, and
SCF, because we found that the presence of FCS decreased the efficiency
of adenoviral gene transfer using a human leukemic cell line (data not
shown). Before the analysis, we confirmed that CD34+ PB
cells expressed very little EGFR on their surface endogenously, and
this was unchanged during ex vivo culture (data not shown). We also
confirmed that these high MOIs had little influence on loss of
viability of CD34+ cells by the Trypan Blue dye exclusion
test under these conditions.
Two-color flow cytometric analysis showed that 60.3% ± 22.4% of the
CD34+ cells expressed EGFR (representative result in Fig
2). However, only 4.61% ± 1.55% of the
CD34+ cells expressed EGFR in the medium containing FCS
without cytokines (data not shown). Cells infected with the mock Ax1w
virus were negative, indicating that the observed staining is not due
to exposure of these cells to adenoviral particles. For the subsequent experiments of ex vivo expansion, we also analyzed surface expression of EGFR after incubation in IL-6 and SCF and observed that it was not
significantly changed (data not shown).
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| Fig 2.
Representative result of two-color flow cytometric
analysis of enriched CD34+ PB cells after 60 hours of
infection with the Ax1w (mock vector) or Ax/hEGFR virus at an MOI of
250 or 1,000 pfu/cell. Enriched CD34+ PB cells (purity,
86.2%) were cultured with each adenovirus vector in a serum-free
medium in the presence of cytokines. After 60 hours of infection, cells
were double-stained simultaneously and analyzed as described in the
Materials and Methods.
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Colony-forming ability of transduced CD34+ cells.
To show directly that adenoviral vectors were transducing functional
progenitors, we sorted infected cells into CD34+
EGFR+ cell versus CD34+ EGFR
cell populations and assayed for clonogenic cultures. As shown in Table
1, CD34+ EGFR+
cells clearly do have colony-forming ability at the almost same frequency as that of the CD34+ EGFR cells.
CD34+ EGFR+ cells could especially produce Mix
colonies, suggesting that more immature progenitors could be transduced
with adenovirus vectors. Thus, the recombinant adenovirus was shown to
be a useful vehicle for readily efficient gene transduction of human
hematopoietic progenitors.
Gene transduction of human LTC-ICs by adenovirus vectors.
Subsequently, we assayed the frequency of LTC-ICs, which are believed
to reflect primitive hematopoietic progenitor cells, in each sorted
population. Purified CD34+ PB cells by immune beads were
infected during 60 hours of incubation in the same way and sorted into
two populations, CD34+ EGFR+ cells versus
CD34+ EGFR cells. Each population was
distributed into microwells containing pre-established irradiated
adherent layer cells. For each evaluation, 4 cell concentrations were
used with 8 replicates per concentration. The frequency of negative
wells (no clonogenic progenitors detectable 7 weeks later) was then
determined. The frequency of LTC-ICs in the starting population was
calculated by Poisson statistics and the weighted mean method with
iterative procedures to determine the best linear fit (Fig
3). The frequency of LTC-ICs in a
population of CD34+ EGFR+ cells was 1:768,
whereas that in a population of CD34+ EGFR
cells was 1:1,206, indicating that the CD34+
EGFR+ population included more primitive progenitor cells
than the CD34+ EGFR population.
Ex vivo expansion of hematopoietic progenitors using EGFR-expressing
recombinant adenovirus.
To examine the feasibility of ex vivo expansion of hematopoietic
progenitors using EGFR-expressing recombinant adenovirus, we performed
the methylcellulose clonal assay to evaluate the proliferation of
colony-producing cells. In 5 experiments, we introduced the Ax1w (a
mock adenovirus vector) or Ax/hEGFR virus to 1.0 × 104
purified CD34+ PB cells at an MOI of 1,000 pfu/cell and
cultured for 5 days in a serum-free medium containing IL-6 and SCF in
the presence or absence of EGF. Under the same conditions, 1.0 × 104 uninfected CD34+ cells were cultured as a
control. In this condition, we observed that transduction efficiency
was equivalent to that in IL-3, IL-6, and SCF (data not shown). After 5 days of incubation, 1/50 of these cells were subjected to the semisolid
culture assay in duplicate. As shown in Table
2, the colony-forming unit pool from
CD34+ PB cells incubated with the Ax/hEGFR virus and EGF
was 5.0- ± 2.4-fold expanded, with a range of 2.6- to 8.2-fold during
the 5 days of culture as compared with that of the uninfected
CD34+ PB cells. However, colony-forming cells from
CD34+ cells incubated with the Ax/hEGFR virus in the
absence of EGF and with the Ax1w virus in the presence or absence of
EGF showed no significant proliferation. No colony was formed from the
suspension culture without cytokines in all conditions of
CD34+ cells (data not shown). Not only the number of
colonies, but also the EGF signal seemed to increase the
size and cell density of colonies (Fig 4).Among proliferated colonies, BFU-E were predominantly expanded.
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| Fig 4.
Photomicrograph of the effect of EGF through
adenovirus-mediated EGFR on the expansion of colony-forming cells after
5 days of suspension culture. Purified CD34+ PB cells
were expanded as described in the Materials and Methods in the presence
or absence of EGF and plated in methylcellulose colony assay. This
figure shows the representative results of day-28 colonies of the
expanded cells from 2,000 CD34+ cells in each fraction.
In the figure, CD34 denotes the results of the uninfected control
fractions.
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Using the cell sorter, we prepared more purified CD34+
cells and repeated the same experiment to exclude the indirect effect of CD34 cells upon the experiment (Fig
5). These results indicated that EGF could
enhance proliferation of hematopoietic progenitors through the
adenovirus-mediated EGFR specifically. Further analysis of CD34+ cells from BM showed similar results (data not shown)
and suggested that the alternative sources of immature cells were also
permissive for adenovirus infection and potentially effective for
expansion.
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DISCUSSION |
To date, recombinant adenovirus vectors have been shown to be effective
vehicles for transient expression of various genes in many
systems.25 Indeed, in clinical trials for gene therapy to
cystic fibrosis patients, replication-deficient adenovirus vectors were
used to transfer the cystic fibrosis transmembrane conductance
regulator gene to the airway epithelial surface.9,10 No
significant toxicity was observed except for the elicited immune response in these studies. Therefore, the high efficiency, safety, and
simplicity of handling in this vector system is very attractive.
However, as for hematopoietic system, only a few studies reported
application of this vector.12,13 This might be due to two
reasons. One is because transduction efficiency of the adenovirus vector to immature human hematopoietic cells has not been adequately evaluated. The other reason is because adenoviral gene transfer causes
transient expression of the inserted gene, and the situation in which
such transient expression is more favorable than stable expression has
not been proposed.
Concerning adenoviral transduction to human leukemic cells, Wattel et
al24 reported that adenovirus could not efficiently be used
for direct gene transfer in leukemic cells. Recently, two studies using
recombinant adenoviruses encoding lacZ or alkaline phosphatase reporter
gene described the efficiency of adenoviral transduction to human
CD34+ cells.26,27 They reported that 6.0% to
20.0% and 25% to 35% of CD34+ cells were infected,
respectively.
In this study, we first analyzed the surface expression of integrin
v3 on human CD34+ PB cells.
Previous works showed that human PB monocytes and T lymphocytes express
a very small amount of integrin v3,
whereas exposure to cytokines or stimulation with mitogens induces its surface expression and renders these cells susceptible to
adenovirus-mediated gene delivery.28,29 However, little is
known about the expression of this molecule on human hematopoietic
progenitors. Our findings showed that CD34+ PB cells did
not natively express integrin v3 on their
surface, whereas exposure of these cells to cytokines induced cell
surface expression of integrin v3 (Fig
1). Therefore, adenoviral gene transfer to the CD34+ PB
cells would require two steps: induction of surface expression of
adenovirus internalization receptor on CD34+ PB cells and
adenovirus attachment and uptake into these cells. However, we observed
that a small population of CD34+ PB cells cultured in
medium without cytokines could also express adenovirus-mediated gene
products (4.61% ± 1.55% of CD34+ PB cells; data not
shown). This is probably because adenovirus vectors would be
internalized via integrin v5 known to
function as another adenoviral receptor or other unidentified
receptors.
Based on our findings described above, we introduced the Ax/hEGFR virus
into the CD34+ PB cells and evaluated the efficiency of
gene transduction. After 60 hours of incubation with the Ax/hEGFR virus
at an MOI of 1,000 pfu/cell, 60.3% ± 22.4% of the CD34+
cells expressed exogenous EGFR with no obvious cell toxicity (Fig 2).
The transduction efficiency of our results was relatively higher than
that of the previous report,26 possibly because we used
cytokines to induce cell surface expression of adenovirus internalization receptors, in addition to the fact that we used the
serum-free medium and the CAG promoter.30
To evaluate the transduction efficiency of immature progenitors with
more accuracy, we sorted CD34+ PB cells expressing
adenovirus-mediated EGFR after 60 hours of incubation and subjected
these cells to semisolid clonal culture and LTC-IC assay. In clonogenic
culture, the transduced CD34+ PB cells could produce
various types of colonies, including CFU-Mix at the nearly same
frequency as the nontransduced CD34+ cells (Table 1),
indicating that immature progenitors such as CFU-Mix are permissive for
adenovirus infection. Moreover, the result of LTC-IC assay showed that
the transduced EGFR-expressing CD34+ cells contained a
number of LTC-ICs (Fig 3). The frequency of LTC-IC in a population of
the transduced CD34+ cells (1:768) was higher than that of
the nontransduced CD34+ cells (1:1,206). This might suggest
that immature progenitors would be more permissive to adenovirus
infection, or LTC-IC expressing adenovirus-mediated EGFR could be
expanded during culture with EGF contained in LTC medium. As a
consequence of these results, adenovirus vectors were shown to be
effective vehicles for gene transduction of human hematopoietic
primitive cells.
Given the features of adenoviral transduction, ie, one is transient
expression which is not subject to integration site-specific effects
and another is unavoidable host immune reaction if used in vivo, we
believed that the adenoviral gene delivery system would be suited for
ex vivo expansion of hematopoietic immature cells. Thus, we tried to
expand hematopoietic progenitors ex vivo through adenovirus-mediated
EGFR.
We used EGF signal for expansion of hematopoietic progenitors with
their immaturity, because EGFR expressed on mouse BM cells is reported
to be able to transduce proliferative signal of EGF with no influence
to differentiation.8 They introduced EGFR into primary
mouse BM cells using retroviral gene transfer and showed that EGF acted
on these cells synergistically with IL-3 in cell proliferation even
under IL-3 saturation condition. In the avian hematopoietic system,
c-erbB, the avian homologue of EGFR, has been shown to be
present in very early avian stem cells and plays a physiological role
in self-renewal.31,32 Using HL60 cells expressing EGFR
introduced by retroviral gene transfer, Chen et al33 showed
that exogenous EGFR confers a self-renewal signal that can shift the
balance of RA-induced terminal differentiation toward self-renewal and
results in a partial block of differentiation. These results previously
reported suggested that EGF signal could have possibility to be used to
amplify hematopoietic primitive cells without differentiation as
neuronal cells, although we did not demonstrate it directly in this
report.
We tried to expand colony-forming cells expressing adenovirus-mediated
EGFR in suspension culture with EGF. As shown in Table 2, EGF could
produce a 2.6- to 8.2-fold increase in colony-forming cells from
CD34+ PB cells expressing adenovirus-mediated EGFR over 5 days of culture, whereas no significant proliferation of colony-forming
cells from CD34+ PB cells incubated in other conditions was
observed. It was also shown that the toxicity of adenovirus vectors
under this condition was negligible, because we observed no significant
difference between the colony-forming unit pools from the
CD34+ PB cells uninfected and infected with the mock Ax1w
adenovirus vector. A similar result was obtained from more purified
CD34+ PB cells (Fig 5).
Interestingly, BFU-E showed more dominant expansion over CFU-GM (Table
2). c-erbB, the avian homologue of EGFR, is known to transmit
the self-renewal signal in avian erythroid
progenitors.31,32 In mammalian hematopoiesis, a recent
study of targeted disruption of mouse EGFR clearly showed that EGFR is
not involved directly.34 However, our results might suggest
that an unidentified signal would control proliferation of human
erythroid progenitors as in avian erythropoiesis, and its intracellular
downstream would be shared with that of EGFR. Further studies are
required to clarify the presence and the characteristics of the
self-renewal signal involved in human erythropoiesis.
Most approaches to ex vivo expansion of hematopoietic immature cells
thus far use IL-3, leaving some unresolved issues. Especially, ex vivo
expansion of more primitive progenitors has not been achieved, because
these cells do not express the IL-3 receptor and IL-3 acts on
relatively late-stage progenitors.35 To address this issue,
Sui et al36 used IL-6 and SCF signal and
succeeded in expansion of immature population of
hematopoietic cells. In this study, we used those early-acting
cytokines for minimal cytokine-induced differentiation and showed that
EGF enhanced the expansion of colony-forming cells with those cytokines
through the ectopic expression of EGFR. However, it is not clarified
directly yet that our approach could proliferate primitive cells such
as LTC-ICs. Indeed, there is a possibility that our results of
expansion were caused from induced differentiation of more primitive
progenitors by our manipulations. Thus, direct evaluation of expansion
of more primitive progenitor populations is required.
Incidentally, the high efficiency and simplicity of adenoviral gene
delivery to human primitive hematopoietic cells is very promising.
Other genes coding for signaling molecules such as transcription
factors or cytokine-dependent kinases could be candidates to be
transferred with adenovirus vectors and promote the proliferation of
hematopoietic progenitors. Moreover, this approach could be applied to
proliferation of other kinds of primary cells derived from various
tissues, such as neuronal or epithelial cells. For the future clinical
application, determination of more effective signaling molecules,
especially capable of expanding the self-renewal HSCs as well as
examination of the safety of adenovirus vectors should be required.
| |
FOOTNOTES |
Submitted March 25, 1997;
accepted January 29, 1998.
Supported by grants from the Ministry of Education, Science, and
Culture and the Ministry of Health and Welfare, Japan.
Address reprint requests to Hisamaru Hirai, MD, PhD, Associate
Professor, The Third Department of Internal Medicine, Faculty of
Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan; e-mail: hhirai-tky{at}umin.u-tokyo.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors appreciate Izumu Saito and Yumi Kanegae for instruction of
an efficient method of constructing recombinant adenoviruses. We thank
Yoichi Shibata and Jyun-ichi Miyazaki for providing clinical samples
and CAG promoter. We appreciate Kirin Brewery Inc and Baxter Healthcare
Corp for generous donation of cytokines and Isolex 50.
| |
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Abstract
Introduction
Abstract