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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2217-2224
One-Day Ex Vivo Culture Allows Effective Gene Transfer Into Human
Nonobese Diabetic/Severe Combined Immune-Deficient Repopulating Cells
Using High-Titer Vesicular Stomatitis Virus G Protein Pseudotyped
Retrovirus
By
Vivienne I. Rebel,
Mayumi Tanaka,
Jeng-Shin Lee,
Sheila Hartnett,
Michael Pulsipher,
David G. Nathan,
Richard C. Mulligan, and
Colin A. Sieff
From the Division of Pediatric Hematology and Oncology, Dana- Farber
Cancer Institute, Boston; and the Departments of Pediatrics and
Genetics, Harvard Medical School and Howard Hughes Medical Institute,
Children's Hospital, Boston MA.
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ABSTRACT |
Retrovirus-mediated gene transfer into long-lived human pluripotent
hematopoietic stem cells (HSCs) is a widely sought but elusive goal. A
major problem is the quiescent nature of most HSCs, with the perceived
requirement for ex vivo prestimulation in cytokines to induce stem cell
cycling and allow stable gene integration. However, ex vivo culture may
impair stem cell function, and could explain the disappointing clinical
results in many current gene transfer trials. To address this
possibility, we examined the ex vivo survival of nonobese
diabetic/severe combined immune-deficient (NOD/SCID) repopulating cells
(SRCs) over 3 days. After 1 day of culture, the SRC number and
proliferation declined twofold, and was further reduced by day 3;
self-renewal was only detectable in noncultured cells. To determine if
the period of ex vivo culture could be shortened, we used a vesicular
stomatitis virus G protein (VSV-G) pseudotyped retrovirus vector that
was concentrated to high titer. The results showed that gene transfer
rates were similar without or with 48 hours prestimulation. Thus, the
use of high-titer VSV-G pseudotyped retrovirus may minimize the loss of
HSCs during culture, because efficient gene transfer can be obtained
without the need for extended ex vivo culture.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
RETROVIRUS-MEDIATED gene transfer into
long-lived pluripotent hematopoietic stem cells (HSCs) could provide
permanent correction of hematopoietic gene dysfunction in a variety of
genetic diseases. However, the results of clinical gene therapy trials are disappointing, due to the low efficiency of gene transfer into HSCs
and/or poor expression in the differentiated progeny of these
cells.1-4 Major problems regarding human
retrovirus-mediated gene transfer include the poor HSC expression of
amphotropic receptors for the retroviral envelope
protein5,6 and the quiescent state of the majority of HSCs,
which does not favor the integration of retroviral vectors, since cell
division is thought to be required.7 Compounding these
problems are the difficulties in assaying human HSCs. Protocols for
clinical trials are based on results from in vitro clonogenic assays of
human progenitors, particularly the long-term culture-initiating cell
(LTC-IC) assay. The development of in vivo xenogeneic models, in which
human cells are transplanted into severe combined immune-deficient
(SCID) mice,8-12 has led to the ability to measure a SCID
reconstituting cell (SRC) in murine bone marrow (BM) months after
transplantation. Cell fractionation and gene-marking studies provide
some evidence that SRCs are more primitive than
LTC-ICs.12,13 Although SRCs may still be a heterogeneous population of cells that include long-term repopulating HSCs, this
assay provides a better measure of stem cell properties than the LTC-IC
assay, especially with regard to the multipotent (ie, myeloid and
lymphoid) and self-renewal properties of HSCs.
One approach to the amphotropic receptor problem has been the
development of new vectors such as Moloney murine leukemia virus-based retrovirus pseudotyped with the vesicular stomatitis virus G protein (VSV-G).14 VSV-G pseudotyped retroviruses, like
conventional retroviruses, can stably integrate into the host genome
but have a much broader host range than conventional retroviruses and
are more stable, thus permitting concentration by ultracentrifugation to greater than 109 infectious particles per
milliliter.15,16 These vectors can efficiently infect
mammalian and nonmammalian cells that are resistant to infection by
conventional amphotropic retroviruses.
The problem of stem cell quiescence is usually approached by using
hematopoietic growth factors (HGFs) ex vivo to stimulate murine17,18 or human19-21 stem and progenitor
cells into cycle, and this paradigm has driven much research in animal
models and clinical trials. The human studies are based on
investigations showing that stimulation with combinations of HGFs such
as interleukin-3 (IL-3), IL-6, and Steel factor (SF) can increase the
efficiency of gene transfer into LTC-ICs in clinically applicable
supernatant infection procedures in the absence of
stroma.22,23 While stromal support has been shown to
increase the efficiency of gene transfer and may be necessary for
optimal stem cell survival,23-25 stroma-based gene transfer
methods are impractical. Despite these laboratory advances, HSC gene
transfer efficiency in primate26,27 and human1-4 clinical studies has been disappointingly low.
Although strategies using combinations of HGFs have increased the
efficiency of gene transfer into in vitro clonogenic progenitors, there
is little evidence that cytokines increase the frequency of gene transfer into long-term repopulating stem cells as measured in blood
cells obtained from patients. Indeed, it is possible that stimulation
with HGFs may irreversibly alter HSC properties, and there is now
strong murine evidence that sustained HSC self-renewal may not be
possible.28-30 If this is also true for human cells, then
the potential of HSCs to self-renew may be finite, determined by
replicative history.
Thus, one of our major goals is to achieve efficient gene transfer into
HSCs after short-term ex vivo culture, to limit progressive cytokine-induced ex vivo proliferation that might lead to stem cell
extinction. The current study was undertaken to address whether the
poor results of clinical gene therapy trials might reflect poor
survival of HSCs and/or poor gene transfer during ex vivo culture, and if the former, whether the period of ex vivo culture could
be shortened. To answer these questions, we used the nonobese diabetic
(NOD)/SCID model to examine the umbilical cord blood (CB) SRC capacity
after different periods of ex vivo culture. We show that the SRC
quantity and repopulation capacity declines rapidly during serum-free
culture in SF, Flt 3 ligand (FL), and either IL-3 or
IL-6/erythropoietin (EPO). However, we also show that prestimulation
with HGFs is unnecessary: we found that a high-titer VSV-G pseudotyped
retrovirus vector could confer efficient gene transfer into SRCs within
1 day of culture.
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MATERIALS AND METHODS |
Preparation of CB cells.
Cells were harvested from placentae after delivery by cesarean section.
The CB was obtained by needle aspiration of the exposed vessels on the
fetal side of the placenta. After diluting the sample (1:6 vol/vol)
with IMDM medium (Life Technologies, Grand Island, NY) containing 2%
fetal bovine serum ([FBS] Sigma Chemical Co, St Louis, MO) and 0.6%
anticoagulant citrate dextrose solution-A ([ACD-A] Cytosol
Laboratory, Braintree, MA),31 the CB cells were centrifuged
over a layer of Histopaque-1.077 (Sigma) to deplete erythrocytes. The
cells were then washed twice in IMDM (2% FBS and 0.6% ACD-A),
incubated on ice with ammonium chloride for 20 minutes, and washed once
more. The processed cells were either frozen down directly (in IMDM
with 50% FBS and 10% dimethyl sulfoxide, Sigma) and further separated
on the day of the experiment or further enriched for progenitor cells
and then frozen down. Human CD34+CD38
progenitor cells were enriched using a positive selection method for
CD34+ cells (Ceprate LC separation system; CellPro,
Bothell, WA) or a negative selection method for lineage-negative cells
(StemSep; StemCell Technologies, Vancouver, BC, Canada), according to
the manufacturer's instructions. CD34+ cells enriched by
the CellPro method were 70% to 90% pure. After negative selection
using the StemSep method, the cell suspension contained 31% ± 9%
CD34+ cells (n = 6). However, with respect to the
purification for CD34+CD38 cells, both
methods were comparable: 11% for the CellPro method and 9% for the
StemSep method when 10 and seven combined CB samples, respectively,
were used for purification of primitive human progenitor cells.
The CB cells enriched for CD34+CD38
progenitor cells (hereafter designated as progenitor cells) were
analyzed for phenotype (flow cytometry) and function (CFC and LTC-IC)
to determine the day 0 values, ie, the quantity and quality of the cell
suspension that was used to initiate cultures for gene transduction.
Infection of hematopoietic cells.
CB progenitor cells were cultured in IMDM medium supplemented with 10%
or 20% BIT (BIT 9500, bovine serum albumin, insulin, and transferrin;
StemCell Technologies). The following human recombinant cytokines were
used: SF (50 ng/mL), FL (100 ng/mL), IL-3 (20 ng/mL), IL-6 (10 ng/mL),
and EPO (2 U/mL). FL was provided by Immunex (Seattle, WA) and IL-3 and
IL-6 by Genetics Institute (Cambridge, MA), and SF and EPO were
purchased from R&D Systems (Minneapolis, MN). Cells were cultured for
24, 48, or 72 hours on non-tissue culture treated plates (Becton
Dickinson, San Jose, CA) previously coated with fibronectin (CH-296; a
generous gift from Takara Shuzo Co, Otsu, Shiga, Japan). During the
last 16 hours of every culture, the cells were exposed to the
retrovirus at a multiplicity of infection (MOI) of 130 or 260 and 4 µg/mL Polybrene (Sigma). Then the cells were harvested, washed once,
and analyzed for phenotype, function, and gene transfer efficiency.
Construction and purification of the VSV-G pseudotyped retrovirus.
The MMP vector is a derivative of the MFG series of retroviral
vectors,32 and pMMP-MDR1 was constructed by inserting the human multidrug resistance 1 (MDR1) gene into the pMMP vector. To
prevent a cryptic splicing event that occurs when the MDR1 cDNA is
inserted into retroviral vectors,33 two sites were
identified and silent mutations were engineered to both of them (J.-S.
Lee, manuscript in preparation).
The pMMP-MDR1 plasmid was transfected into the 293GPG packaging cell
line34 by the calcium phosphate method. 293GPG cells constitutively express murine leukemia virus gag-pol gene products and
produce VSV-G proteins in a tetracycline-inducible fashion. After
transfection and selection with 60 ng/mL colchicine, viruses produced
from about 60 individual clones were titered by infection on 3T3 cells.
The viral supernatants from the clone with the highest titer (up to
1 × 107 infection particles/mL) were concentrated by
ultracentrifugation at 50,000g at 4°C for 1.5 hours. The
virus pellets were resuspended in small volumes of DME overnight at
4°C. The titer of the concentrated viruses, determined by Southern
blot analyses of the infected cells, was normally in the range of
109/mL.
Animals.
NOD/LtSz-scid/scid (NOD/SCID) mice were purchased from Jackson
Laboratory (Bar Harbor, ME). They were bred and maintained in the
Redstone animal facility of the Dana-Farber Cancer Institute in
microisolator cages under well-defined sterile conditions. The mice
were used as recipients for human cell transplantation at 8 to 12 weeks
of age. Whole-body radiation was given in a single dose (325 to 350 cGy, 110 cGy/min) using a 137Cs source 1 to 4 hours before
transplantation. The indicated human cell numbers were injected in a
final volume of 0.25 to 0.35 mL phosphate-buffered saline ([PBS] Life
Technologies) with 2% FBS. Six to 8 weeks later, the animals were
killed by CO2 asphyxiation, after which the BM cells from
both the femur and tibia were harvested for further analysis of human
cell engraftment.
Analysis of human cell engraftment.
BM cells were harvested by flushing the hindlimb bones with PBS/2% FBS
using a 3-mL syringe and a 21-gauge needle. The cell suspension was
washed once and then resuspended in PBS/2% FBS. Cells were counted
(using trypan blue to exclude dead cells) and assayed by flow cytometry
to determine the proportion of human cells. When greater than 0.1% of
the cells were of human origin, the samples were further analyzed for
the presence of different myeloid and lymphoid lineages by flow
cytometry and CFC assays.
Flow cytometric analysis.
For phenotyping the CB cell suspension before and after each culture,
cells were labeled with the following antibodies (Abs): CD34-phycoerythrin (PE) (PharMingen, San Diego, CA) and
CD38-fluorescein isothiocyanate (FITC) (Immunotech, Westbrook, ME).
Irrelevant, isotype-controlled Abs were used in every experiment to
determine background staining. For detection of human cells in mouse
BM, an anti-CD45 Ab (HLe-1; Becton Dickinson) was used. To further define the different lineages in the total human cell population, BM
cells were simultaneously stained with anti-CD45-FITC and an Ab
against one of the following lineage markers: CD34-PE, CD33-PE, CD14-PE
(all from Becton Dickinson), CD4-PE, CD8-PE, and CD19-PE (all from
PharMingen). As part of the analysis of human cell engraftment in mice,
BM cells from a naive NOD/SCID mouse were labeled to ensure that the
Abs used were specific for human cells. All staining procedures were
performed in PBS/2% FBS. Cell labeling was performed on ice (35 minutes), after which the cells were washed twice. Propidium iodide
(Sigma) 2 µg/mL was added during the final wash. The flow cytometric
analysis was performed on a FACScan (Becton Dickinson).
CFC assay.
To determine the human CFC content of the murine BM after
transplantation, marrow cells were plated in IMDM/0.9% methylcellulose media (Methocel MC; Fluka, Buchs, Switzerland) containing 15% defined
FBS (Hyclone Laboratories Inc, Logan, UT), 15% human plasma, and the
following cytokines: SF (50 ng/mL), IL-3 (20 ng/mL),
granulocyte/macrophage colony-stimulating factor ([GM-CSF], 20 ng/mL;
a generous gift from Genetics Institute), and EPO (2 U/mL).35 A maximum of 2 × 105 BM cells were
plated per dish (Fisher, Pittsburgh, PA). Colonies were scored in situ
after 14 to 20 days of incubation at 37°C in a humidified atmosphere
of 5% CO2 in air using well-established criteria. Control
dishes with 2 × 105 BM cells from nontransplanted
NOD/SCID mice were plated to ensure that murine colonies did not form
under these culture conditions; apart from an occasional small
macrophage colony, these dishes were always empty.
LTC-IC assay.
Test cells, ie, noncultured and cultured CB progenitor cells, were
cultured in Myelocult medium (StemCell Technologies) to which
106 mol/L freshly dissolved hydrocortisone (Sigma) was
added before use. The cultures were initiated by seeding the test cells
on an irradiated (3,300 cGy) stroma layer, which was previously
established from human BM cells obtained from discarded filters after
BM harvests. The cultures were maintained at 37°C in a humidified
atmosphere of 5% CO2 in air with weekly half-medium
changes. After 5 to 6 weeks, the cultures were harvested and assayed
for CFCs. The LTC-IC cultures were either set up in limiting dilution,
which permits the frequency of LTC-ICs in the test population to be
calculated using Poisson statistics,36 or in bulk cultures.
Analysis of gene transfer.
The presence of the gene from the viral construct was determined by
polymerase chain reaction (PCR) in human CFCs grown from the harvested
mouse BM cells. After scoring the plates, 20 colonies per mouse (or
less, if fewer were present) were picked at random from the
methylcellulose and directly deposited into Eppendorf tubes containing
500 µL PBS. The colonies were left for 1 hour at room temperature to
allow the methylcellulose to dissolve. The cells were then washed twice
and resuspended in lysis buffer containing 10 mmol/L Tris hydrochloride
(pH 8.3), 2 mmol/L MgCl2, 50 mmol/L KCl, 0.45% Nonidet
P40, 0.45% Tween 20, and 1 mg/mL proteinase K, as previously
described.25 The samples were left overnight at 37°C or
for 2 hours at 56°C. To inactivate the proteinase K, the samples were
then heated at 94°C for 10 minutes. DNA was amplified using a
DeltacyclerII PCR apparatus (Ericomp Inc, San Diego, CA). The primer
set to demonstrate the presence of the transduced gene was chosen from
the MMP vector within the proviral construct:
5'-TCTGCTCCCCGAGCTCAATA-3' and 5'-CCGACTGGTTGTGAGCGAT-3'. GM-CSF or EPO
genes were used as internal controls, and fragments were amplified
using the primers 5'-TGAAATTTGTCTGCATGAAGGAGT-3' and
5'-GCATTGTAGATGAAACAGGAGAAA-3' and 5'-ACGCCTCTTCACCACCCACAA-3' and
5'-TTCGAGGCCAAAGCAGATGAG-3', respectively. Samples that failed to show
a PCR product of either internal control were not included in the
calculation of gene transfer efficiency.
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RESULTS |
VSV-G pseudotyped retrovirus does not decrease the survival or in vitro
function of hematopoietic progenitor cells.
To examine whether ex vivo culture and addition of VSV-G pseudotyped
retrovirus affected the survival or in vitro function of primitive
hematopoietic cells, CD34+ cells were isolated from
approximately 10 combined CBs, and cultured on fibronectin-coated
plates in serum-free medium containing SF, FL, and IL-3 for 1, 2, or 3 days. During the last 16 hours of each culture period, the cells were
exposed to VSV-G pseudotyped MMP-MDR1 viral supernatant that was
produced from a human 293-based packaging cell line and concentrated
to very high titer by centrifugation (108 to
109 particles/mL). At the end of each culture period, the
cells were analyzed for both phenotype and function and compared with
noncultured CB cells. At a MOI of 130 (130 virus particles per cell),
the number of total CD34+ and
CD34+CD38 cells remained constant during the
3-day culture period, despite a small decrease in total cell number
after 2 days of culture (Fig 1). The
addition of a twofold higher dose of virus (MOI 260) during the last 16 hours of a 1- or 2-day culture resulted in a slightly greater
decrease in cell number (including the most primitive
CD34+CD38 cells), although similar or
greater recovery was noted by day 3 (data not shown). An in vitro
limiting dilution analysis of the number of LTC-ICs before and after
culture confirmed this phenotypic analysis
(Table 1). In two experiments at an MOI of 130, the number of LTC-ICs was not significantly affected after 3 days
of culture. In a third experiment, in which twice the amount of virus
was used (MOI 260), the LTC-IC assay was set up in bulk cultures and
measured before and after 1 or 3 days of culture. The number of CFCs
obtained from noncultured LTC-ICs was similar to the number observed
for LTC-ICs established after 1 and 3 days of ex vivo culture (data not
shown).
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| Fig 1.
Maintenance of human
CD34+CD38 cells in serum-free cultures
initiated with CB cells enriched for CD34+ cells. The
number of total cells ( ), CD34+ cells ( ), and
CD34+CD38 cells ( ) are shown as a
function of time in culture. To calculate the number of
CD34+ cells or CD34+CD38
cells at each time point, the number of total live cells (counted with
a hemocytometer) was multiplied by the percentage of
CD34+ cells or CD34+CD38
cells obtained by FACS. Shown is a representative figure of one of
three experiments.
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Ex vivo culture of CB CD34+ cells reduces both the
number and quality of SRCs.
Engraftment in the NOD/SCID mice was defined as the presence of greater
than 0.1% human cells in the murine BM with evidence of both myeloid
and lymphoid reconstitution, as well as the capacity to form colonies
in methylcellulose. To determine whether ex vivo culture affected these
SRC parameters, CD34+ cells were cultured for 1, 2, or 3 days and transplanted into NOD/SCID mice. At limiting cell doses, the
number of mice with detectable human myeloid and lymphoid cells in the
BM (ie, the number of positive mice) was reduced after transplantation
with cultured cells. Poisson analysis of the proportion of negative mice shows that the number of SRCs declined over the 3-day culture period (Table 2). This reduction in SRCs
was accompanied by a diminished number of human CFCs obtained from mice
transplanted with cultured cells (Fig 2).
The quality of SRCs was measured by the level of human cell engraftment
and by their ability to give rise to SRCs that could reconstitute
secondary NOD/SCID recipients. The relative contribution to the
different lineages (measured by CD34 [progenitor cells], CD33 and
CD14 [myeloid cells], and CD4/CD8 and CD19 [lymphoid cells]) was
unaffected by time in culture (data not shown). However, the overall
level of human cell engraftment decreased dramatically with time in
culture (Fig 3). Whereas the transplantation of 105 noncultured cells resulted, on
average, in 13% human cells in the BM of transplanted NOD/SCID mice,
after 1 day of culture, this was reduced to less than half, and after 3 days of culture, only 4% of the input level was observed. Thus, the
level of engraftment declined 25-fold after 3 days in culture. This
could be due in part to a decline in the number of SRCs, although the
reduction in SRCs was much less than that, ie, fourfold. The results of the secondary transplantation experiments show that BM cells from a
small number of primary recipients of noncultured transplants (14%)
could reconstitute secondary recipients (Table
3). In contrast, none of the cells
harvested from mice transplanted with cultured cells were able to
reconstitute secondary recipients. If culture does not affect the
self-renewal capacity of SRCs, we would expect 14%, ie, approximately
two mice, of all secondary recipients transplanted with cultured cells
(16 mice in total) to be reconstituted with human cells. However, no
human cell reconstitution was detectable in any of the secondary
transplanted mice. It is possible that 1 day in culture may still
preserve the self-renewal of SRCs but the number of mice analyzed in
this group was too small for detection. Taken together, these results
show that culture affects the quality of SRCs in that the proliferative
and self-renewal ability diminishes with time in culture.
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| Fig 2.
Number of human CFCs harvested from transplanted animals
is lower in animals that received cultured cells versus noncultured
cells. Circles represent individual mice that received noncultured
cells (day 0) or cells that were cultured for 1, 2, or 3 days in
serum-free medium supplemented with SF, FL, and IL-3. The total number
of human colonies per femur was calculated by dividing the number of
total cells obtained per femur by the number of cells plated per
methylcellulose dish (usually 2 × 105 cells),
multiplied by the average number of colonies scored in a duplicate set
of dishes. There were no significant differences in the relative
contribution of the types of colonies between cultured and noncultured
cells. Shown are the combined data from three experiments.
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| Fig 3.
Reconstituting ability of the graft decreases with time
in culture. The percentage of human CD45+ cells in the BM
of reconstituted recipients transplanted with 105 or 5 × 104 human CB cells that were either not cultured or
cultured for 1, 2, or 3 days is illustrated. Open circles represent
individual mice; the horizontal bar is the average value of the group.
Data from three experiments were combined.
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The VSV-G protein is known to be toxic to cells.16 Although
the phenotypic analysis and the in vitro functional data do not suggest
that this was the case in our experiments, we wished to exclude VSV-G
toxicity as the cause of the decline in SRCs. Groups of five mice were
transplanted with 105 CD34+ CB cells cultured
for 3 days in the same cytokines with or without the addition of virus.
All mice were reconstituted, and the level of human reconstitution was
comparable in both groups, ie, 0.9% ± 1.2% and 0.4% ± 0.4%, respectively.
Recent reports demonstrate that IL-6 may be an important cytokine for
the survival of human progenitor cells,37 and confirm earlier reports of the maintenance of murine HSCs in a combination of
SF, IL-6, and EPO.38 Therefore, we sought to investigate whether the quality of SRCs would be better preserved in a cytokine combination that, in addition to SF and FL, contained IL-6 and EPO
instead of IL-3. Although the level of human cell engraftment was low
in this experiment (Table 4), it was
threefold to fourfold higher in recipients of cells cultured in SF, FL,
IL-6, and EPO versus SF, FL, and IL-3. However, the number of SRCs
after 1 and 3 days of culture in the IL-6 growth factor combination was
decreased as much as in the IL-3 combination. Interestingly, when BM
cells from four primary recipients that received cells cultured in SF, FL, IL-6, and EPO were transplanted into irradiated secondary recipients (three each), human cell engraftment was observed in two of
three secondary recipients from one of the primary recipients (data not
shown). These results suggest that SRCs cultured in SF, FL, IL-6, and
EPO survive no better than SRCs in SF, FL, and IL-3, while the
self-renewal capacity of the surviving SRCs may be better preserved.
Prestimulation of SRCs is unnecessary for efficient infection with
VSV-G pseudotyped retrovirus.
In light of the decrease in SRC number and function during ex vivo
culture, we next wished to determine whether the VSV-G pseudotyped
retrovirus could infect SRCs without an extended period of
prestimulation. We therefore compared gene transfer efficiency in CB
cells cultured for 1 or 3 days, ie, no prestimulation or 48 hours
prestimulation in serum-free medium with SF, FL, and IL-3. To assess
gene transfer efficiency in SRCs using VSV-G pseudotyped retrovirus, we
used PCR to detect the transduced retroviral sequence in individual
CFCs from the BM of positive mice. Figure 4
shows gene transfer efficiency as a function of time in culture (1 or 3 days). The results clearly show that the percentage of CFCs transduced
with the retroviral vector after 1 day in culture was not significantly
different from that after 3 days in culture. When a similar experiment
was performed with SF, FL, IL-6, and EPO, we found that the gene
transfer efficiency after 3 days of culture was 2.4-fold higher than
after 1 day, ie, 48% versus 20%, respectively.
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| Fig 4.
Gene transfer efficiency with a 1-day infection protocol
equals that of a 3-day protocol. The percent gene transfer represents
the proportion of methyl cellulose colonies formed by human cells
harvested from the BM of reconstituted recipients that were
PCR-positive for the MDR gene from the retroviral construct. The days
in culture indicate the total number of days human cells were cultured
before transplantation into irradiated NOD/SCID recipients. The data
represent three combined experiments in which the analysis of day 1 was
performed in 2 separate experiments. A MOI of 130 or 260 did not show
differences in gene transfer efficiency.
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DISCUSSION |
Initial studies of the effects of culture on SRCs showed a sixfold
decrease of BM but no decrease of CB SRCs after 1 week on allogeneic
stroma.39 More recent reports suggest that it may be
possible to maintain or increase SRC numbers in serum-free cultures
supplemented with a cytokine combination of SF, FL, IL-3, IL-6, and
G-CSF.37,40 These findings appear to conflict with our data
that the SRC number and quality decline rapidly in culture. However,
Conneally et al37 report that after culture, the number of
CFCs and LTC-ICs generated per SRC is lower than before culture, despite correction for the observed twofold increase in the number of
SRCs. Data from Bhatia et al40 suggest a decline in the
quality of SRCs, as well. They calculate a fourfold increase in SRCs
after 4 days in culture; however, despite this increase, the level of engraftment of 500 CD34+CD38 cells was low
(<1%), similar to the level obtained with fourfold fewer day 0 cells.13 This is consistent with their further observation that by day 9 no SRC activity was detectable at all. In summary, both
of these studies confirm our own in that they show a decline in SRC
function over time.
Previous studies with amphotropic retroviruses have shown that it is
possible to obtain efficient gene transfer into primitive hematopoietic
progenitors assayed in vitro by CFC and LTC-IC
assays.22,24,41 Despite these encouraging data, the results
of most clinical studies have been disappointing in that very few HSCs
(<1%) appear to be transduced.1-4 Therefore, CFC and
LTC-IC data do not predict clinical outcome, and it was of considerable
interest to determine the efficiency of retrovirus transduction into
SRCs. It was recently shown that the combination of SF, FL, IL-3, IL-6,
and G-CSF resulted in a mean gene transfer rate of 32% into SRCs
(range, 11% to 43%)42; in this series of experiments, it
was necessary to extend the period of prestimulation to 72 hours, with
two subsequent 24-hour exposures to virus supernatant, ie, 120 hours ex
vivo. Larochelle et al12 reported very poor gene transfer
into SRCs using fibronectin-coated dishes and supernatant infection.
Better rates of gene transfer could only be obtained when very large
numbers of CB cells (3 to 5 × 108) were directly
cocultured with retrovirus packaging cells. Dao et al43
used the beige/nude/xid mouse model to show that a mean of 5% of SRCs
in the CD34+ cell fraction could be transduced in a
protocol that requires addition of virus supernatant during 72 hours of
culture on a stromal support supplemented with SF, IL-6, and IL-3.
Transduction of SRCs in the CD34+CD38
fraction required 7 days of culture and the addition of FL; under these
conditions, in which the transferred gene conferred neomycin resistance, low numbers of G418-resistant colonies (2% to 4%) were
observed in two of four mice. Gene transfer rates similar to those
reported here were observed in one of the above-mentioned studies.42 However, all of these studies required prolonged periods of incubation of the cells in culture, and our data clearly show that this is undesirable.
Although 3 days in SF, FL, IL-6, and EPO resulted in a 2.4-fold
increase in gene transfer efficiency compared with 1 day ex vivo, the
extended culture was accompanied by a fivefold loss in SRC number and
an even greater loss in proliferative potential (Table 4). Thus, the
absolute number of transduced SRCs in mice transplanted after 3 days in
SF, FL, IL-6, and EPO is lower than in mice transplanted after 1 day,
despite the higher gene transfer efficiency. This finding that both the
number and quality of SRCs are reduced during ex vivo culture may be an
important consideration in the evaluation of results of clinical gene
therapy trials. In this respect, it is of particular interest that in
the one clinical study with good gene transfer results, BM harvested
from children during recovery from chemotherapy was cultured for only 6 hours ex vivo and exposed to retrovirus supernatant in the absence of
cytokines; gene transfer rates of 20% were observed in CFCs up to 42 months posttransplant.44,45
Perhaps the most surprising result of this study is that comparable
gene transfer rates were observed without or with prestimulation in
culture. Cell cycle analysis shows that after isolation the majority
(97.5%) of CD34+ CB cells are in
G0/G1, and that after 14 to 24 hours in IL-3 and SF approximately 80% remain in this state; by 48 to 72 hours, the
proportion of cells remaining in G0/G1 has
decreased to about 50%.46 These data suggest that cells
that exit G0/G1 would be the preferred targets
for retrovirus integration and stable gene transfer. However, recent
data from the same group47 show that CD34+
mobilized blood progenitor cells stimulated to exit G0 into
G1 with IL-3, SF, and FL are severely compromised in their
SRC content but cells that remain in G0 retain their SRC
activity. The finding of a loss of SRC activity after culture is
consistent with our own results. We therefore speculate that the VSV-G
pseudotyped particles target many cells, including G0 cells
ex vivo, and after transplantation and homing, cell division in vivo in
the BM microenvironment leads to integration and stable gene transfer.
In conclusion, our studies show that prolonged ex vivo culture of
hematopoietic cells compromises critical aspects of SRC function. We
show that the use of a VSV-G pseudotyped retrovirus can obviate the
damaging effect of culture, since it allows efficient gene transfer
into CB SRCs after only 20 hours ex vivo culture. Whether the same
approach will be as successful with BM or mobilized stem/progenitor
cells remains to be determined. Since it is impossible to determine in
the laboratory whether the SRC assay truly reflects HSC with
multipotent and self-renewal capacity, a clinical trial to test the
VSV-G pseudotyped virus in a short-term infection protocol is needed.
| |
ACKNOWLEDGMENT |
The authors thank Jessica Denham for excellent technical assistance and
the personnel of the Redstone animal facility for the care of the
animals. We thank Amy Perrault for help in preparing the manuscript.
| |
FOOTNOTES |
Submitted August 19, 1998; accepted November 22, 1998.
Supported by Grants No. RO1 HL55709 and 1 P50 HL54785 from the National
Institutes of Health.
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 Colin A. Sieff, MB, BCh,
Dana-Farber Cancer Institute, 44 Binney St, Boston MA 02115.
| |
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ABSTRACT
INTRODUCTION