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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2217-2224
By
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.
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.
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.
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 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).
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
10 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.
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
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.
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.
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 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.
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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Retroviral vector-mediated gene t |
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ABSTRACT
INTRODUCTION
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
), CD34+ cells (
), and
CD34+CD38
) are shown as a
function of time in culture. To calculate the number of
CD34+ cells or CD34+CD38
