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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3487-3493
By
From the Terry Fox Laboratory; BC Cancer Agency; and the Departments
of Pathology and Laboratory Medicine, Medical Genetics, and Medicine of
the University of British Columbia, Vancouver, BC, Canada.
Recent studies have shown efficient gene transfer to primitive
progenitors in human cord blood (CB) when the cells are incubated in
retrovirus-containing supernatants on fibronectin-coated dishes. We
have now used this approach to achieve efficient gene transfer to human
CB cells with the capacity to regenerate lymphoid and myeloid progeny
in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34+ cell-enriched populations
were first cultured for 3 days in serum-free medium containing
interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor,
Flt3-ligand, and Steel factor followed by two 24-hour incubations with
a MSCV-NEO virus-containing medium obtained under either
serum-free or serum-replete conditions. The presence of serum during
the latter 2 days made no consistent difference to the total number of
cells, colony-forming cells (CFC), or long-term culture-initiating
cells (LTC-IC) recovered at the end of the 5-day culture period, and
the cells infected under either condition regenerated similar numbers
of human CD34+ (myeloid) CFC and human
CD19+ (B lymphoid) cells for up to 20 weeks in NOD/SCID
recipients. However, the presence of serum increased the viral titer in
the producer cell-conditioned medium and this was correlated with a
twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% ± 3%
and 17% ± 8%, G418-resistant in vivo repopulating cells and LTC-IC
were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% ± 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively),
whereas values for LTC-IC and NOD/SCID repopulating cells were highly
and significantly correlated (r2 = 0.85). These findings
provide further evidence of a close relationship between human LTC-IC
and NOD/SCID repopulating cells (assessed using a
TRANSDUCTION OF PLURIPOTENT hematopoietic
stem cells using recombinant retroviruses forms the basis of most
current strategies for the correction of single gene defects. Efficient
transfer of genes into murine hematopoietic stem cells with long-term
in vivo repopulating ability can now be routinely achieved using this
approach.1-4 Encouraging results have also been obtained with human progenitors detectable in vitro as colony-forming cells (CFC) and their more primitive precursors identified as long-term culture-initiating cells (LTC-IC).5-9 More recent findings
indicate the possibility of gene transfer to human hematopoietic cells capable of engrafting immune-deficient mice.10-12 However,
the application of this technology to clinical transplants has,
overall, yielded disappointing results with a few notable exceptions.
The latter include results obtained using bone marrow cells from
children undergoing hematopoietic recovery13 and a
preliminary report of improved gene transfer under conditions that may
favor maintenance of proliferating hematopoietic stem cells in
vitro.14,15
Our approach has focused on the identification of factors that rapidly
stimulate the proliferation of human cell populations that include
transplantable progenitors without loss of their original functional
potential. Recently, we showed that LTC-IC (defined using a 6-week CFC
output endpoint16) and cells able to regenerate human
lymphomyelopoiesis in sublethally irradiated nonobese diabetic
(NOD)/severe combined immunodeficiency (SCID) mice (referred to as
competitive repopulating units [CRU]) are similarly amplified in
short-term cultures of CD34+CD38lo human cord
blood (CB) cells stimulated by high concentrations of Flt3-ligand (FL),
Steel factor (SF), interleukin-3 (IL-3), IL-6, and granulocyte
colony-stimulating factor (G-CSF).17 In addition, we found
that LTC-IC and CRU in freshly isolated CB cells are similarly
distributed between the CD38+ and CD38- subsets
of the CD34+ CB population. These findings suggested a
close relationship between the cells identified by these two assays and
encouraged us to continue to use the LTC-IC assay to identify
conditions for optimizing retroviral-mediated gene transfer to CRU.
This allowed the development of a supernatant infection protocol that gives reproducibly high levels of retroviral-mediated gene transfer to
human CB CRU ( Human cells.
Samples of CB from normal, full-term infants delivered by cesarean
section were collected in heparin according to protocols approved by
the University of British Columbia Clinical Screening Committee for
Research Involving Human Subjects. This included obtaining informed
consent from the mother before delivery. A light density (<1.077
g/mL) cell fraction was first isolated by centrifugation of the CB
cells on ficoll-hypaque (Pharmacia, Uppsala, Sweden). These cells were
then either used directly or were further fractionated on a
StemSepÔ column (StemCell Technologies Inc, Vancouver, BC) to
isolate a CD34+ cell-enriched population (by removal of
cells expressing surface antigens characteristic of various mature
hematopoietic cells18), according to the manufacturer's
instructions. In some experiments, highly purified (>99.9%)
CD34+ or CD34+CD38lo cells were
isolated from these lin- cells by fluorescence-activated cell sorting (FACS), as described in detail elsewhere.17 In the remainder, the enriched CD34+ cells were used without
further purification. Surplus human bone marrow (BM) cells were
obtained with informed consent from normal adult donors of allogeneic
BM transplants or were cadaveric samples obtained from the Northwest
Tissue Centre (Seattle, WA). To generate marrow fibroblasts for the
infection experiments, fresh BM cells were first cultured for at least
5 weeks in Iscove's medium with 20% fetal calf serum (FCS; StemCell)
and then subcultured repeatedly until a pure fibroblast monolayer was
obtained.
Human cytokines.
Highly purified recombinant IL-3 and granulocyte-macrophage CSF
(GM-CSF) were gifts from Novartis (formerly Sandoz, Basel, Switzerland). IL-6 and SF were purified from media conditioned by COS
cells that had been transiently transfected in the Terry Fox Laboratory
with the corresponding human cDNAs. FL was a gift from Immunex Corp
(Seattle, WA) and purified human erythropoietin (Ep) and G-CSF were
kindly provided by StemCell.
Retroviral vector.
An MSCV-NEO virus19 constructed using the MSCV 2.1 vector
(kindly provided by Dr R. Hawley, University of Toronto, Toronto, Canada) was used to establish a GP-env AM12 MSCV-NEO producer cell line
as described.18 The titer of these producer cells was
107 colony-forming units/mL as assessed by the transfer of
G418 resistance to NIH-3T3 cells.20 The producer cells were
shown to be free of helper virus, as indicated by the inability to
recover infectious virus from MSCV-NEO-infected NIH-3T3 cells (capable
of transferring G418 resistance to a culture of naive NIH-3T3 cells).
Supernatants were collected from confluent cultures of MSCV-NEO
virus-producing cells after incubation of these overnight with fresh
Iscove's medium containing 20% FCS or bovine serum albumin, insulin,
and transferrin (BIT, StemCell) as indicated. The medium was then obtained, filtered through 0.4-mm filters, and stored frozen at CFC and LTC-IC assays.
Methylcellulose assays (all reagents from StemCell) were performed
essentially as previously described.16 After infection, some cells were plated in methylcellulose both with and without G418
(1.6 mg/mL, dry weight, GIBCO-BRL, Burlington, Canada). At these
concentrations of G418, no colony growth from uninfected cells was seen
in control groups (mock-infected cells) included in every experiment.
LTC-IC assays were performed also as described16 with
maintenance of the cultures at 37°C with weekly half-medium changes
for 6 weeks, at the end of which the nonadherent and adherent fractions
were obtained, pooled, and plated in methylcellulose with and without
G418 as indicated.
Assessment of mice transplanted with human cells.
NOD/LtSZ-scid/scid mice21 bred in the animal
facility at our institution were housed in microisolator cages and
given autoclaved food and water, acidified just before and after total
body irradiation (350 cGy). Human CB cells plus 106
irradiated (1,500 cGy) normal human BM cells as carrier cells were then
injected intravenously. Mice were maintained at least 6 weeks after
transplantation, at which time they were killed and the cellular
contents of both femurs and both tibias flushed out with HFN (Hanks'
buffered salt solution containing 2% fetal calf serum and 0.1% sodium
azide) and a single cell suspension obtained from each mouse. Aliquots
were stained as previously described17 with
anti-CD45-fluorescein isothiocyanate (FITC; HLe 1; Becton Dickinson,
Mountain View, CA) and anti-CD71-FITC (OKT9),22 anti-CD19-phycoerythrin (PE; Leu12; Becton
Dickinson) and anti-CD34-CY5 (8G12),23 or FITC-conjugated
and PE-conjugated mouse Ig as negative controls. Normal mouse BM cells
showed less than 0.1% nonspecific staining with these antibodies. In
animals containing both human lymphoid (CD19+) and human
CD34+ populations, the human CD34+ cells were
sorted and plated in CFC assays with and without G418 as described
above.
Polymerase chain reaction (PCR) analysis.
Colonies generated in CFC assays were plucked and analyzed individually
using the PCR and Southern blotting with a NEOr probe to
amplify and identify incorporated NEO-specific sequences as previously
described.24
Validation of the supernatant infection protocol.
In an initial series of experiments, the efficiency of infecting human
CB CFC and LTC-IC when the target cells were incubated with MSCV-NEO
virus-containing supernatants under various culture conditions was
compared with the levels of gene transfer obtained by cocultivation
with MSCV-NEO viral-producer cells. The conditions chosen were based on
previously reported findings that coincubation of the target cells on
fibronectin25,26 or fibroblasts5,27 could
improve the efficiency of gene transfer to primitive human hematopoietic cells. Either light density (106/mL) or
lin- (36% ± 6% CD34+, 105/mL)
CB cells were first incubated in the absence of virus for 48 hours in
Iscove's medium with 20% FCS and 20 ng/mL IL-3, 10 ng/mL IL-6, and 50 ng/mL SF. Aliquots of these prestimulated cells were then incubated in
the same culture volume for an additional 48 hours either in cell-free
virus-containing medium supplemented with the same cytokines in petri
dishes or in dishes that had been precoated with human full-length
fibronectin (Sigma, St Louis, MO) at a concentration of 5 µg/cm2, or on top of a monolayer of irradiated (1,500 cGy) allogeneic human marrow-derived fibroblasts, or in fresh medium
containing the same cytokines on top of a monolayer of irradiated (150 cGy) producer cells, as indicated. Polybrene was added to all media to
give a final concentration of 4 µg/mL. The cytokine-supplemented viral supernatants (and control media) were replaced halfway through the 48-hour infection period, at the end of which all nonadherent cells
were obtained, washed, and assessed for G418-resistant CFC and LTC-IC.
The results are summarized in Table 1.
Supernatant infection on fibronectin-coated plates gave similarly high
levels of gene transfer to LTC-IC, as were obtained by cocultivation (44% v 39%) and both conditions also gave a high level of
gene transfer to CFC. Supernatant infection in the absence of either fibronectin or human marrow fibroblasts produced very low levels of
gene transfer to any type of progenitor. The presence of human fibroblasts improved gene transfer efficiencies to CFC, but the gene
transfer efficiencies and recoveries of LTC-IC were reduced to levels
that precluded their assessment.
Retention of CRU activity during infection.
A series of six experiments were then undertaken to determine how the
maintenance of CRU activity might be influenced by incubation of the
cells with a retroviral supernatant generated in medium containing 20%
FCS or medium supplemented with a defined serum substitute (BIT;
StemCell). At the time of starting these experiments, we had just
determined that FL in addition to SF, IL-3, and IL-6 (or G-CSF) is
important for achieving optimal expansion of LTC-IC and CFC in
short-term cultures of normal adult human BM,28 and that
this combination of cytokines would also support some expansion of CB
LTC-IC and CRU (fourfold and twofold, respectively), in 5- to 8-day
cultures.17 Therefore, the cytokines selected for use in
this next set of gene transfer experiments were changed from the
previous combination to FL and SF (100 ng/mL each) plus IL-3, IL-6, and
G-CSF (20 ng/mL each). To avoid the toxicity that polybrene had been
found to have on primitive cells29 (which we confirmed),
the polybrene was replaced with 5 µg/mL of protamine sulphate. In
addition, the period of prestimulation was extended from 48 to 72 hours. This latter change was based on our observations of single
CD34+CD38- CB cells, which showed that under
the conditions used, all viable cells would divide within 5 days, but
not before.17 Four of the experiments were set up with
lin- CB cells (at 105 cells/mL), one with
FACS-purified CD34+ CB cells (at 105 cells/mL),
and one with FACS-purified CD34+CD38lo CB cells
(at 104 cells/mL). The rest of the protocol was the same as
had been found to be optimal in the previous experiments, ie, the cells were prestimulated in cytokine-supplemented, serum-free medium followed
by 48 hours of infection on fibronectin-coated petri dishes with
replacement of the cytokine-supplemented viral supernatants (prepared
either in medium plus 20% FCS or serum-free plus BIT) after the first
24 hours. At the end of the second 24 hours of infection, the cells
were obtained and assayed for CFC, LTC-IC, and for their ability to
generate lymphoid and myeloid progeny after their transplantation into
sublethally irradiated NOD/SCID mice. The input cell type and numbers
and the number of resulting positive mice for human CFC and
CD19+ cells is shown in Table
2. Figure 1 shows a representative dot plot
of the relative numbers of total human cells and human
CD34+ cells present in the marrow of one of the mice
transplanted with human cells from these experiments. As shown in
Table 3, the presence or absence of serum
in the cultures from which the cells transplanted were obtained made no
consistent difference to any of the endpoints of human engraftment
assessed in mice up to 15 weeks posttransplant. In addition, there was
also no difference in the total numbers of cells, CFC, or LTC-IC
recovered from the two types of infection cultures (ie, viral
supernatants prepared in serum-free or serum-replete medium, data not
shown). The results from both procedures were therefore pooled to
derive mean (± standard error of mean [SEM]) yields of each
progenitor cell type at the end of the 5-day infection culture period
(per 105 input CD34+ cells) as follows: 3.4 ± 1.1 × 106 total cells, 1.4 ± 0.4 × 106 CFC, and 790 ± 290 LTC-IC (the results from the
experiment that was performed with CD34+CD38lo
cells was excluded from this analysis).
Gene transfer to human CB progenitors.
To assess gene transfer efficiencies in these latter experiments, the
proportion of G418-resistant CFC, or progeny CFC derived from LTC-IC or
(in vivo) from the CRU injected into the NOD/SCID mice was determined.
The results, shown in Fig 2, show average gene transfer efficiencies that range from a maximum of 68%
(burst-forming unit-erythroid [BFU-E] exposed to
FCS-containing supernatants) to a low of 8% (CRU exposed to
BIT-containing supernatants). However, for each progenitor type, there
was an approximately twofold to threefold higher proportion of
G418-resistant cells when these were infected with FCS-containing
supernatants, despite the fact that the total number of progenitors
present had not been affected. Assessment of the viral titer of the
supernatants prepared with FCS and BIT showed a threefold difference (6 × 106 in FCS v 2 × 106 in
BIT, n = 2). Thus, the most likely cause of the reduced gene transfer
obtained with the BIT-containing supernatants was simply their reduced
content of virus.
The studies described in this report identify conditions that allow
human in vivo repopulating cells to be reproducibly infected by
recombinant retroviruses at high efficiency ( Submitted August 4, 1997;
accepted December 15, 1997.
The authors thank Dr R. Hawley (University of Toronto); Dr P. Lansdorp
(Terry Fox Laboratory, Vancouver, BC); and StemCell and Novartis for
valuable gifts of vectors, antibodies, and other reagents. The expert
technical help of Margaret Hale, Gayle Thornbury, Jessyca Maltman, and
Maya Sinclaire, and the secretarial assistance of Bernadine Fox are
also acknowledged.
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