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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3163-3171
By
From the Molecular Virology Lab, Georg-Speyer-Haus, Frankfurt,
Germany; the Department of Hematology/Oncology, Bone Marrow
Transplantation Hospital, Idar-Oberstein, Germany; the Molecular
Immunology Unit, Institute of Child Health, London, UK; and the
Department of Hematology/Oncology, School of Medicine, University of
Frankfurt, Frankfurt, Germany.
Stable gene transfer to human pluripotent hematopoietic stem cells
(PHSCs) is an attractive strategy for the curative treatment of many
genetic hematologic disorders. In clinical trials, the levels of gene
transfer to this cell population have generally been low, reflecting
deficiencies in both the vector systems and transduction conditions. In
this study, we have used a pseudotyped murine retroviral vector to
transduce human CD34+ cells purified from bone marrow
(BM) and umbilical cord blood (CB) under optimized conditions. After
transduction, 71% to 97% of the hematopoietic cells were found to
express a low-affinity nerve growth factor receptor (LNGFR) marker
gene. Six weeks after transplantation into immunodeficient
NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was
detected in 6% to 57% of CD45+ cells in eight of nine
engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45%
of secondary colonies derived from BM cells of engrafted NOD/SCID mice.
Our data show consistent transduction of SCID-repopulating cells (SRCs)
and suggest that the efficiency of gene transfer to human hematopoietic
repopulating cells can be improved using existing retroviral vector
systems and carefully optimized transduction conditions.
© 1998 by The American Society of Hematology.
GENE TRANSFER INTO pluripotent
hematopoietic stem cells (PHSCs) is one of the most promising
alternatives for the curative treatment of a variety of inherited and
acquired disorders of blood cells. In murine syngeneic bone marrow (BM)
transplantation models, a significant proportion of cells participating
in long-term engraftment of lethally irradiated mice can be
reproducibly and stably transduced ex vivo by the current generation of
retroviral vectors.1-3 However, transfer of this technology
to humans, nonhuman primates, and other large outbred animals has been
much less successful.4-13 The reasons for this discrepancy
are uncertain, but probably reflect incomplete understanding of culture
conditions required to maintain the integrity and functionality of the
PHSC, an inability of the current generation of murine retroviral
vectors to transduce quiescent cells,14,15 and a deficiency
of receptors on the PHSC surface for the commonly used amphotropic
retroviral envelope.16,17
In animal models and human trials, high levels of gene transfer to
clonogenic progenitor cells and long-term culture-initiating cells
(LTC-ICs) in vitro have not been predictive of long-term reconstitution. The development of efficient protocols for PHSC gene
transfer has therefore been limited by the failure of in vitro
surrogate progenitor assays to represent the repopulating cell
fractions of the human hematopoietic system. To address this problem,
alternative assay systems have been developed that test the ability of
human hematopoietic cells to engraft immunodeficient mice.18-24 In one such model, which is based on the
engraftment of severe combined immunodeficiency disease (SCID) and
nonobese diabetic/SCID (NOD/SCID) mice, a novel population of human
hematopoietic cells, defined as SCID-repopulating cells (SRCs), have
been shown to be capable of extensive proliferation and multilineage
(lymphoid and myeloid) differentiation in vivo.25
Furthermore, this activity is highly enriched in
CD34+CD38 In this study, we have evaluated the efficiency of gene transfer to
primitive human hematopoietic cells using a GALV-pseudotyped murine
retroviral vector, and optimized ex vivo transduction conditions. We
show here that these cells retain their ability to repopulate NOD/SCID
mice and can be transduced relatively efficiently.
Recombinant human cytokines and growth factors.
Stem cell factor (SCF), interleukin (IL)-3, IL-6, Flt3-Ligand (Flt3-L),
and anti-transforming growth factor (TGF) Purification of hematopoietic CD34+ cells.
Human BM was obtained under local anesthesia from the iliac crest of
healthy adult volunteers after informed consent and ethical approval.
Samples of umbilical cord blood (CB) were obtained from discarded
placental and umbilical tissues by drainage of the blood into sterile
collection bags. The BM and CB samples were diluted 1:3 in
phosphate-buffered saline (PBS) and enriched for mononuclear cells by
density gradient over Ficoll-Paque (1.077 g/mL; Seromed, Berlin, Germany). The BM-derived low density cell fraction
was subjected to one cycle of plastic adherence (1 to 2 hours) before the isolation of CD34+ cells. CD34+ cells were
isolated by superparamagnetic microbeads selection using the miniMACS
system according to the manufacturer's instructions (Miltenyi Biotec,
Inc, Gladbach, Germany). The purity of the cell population ranged
between 75% and 97% CD34+ cells as estimated by
fluorescence-activated cell sorting (FACS) analysis using either a
phycoerythrin (PE)- or a fluorescein isothiocyanate (FITC)-conjugated
mouse monoclonal antibody against the human CD34 antigen
(anti-hematopoietic-progenitor-cell-antigen-2[anti-HPCA-2], Becton
Dickinson; San Jose, CA).
Transduction of human hematopoietic CD34+ cells.
For the transduction of human CD34+ cells, retroviral
supernatant was harvested from 80% confluent PG-13 monolayers after 12 to 16 hours cultivation in serum-free X-VIVO10 medium (Boehringer Ingelheim; Heidelberg, Germany) supplemented with 1% bovine serum albumin (BSA; Stem Cell Technologies, Vancouver, Canada), 2 mmol/L L-glutamine and 1% penicillin/streptomycin, filtered (0.45 µm) and
kept frozen at Flow cytometric analysis of transduced cells.
After transduction, cells were washed twice in PBS containing 1% heat
inactivated fetal calf serum (FCS) and 0.1% sodium azide. To assess
for LNGFR expression, cells were incubated with an unconjugated mouse
antihuman LNGFR antibody (Boehringer Mannheim, Mannheim, Germany),
which was detected with a goat antimouse F(ab)-fluorescein isothiocyanate (FITC) (Dianova; Hamburg, Germany). Alternatively, a
biotinylated primary LNGFR antibody (kindly provided by Dr S. Seeber,
Boehringer Mannheim, Penzberg, Germany) was used and subsequently detected with PE-conjugated streptavidin (Dianova). For the flow cytometric analysis of engrafted NOD/SCID mice, directly conjugated antibodies against human cell surface antigens were purchased from
Becton Dickinson (Oxford, UK) (CD19-FITC, CD34-FITC, CD38-PE, CD45-PerCP) or DAKO, Ltd (High Wycombe, UK) (CD2-PE, CD3-FITC, CD13-PE). A total of 1 × 106 cells obtained from the
BM of injected mice were incubated for 30 minutes on ice with
saturating amounts of antibodies in staining buffer (PBS, 5% FCS,
0.01% sodium azide). A sample from each mouse was also stained with
directly conjugated isotype-matched control antibodies (Becton
Dickinson). After incubation, cells were washed three times and fixed
in 1% paraformaldehyde. Flow cytometric analysis was performed on a
FACScan or FACSCalibur using the CellQuest software package (Becton
Dickinson). In all experiments, isotype controls were used to set the
quadrant markers such that the quadrant defining negative PE and FITC
fluorescence contained at least 97% of the isotype control cells. The
engrafted human cells were detected by CD45 positivity and the
expression of the lineage markers. LNGFR expression was determined on
the CD45-gated population.
Progenitor cell assays.
For clonogenic assays, transduced cells were plated in 35-mm dishes
containing 0.35% agar, 25% FCS (Hyclone; Erembodegem-Aalst, Belgium),
50 ng/mL SCF, 20 ng/mL IL 3, 10 ng/mL G-CSF, and 10 ng/mL GM-CSF in
McCoy's medium (Life Technologies; Gaithersburg, MD). Cultures were
incubated at 37°C in a 5% CO2 humidified atmosphere and colonies were enumerated after 10 to 15 days. For LTC-IC assay, 500 or 1,000 cells were seeded on a preestablished monolayer of the murine
FBMD-1 cell line32 (kindly provided by R.E.
Ploemacher, Rotterdam, The Netherlands) in MyeloCult (Stem Cell
Technologies) containing 20 ng/mL IL-3, 100 U/mL IL-6, and 50 ng/mL
SCF. Cultures were incubated for 5 weeks at 37°C, 5%
CO2 with weekly changes of half of the medium. At the end
of the 5-week LTC-IC assay period, the nonadherent and adherent
fractions were harvested and assayed for the content of hematopoietic
progenitors by plating 100,000 hematopoietic cells in clonogenic
assays, as described above and scored 14 days later. LNGFR-positive
colonies were detected by immunostaining techniques (manuscript in
preparation). Similarly, 2 × 105 cells
derived from the BM of engrafted cells 6 weeks after injection were
analyzed for the presence of human hematopoietic progenitors. Cells
were plated in Methocult (Stem Cell Technologies), supplemented with
Iscove's modified Dulbecco's medium (IMDM), 30% FCS, human growth
factors (25 ng/mL SCF, 10 U/mL IL-3, 9 U/mL GM-CSF, 2 U/mL erythropoietin [Epo]; all R&D Systems), 2 mmol/L L-glutamine, and 50 µmol/L 2-mercapto-ethanol, resulting in 0.9% final concentration of
methylcellulose. The cultures were incubated in a fully humidified atmosphere at 5% CO2.
NOD/SCID mouse reconstitution assay.
The NOD/LtSz-scid/scid (NOD/ SCID) mice (original stocks kindly
provided by John E. Dick, Hospital for Sick Children, Toronto, Canada) were housed in sterile microisolator cages in a
laminar flow caging system (Thoren, Hazleton, PA) and supplied with
sterile food, acidified water, and bedding. All manipulations were
conducted in a laminar flow hood. Transduced CD34+ cells
were injected intravenously via the tail vein of 6- to 8-week-old mice,
which had been sublethally irradiated with 325 cGy (137Cs
source). Mice were killed by CO2 inhalation 6 weeks after
injection and BM cells were harvested for flow cytometric analysis and
growth of hematopoietic progenitors.
Polymerase chain reaction (PCR) for human LNGFR.
The presence of LNGFR provirus in secondary colonies was determined
using the primers 5 Optimized transduction of CD34+cells using
LNGFR expression.
On the basis of previous studies suggesting that retroviral vectors
generated on the PG13 packaging cell line may have advantages over
amphotropic vectors for transduction of human hematopoietic cells,13,33-35 a GALV-pseudotyped LNSN retroviral vector
was used in all experiments. The LNSN construct, which contains the
full-length low-affinity receptor for human nerve growth factor (LNGFR)
under the transcriptional control of the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR),36 was selected for
these studies because LNGFR expression allows rapid evaluation of gene transfer by flow cytometric analysis and
immunocytochemistry.36-39 To optimize gene transfer to
human CD34+ cell populations, a detailed study of several
parameters that could improve efficiency was performed (manuscript in
preparation). The optimized transduction protocol included
prestimulation of the CD34+ cells for 20 hours in
serum-free medium (X-VIVO10) supplemented with 1% BSA, 2 mmol/L
L-glutamine, IL-3, IL-6, SCF, FLT3-L, and anti-TGF
Gene transfer into CFCs and LTC-ICs.
Immunocytochemical detection of LNGFR expression in hematopoietic
colonies indicated an efficiency of gene transfer to colony-forming progenitors (CFCs) of 63.4% ± 11.7% (range, 49.0% to 84.0%)
(Fig 2). Absence of background staining in mock-transduced preparations confirmed the specificity of detection (not shown). No difference in
the efficiency of CFC transduction was observed between
CD34+ cells derived from BM (63.6% ± 10.1%) or from
CB (63.1% ± 13.4%). To assess gene transfer into more primitive
progenitors, transduced cells were maintained under long-term culture
conditions on FBMD-1 cells.32 Of colonies derived from
progenitor cells removed from the culture after 5 weeks, 23.6% ± 4.3% (range, 19.3 to 28.0%) expressed LNGFR by immunostaining.
Gene transfer into SRCs.
To test for transduction of primitive human cells with repopulating
ability, hematopoietic cells (derived from BM or CB) were injected into
the tail vein of sublethally irradiated NOD/SCID mice after
transduction under the optimized conditions outlined above. Cells
recovered from mouse femurs 6 weeks after engraftment were analyzed for
the percentage of human cells (CD45 expression) and expression of the
LNGFR marker gene by flow cytometry and for the level of gene transfer
to CFCs by PCR. Results are summarized in Table 1. After
transplantation of between 0.5 and 4.2 × 106 cells,
nine of 14 animals showed detectable levels (0.3% to 33.3%) of human
cell engraftment measured by flow cytometric detection of CD45
expression (Figs 3A and
4). Multilineage engraftment determined by surface immunophenotype (CD19, CD13, CD2), and CFC profile (not
shown) was observed in all nine animals (Fig 3B). In five animals, no
CD45+ cells were detectable, although engraftment at levels
below those measurable in these studies is possible.
Efficient gene transfer to human PHSCs has been limited by incomplete
understanding of their biologic properties and by deficiencies of
vector systems and ex vivo transduction conditions. These confounding factors are reflected in data from clinical trials and from studies in
which transduced cells are engrafted in immunodeficient
mice.4,6,9,10-12,23,24,44 The NOD/SCID model system has
been shown to support the engraftment and retention of primitive human
hematopoietic cells with the potential for extensive proliferation and
multilineage differentiation.23,25-27,45,46 Unlike the
majority of LTC-ICs, which are incapable of repopulation, SRCs are
found exclusively in the CD34+CD38- cell
fraction at a calculated frequency of approximately 1 in 600 in CB and
BM and are therefore phenotypically and functionally distinct.26 Furthermore, kinetic experiments indicate that
engraftment of SRCs is followed by a large expansion of LTC-ICs in
vivo, suggesting that these are derived from a more primitive
cell.27 Although both CFCs and LTC-ICs are readily
transduced, the efficiency of gene transfer to SRCs has generally been
very low, and the repopulating potential is markedly compromised by ex
vivo culture.13,23,29 Similar findings have been reported
for pluripotent cells engrafting bg/nu/xid mice, although there are
qualitative and quantitative differences in repopulating cell
engraftment patterns in this model compared with that of
SRCs.24,30
Submitted March 4, 1998;
accepted June 21, 1998.
We are indebted to S. Seeber (Boehringer Mannheim, Penzberg) for the
LNSN construct and LNGFR antibodies, R.E. Ploemacher (Erasmus
University, Rotterdam, The Netherlands) for the FBMD-1 cell line, T. Tonn (Blood Bank, Frankfurt) for CB samples, and Mike Blundell for
technical assistance.
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,
Abstract
Introduction
Abstract
fractions, and based on the
kinetics of engraftment, represents a more primitive cell population
than most LTC-ICs and CFCs.
80°C until use. The
CD34+-enriched target cell population was prestimulated at
a cell concentration of 1 × 105 cells/mL for 20 hours
in serum-free X-VIVO10 supplemented with 1% BSA, 2 mmol/L L-glutamine
and 1% penicillin/streptomycin in the presence of IL-3 (20 ng/mL),
IL-6 (100 U/mL), SCF (50 ng/mL), Flt3-L (100 ng/mL), and anti-TGF
-TGTGTGAGCCCTGCCTGGAC, beginning at position
300 in exon 2 and 5
LNGFR) gene.
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