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Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 431-440
Retrovirally Transduced CD34++ Human Cord Blood Cells
Generate T Cells Expressing High Levels of the Retroviral Encoded Green
Fluorescent Protein Marker In Vitro
By
Bruno Verhasselt,
Magda De Smedt,
Rita Verhelst,
Evelien Naessens, and
Jean Plum
From the Department of Clinical Chemistry, Microbiology and
Immunology, University of Ghent, University Hospital of Ghent, Ghent,
Belgium.
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ABSTRACT |
Human umbilical cord blood (UCB) hematopoietic stem cells (HSC)
receive increased attention as a possible target for gene-transfer in
gene therapy trials. Diseases affecting the lymphoid lineage, as
adenosine deaminase (ADA) deficiency and acquired immunodeficiency syndrome (AIDS) could be cured by gene therapy. However, the T-cell progenitor potential of these HSC after gene-transfer is largely unknown and was up to now not testable in vitro. We show here that
highly purified CD34++ Lineage marker-negative
(CD34++Lin ) UCB cells generate T,
natural killer (NK), and dendritic cells in a severe combined
immunodeficient mouse fetal thymus organ culture (FTOC).
CD34++Lin and
CD34++CD38Lin UCB cells
express the retroviral encoded marker gene Green Fluorescent Protein
(GFP) after in vitro transduction with MFG-GFP retroviral supernatant.
Transduced cells were still capable of generating T, NK, and dendritic
cells in the FTOC, all expressing high levels of GFP under control of
the Moloney murine leukemia virus (MoMuLV) long terminal repeat
promotor. We thus present an in vitro assay for thymic T-cell
development out of transduced UCB HSC, using GFP as a marker gene.
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INTRODUCTION |
IN HUMANS AND other mammals, mature cells
of the erythroid, myelomonocytic, and lymphoid lineages are the progeny
of hematopoietic progenitor cells (HPC), the lineage-committed
descendants of hematopoietic stem cells (HSC).1 This
concept was clearly supported by use of retroviral
marking.2 The self-renewing HSC are the ultimate target of
gene therapy for long-lasting gene-transfer to the hematopoietic system.3-12 During human ontogeny, HSC are first found to
be associated with embryonic aortic endothelium and later home to fetal
liver and eventually bone marrow.13 At least during
prenatal and early postnatal life, HSC or HPC seed the thymus to
generate T, natural killer (NK), and dendritic cells.1,14
At birth, a substantial number of HSC/HPC are present in umbilical cord
blood (UCB).5,10,15 Several reports7,8 have
shown that UCB HSC can be used for allologous or autologous
transplantation to reconstitute the hematopoietic system after bone
marrow ablation in children. The potential use of UCB HSC in adults is
currently being studied.16 Kohn et al8 have
shown that, in the absence of cytoablative therapy, retrovirally transduced autologous UCB HSC/HPC expressing adenosine deaminase (ADA)
can engraft ADA-deficient newborns. Although this study showed the
presence of ADA in selected granulocyte-macrophage colony-forming unit
(GM-CFU) colonies 1 year after transplantation, it failed to show
activity or presence of ADA in ex vivo expanded peripheral T
lymphocytes. However, the investigators communicated that transduced
circulating T cells were observed in these patients upon reduction of
the PEG-ADA substitution therapy dosage.17 Because
lymphocytes are the lineage affected by the ADA deficiency, gene
therapy may fail if transduced UCB HSC can not generate T cells. This
shows the need to assay T progenitor potential of ex vivo transduced
HSC. In vivo assays using SCID-hu,3,12,18 NOD/SCID,19 and beige/nude/XID (bnx) mice20
have been used to demonstrate T-cell development out of transduced
human UCB HSC/HPC. In contrast to assays for myelopoiesis and
B-lymphocyte development, a simple in vitro culture assay for thymic
T-cell development from HSC after gene-transfer was still lacking.
We previously described a SCID mouse fetal thymus organ culture (FTOC)
that supports differentiation of human CD34++ Lineage
marker-negative (Lin) fetal liver cells into mature
thymocytes.21,22 In this report, we show that this organ
culture supports T-cell, NK cell, and dendritic cell development from
highly purified cord blood CD34++Lin
cells. We also show that purified UCB
CD34++Lin and
CD34++CD38Lin cells
express the retroviral encoded marker gene Green Fluorescent Protein23 (GFP) after in vitro transduction. Finally, we
show that transduced UCB CD34++ cells can still generate
mature thymocytes in vitro expressing high levels of GFP. The Moloney
murine leukemia virus (MoMuLV) long terminal repeat (LTR) promotor was
shown to be sufficiently active in transduced UCB CD34++
cells and thymocytes. In this report, an in vitro assay for thymic T-cell development out of transduced UCB HSC/HPC using GFP as a marker
gene is presented as a versatile alternative to the laborious in vivo
murine models. Without the need for human fetal tissue and in vivo
testing, this assay will allow us to define transduction protocols for
human cord blood HSC that combine optimal gene-transfer and
preservation of T-lymphoid progenitor potential.
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MATERIALS AND METHODS |
Monoclonal antibodies (MoAbs).
Mouse antihuman MoAbs used were CD1a (OKT6, fluoresceïn
isothiocyanate [FITC]-labeled, from Ortho Diagnostic Systems
[Ortho], Raritan, NJ; or T6-RD1, phycoerythrin [PE]-labeled, from
Coulter, Hialeah, FL), CD2 (Leu-5b FITC, from Becton Dickinson
Immunocytometry Systems [BDIS], Mountain View, CA), CD3 (Leu-4 FITC
or PE, from BDIS; or S4.1 tricolor [TC] from Caltag Laboratories
[Caltag], San Francisco, CA), CD4 (Leu-3a FITC, or PE from BDIS or
OKT4 FITC from American Tissue Type Collection [ATCC], Rockville, MD; or S3.5 TC from Caltag), CD5 (Leu-1 FITC from BDIS), CD7 (Leu-9 FITC
from BDIS), CD8 (OKT8 FITC, from ATCC; or Leu2a FITC or PE from
BDIS), CD10 (anti-CALLA FITC from BDIS); CD16 (3G8 FITC from
PharMingen, San Diego, CA), CD19 (Leu-12 from ATCC), CD33 (WM-54 FITC
from Dako, Glostrup, Denmark), CD34 (HPCA-2 PE from BDIS), CD38 (OKT10
FITC from Ortho or OKT10 biotin [BIO] from ATCC), CD44 (3F12 FITC,
kind gift of Dr W. De Smet, Innogenetics, Ghent, Belgium), CD45
(anti-HLe-1 FITC from BDIS), CD45RA (MD4.3 BIO, kind gift of Dr F. Koning, University Hospital, Leiden, The Netherlands), CD56 (NCAM 16-2 PE from BDIS), anti-c-kit (CD117, 95C3 BIO from Caltag),
anti-glycophorin-A (10F7MN, kind gift of Dr L. Lanier, DNAX, Palo
Alto, CA), anti-T-cell receptor (TCR)-  (anti-TCR-/-1 PE
from BDIS), and anti-HLA-DR (L243 BIO from ATCC)
Rat antimouse MoAb CD45-Cychrome (30F11.1 from PharMingen) was used to
gate out mouse cells in flow cytometry and anti-FcRII/III MoAb
(Clone 2.4.G224; kind gift of Dr J. Unkeless, Mount Sinai
School of Medicine, New York, NY) was used to block murine Fc
receptors.
Isotypic control antibodies were IgG1 (FITC and PE from BDIS or BIO
from Caltag) and IgG2a (FITC and PE from BDIS or BIO from Caltag).
Biotinylated antibodies were shown by streptavidin-TC (SA-TC;
Caltag). MoAbs from ATCC were FITC or BIO conjugated in our laboratory
using standard methods.
SCID mice.
C.B.-17 scid/scid (SCID) mice, originally purchased from Iffa
Credo (L'arbresle, France), were bred in our specific pathogen-free breeding facility. After overnight mating, the appearance of vaginal plugs was noted as day 0 of pregnancy. Fourteen- to 15-day pregnant mice were killed by cervical dislocation, and fetal thymic lobes were
dissected out.
Animals were treated according to the guidelines of the Laboratory
Animal Ethical Commission of the University Hospital of Ghent.
Purification of CD34++ cord blood cells.
UCB was obtained from full-term, normal newborns and used following the
guidelines of the Medical Ethical Commission of the University Hospital
of Ghent. Within 6 hours after collection, the mononuclear cell
fraction (median, 1.4 × 106 CD45+
cells/mL EDTA cord blood) was isolated over a Lymphoprep
density-gradient (Nyegaard, Oslo, Norway), resuspended in 9 vol fetal
calf serum (FCS; Life Sciences, Paisley, UK) and 1 vol dimethyl
sulfoxide (Serva, Heidelberg, Germany), and cryopreserved in liquid
N2 until use. After thawing, cells were kept at 4°C at
all time until the initiation of culture. Washed cells were resuspended
in phosphate-buffered saline/2% (vol/vol) FCS and stained with
glycophorin-A, CD19, and CD7 FITC. Labeled cells were depleted using
sheep antimouse Ig-coated Dynabeads (Dynal AS, Oslo, Norway; 4 beads
per cell) in a magnetic particle concentrator (Dynal AS). Unlabeled
cells were recovered and stained with CD1 FITC, CD3 FITC, CD4 FITC, CD8
FITC, CD34 PE, and, in some experiments, CD38 BIO. Cells that were CD34
PE++ and FITC (called
CD34++Lin) were sorted on a FACS Vantage
(BDIS) cell sorter equipped with an argon-ion laser (488 nm). On the
average, 15 CD34++Lin cells were
obtained per 104 mononuclear cells. In some experiments,
cells sorted were CD34 PE++, FITC, and
CD38 BIO/SA-TC or CD38 BIO/SA-TC+
(called CD34++CD38Lin
or CD34++CD38+Lin,
respectively). Sorted populations are collectively called
CD34++ cells. Sorted cells were checked for purity, which
was always at least 99.5%.
Cell culture.
All cultures were performed at 37°C in a humidified atmosphere
containing 7.5% (vol/vol) CO2 in air. The cells were
cultured in Iscove's modified Dulbecco's medium (IMDM), supplemented
with penicillin (100 IU/mL), streptomycin (100 µg/mL), and 10%
heat-inactivated FCS (complete IMDM; all products from Life Sciences).
FTOC was performed in complete IMDM containing 10% heat-inactivated
human AB serum (BioWhittaker, Walkersville, MD) instead of FCS.
MFG-GFP retrovirus.
The marker gene Green Fluorescent Protein23 (EGFP;
Clontech, Palo Alto, CA) was Nco I-Not I cloned in the
pBluescript vector (Stratagene, La Jolla, CA). An Nco
I-BamHI fragment containing GFP was inserted between the unique
corresponding restriction sites of the MFG retroviral vector
backbone.25 All enzymes used and DH5 an Escherichia
coli host bacteria were bought from Life Sciences. Propagated
plasmids were purified with resin columns (Qiagen, Hilden, Germany).
The Phoenix-A, a 293 cell line (human fetal kidney)-based amphotropic
packaging cell line that was kindly provided by Dr P. Achacoso and Dr
G.P. Nolan (Stanford University School of Medicine, Stanford, CA), was
transfected with the MFG-GFP plasmid using calcium-phosphate
precipitation (Life Sciences). GFP-expressing cells (usually ~70% of
the transfected cells, 2 days posttransfection) were selected by
electronic cell sorting using a FACS Vantage cell sorter (BDIS). After
3 rounds of sorting over 7 weeks of time, a stable transfected cell
line was established. Cell culture supernatant was harvested from
confluent cultures in 150 cm2 tissue culture flasks
(Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) 24 hours after
refreshment of the medium. Pooled supernatants were spun (10 minutes
for 350g at 21°C) and aliquots were stored at
 70°C until use. The batch used in this report was shown to be free of replication competent retrovirus and contained after thawing
5 × 106 transforming units/mL (titrated on Jurkat T
cells [ATCC]; data not shown).
Gene transduction into CD34++ cord blood cells.
Sorted CD34++ cord blood cells were resuspended in complete
IMDM supplemented with 100 ng/mL recombinant human c-kit ligand (stem
cell factor [SCF]; R&D Systems Europe, Abingdon, UK) and 500 U/mL (50 U/mL in the case of
CD34++CD38Lin and
CD34++CD38+Lin cells)
recombinant human IL-3 (Innogenetics, Antwerp, Belgium) and seeded in
96-well round bottom tissue culture plates (Falcon) at 1.5 to 2 × 104 cells in 150 µL medium per well. After 24 hours of
culture, half of the medium volume was replaced with viral supernatant,
supplemented with cytokines (to keep final cytokine concentration
unchanged) and with the liposome complex DOTAP (Boehringer Mannheim,
Mannheim, Germany) to a final concentration of 20 µg/mL. After
resuspension of the cells, the 96-well plates were spun26
(1.5 hours for 950g at 32°C) and put back in culture for 48 hours. After this period, a fraction of the cells was used to assay
transduction efficiencies, while the remainder was used in FTOC. In
certain experiments, cells were sorted for GFP expression before
transfer to FTOC.
FTOC.
In Terasaki plates, hanging drops were prepared by adding per well 25 µL complete IMDM containing 1 × 104 freshly sorted
CD34++ UCB cells or the progeny of 1 to 1.5 × 104 CD34++ UCB cells transduced as described
above. In some experiments, 104 transduced
CD34++Lin UCB cells, sorted after
transduction based on GFP expression, were used. To each of these
wells, 1 fetal thymic lobe was added and the plates were inverted to
form hanging drops and incubated during 48 or 72 hours. After this
incubation, at day 0 of FTOC, the lobes were removed, washed in
complete medium, put on the surface of a nuclepore filter (Nuclepore,
Costar, Cambridge, MA), resting on a Gelfoam sponge (Upjohn, Kalamazoo,
MI) soaked in complete medium in a 6-well tissue culture plate
(Falcon), and cultured for 11 to 38 days. At the end of the culture,
lobes were mechanically disrupted with a small tissue grinder to obtain
a single cell suspension. Thymocytes were stained with Trypan Blue and
counted with a hematocytometer. The cells, of which at least 80% were
viable, were used for flow cytometry.
Flow cytometry.
Before labeling, cells were suspended in phosphate-buffered saline
containing 1% (wt/vol) bovine serum albumin and 0.1% (wt/vol) NaN3. In all cases in which human cells were stained in the
presence of mouse cells, the mixture of cells was preincubated for 15 minutes with saturating amounts of anti-FcRII/III MoAb (Clone
2.4.G2)24 to avoid nonspecific binding of MoAbs by the
murine cells. Subsequently, the cells were stained with a panel of
MoAbs, as indicated. If present, murine and dead cells were gated out
by mouse CD45 Cychrome and propidium iodide, respectively. Negative
controls included isotype MoAbs conjugated with the corresponding
fluorochrome. The cells were analyzed on a FACScan flow cytometer
(FACS; BDIS) with an argon-ion laser tuned at 488 nm. Forward light
scattering, orthogonal scattering, and three fluorescence signals were
determined and stored in list mode data files. Data acquisition and
analysis was performed with Lysis 2.0 software (BDIS).
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RESULTS |
Human CD34++Lin cord blood cells
differentiate into mature T, NK, and dendritic cells in a mouse
fetal thymus organ culture.
On the average, 1.6% of the CD45+ cells in the UCB
mononuclear cell fraction were CD34+. About 10% of
these CD34+ cells were recovered after sorting for high
expression of CD34 and absence of lineage markers. Immunophenotyping of
highly purified CD34++CD1CD3CD4CD7CD8CD19gpA
cells showed no expression of other lineage surface markers CD2, CD5,
CD16, or CD33 (data not shown). CD38 expression was low on most cells
and undetectable on some. In contrast, the cells were almost
homogeneous HLA-DR+ and c-kitlow
(Fig 1). This
CD34++Lin phenotype includes the most
primitive HSC found in cord blood5,15,19 but does not
contain cells with rearranged TCR loci27 and therefore no
pro-T cells.1 A minor fraction of the purified
CD34++Lin cells expressed CD45RA (Fig
1). After transfer to fetal thymic lobes of SCID mice, most cells
downregulated CD34 and upregulated CD38, so that after 11 days, 80% of
the human cells were CD34CD38+
(Fig 2A). About 20% of the human cells
expressed the marker CD1, most of them in coexpression (not shown) with
CD4 but not with CD2 (Fig 2A). The cells with highest expression of CD4
coexpressed high levels of HLA-DR. Cells with this phenotype in FTOC
cultures were shown to have a dendritic morphology21 and
are high efficient stimulators of allogenic mature lymphocytes
(manuscript in preparation). Together with the absence of
both the myeloid marker CD14 and the T-cell marker CD3, this indicates
that these CD4++HLA-DR++ cells represent thymic
dendritic cells. At this time of FTOC (days 11 to 14), the dendritic
cells are a major component of the thymocyte population (Fig 2A). Their
absolute number does not increase later in culture, so that their
contribution to the number of thymocytes recovered decreases to less
than 5% at days 35 to 38 of FTOC (data not shown). As we showed using
this model with fetal liver CD34++Lin
cells,21 with increasing duration of culture,
CD4+CD8+ double-positive cells start to appear,
the TCR-associated antigen CD3 is gradually upregulated and some CD4 or
CD8 single-positive cells expressing high levels of CD3 are generated
(Fig 2B). After 35 days of culture, on the average 4 × 104 human cells were recovered per lobe. Most of the cells
expressing CD3 were TCR positive, but some were TCR
positive (Fig 2B). After this time of culture, some of the
TCRhi, CD4, or CD8 single-positive cells have
downregulated CD1 (data not shown) and thus represent a mature
thymocyte phenotype in human T-cell development.28 As shown
in Fig 2B, the TCR+ cells were also either
CD1+ or CD1. We recently showed that the
latter population represents the most mature human TCR T
cells.29 Some of the human cells were CD3CD56+ NK cells. This population,
which becomes predominant if FTOC is performed in the presence of
interleukin-15 (IL-15; manuscript in preparation), has an
NK-like morphology upon isolation from FTOC (not shown).
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| Fig 1.
Phenotype of sorted
CD34++Lin UCB cells. Flow cytometric
analysis of sorted CD34
PE++CD1CD3CD4CD7CD8CD19gpA
cells, stained after sorting with biotinylated antibodies recognizing CD38, c-kit, HLA-DR, and CD45RA, shown in a second step by SA-TC. Dot
plots shows CD34 PE versus TC staining, gated on live cells. Values in
the quadrants indicate the percentage of cells present in the
corresponding quadrant. Quadrants were set to include 99.5% of sorted
cells stained, with isotypic control antibody in the upper left
quadrant. The data shown are representative of four independent
experiments.
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| Fig 2.
CD34++Lin UCB cells
generate T, NK, and dendritic cells in the FTOC. Flow cytometric
analysis of thymocytes recovered from FTOC initiated with freshly
sorted CD34++Lin UCB cells. Cells were
stained with CD1 PE, CD2 FITC, CD3 PE, CD4 PE, CD8 FITC, CD34 PE,
CD38 FITC, CD45 FITC, CD56 PE, HLA-DR FITC, and anti-TCR FITC.
Dot plots show staining of live, human cells recovered after 11 (A) and
35 (B) days of culture. The values in the crosses indicate the
percentage of cells present in the corresponding quadrant. Quadrants
were set to include 99% of the cells stained, with isotypic control
antibody in the lower left quadrant, except for the CD4 PE versus
HLA-DR FITC dot plot in (A), in which quadrants are set to contain the
CD4++HLA-DR++ dendritic cells in the
upper right quadrant. The data shown are representative of eight
independent experiments.
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Collectively, these data indicate that purified UCB
CD34++Lin cells give rise to mature
TCR+ and TCR+ T, NK, and thymic
dendritic cells after introduction in a fetal SCID thymus in vitro. In
this way, we have at our disposal an in vitro model that allows the
study of precursor potential of purified UCB HSC/HPC for the lineages
cited above. This is of particular interest for HSC, residing in the
CD34++CD38 fraction,15,19
genetically modified for fundamental research or gene therapy purposes.
Therefore, to follow the fate of such cells in the FTOC, we next
examined the ability of retrovirally transduced UCB
CD34++Lin cells to express a marker gene
after retroviral transduction in vitro. These experiments were followed
by a comparison between CD34++CD38Lin and
CD34++CD38+Lin cells in FTOC
after transduction.
Human CD34++Lin cord blood cells
express the retroviral encoded GFP after transduction in vitro.
Sorted CD34++Lin cord blood cells were
cultured in medium supplemented or not with the cytokines IL-3
and SCF. After 1 day of culture, cell-free MFG-GFP retroviral
supernatant was added. This retrovirus encodes a GFP
variant23 under control of the MoMuLV LTR promotor.
After another 2 days of culture, the cells were restained with CD34 PE
and assayed for GFP expression. As shown in
Fig 3, cells grown in complete medium
without cytokine or virus were still uniformly CD34+ and
not fluorescent above autofluorescence level in the FITC channel (FL1).
If no cytokines, but only viral supernatant was added, all cells were
still CD34+, but only a few cells expressed the marker gene
48 hours postinfection. This observation is in line with earlier
reports that show that more than 90% of
CD34++CD38/dim cord blood
cells are not in cell cycle upon isolation15 and need
cytokine stimulation to allow retroviral transduction.10 Cells grown in medium supplemented with IL-3 and SCF could be transduced at significant efficiencies (Fig 3). The marker gene expression level was sufficient to delineate a clear distinct population of transduced cells on FACS analysis. Cord blood donor to
donor variations were observed in transduction percentages (average,
40%; range, 15% to 51%; number of experiments [n] = 8). After 3 days of culture, cytokine-stimulated cells always downregulated CD34 in
at least a part of the population (Fig 3). On the average, 3 times
(range, 2.3 to 4; n = 3) more cells were recovered in the
cytokine-stimulated cultures compared with nonstimulated cultures after
3 days of culture. There was no obvious correlation between cell number
expansion and transduction percentages. An additional round of
transduction 2 days after initiation of the culture increased the
average percentage of GFP-expressing cells to 45%. However, a
decreased cell number expansion was observed compared with a single
round of transduction, despite medium replacement with fresh,
cytokine-supplemented complete IMDM, 4 hours postinfection after each
round of transduction. Therefore, only one round of transduction was
used in all subsequent experiments.
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| Fig 3.
GFP is expressed by cytokine-stimulated
CD34++Lin UCB cells transduced with
MFG-GFP retroviral supernatant. Flow cytometric analysis of the progeny
of CD34++Lin UCB cells after culture for
3 days, either without cytokines and retroviral supernatant (A),
without cytokines but with retroviral supernatant (B), or with both
cytokines (100 ng/mL SCF and 500 U/mL IL-3) and retroviral supernatant
(C). Retroviral supernatant was added after 1 day of culture. Dot plots
shows CD34 PE staining versus GFP expression gated on live cells. The
values in the crosses indicate the percentage of cells present in the
corresponding quadrant. Quadrants were set to include 99.5% of the
cells from staining showed in (A) in the upper left quadrant. The data
shown are representative of six independent experiments.
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These data show that CD34++Lin UCB cells
can be transduced at high frequency using cell-free, high-titer
supernatant after 1 day of stimulation with IL-3 and SCF. Furthermore,
the MoMuLV LTR promotor activity in these HSC/HPC was sufficient to
generate an intense fluorescence of the GFP marker.
Transduced human CD34++Lin cord blood
cells generate mature T, NK, and dendritic cells expressing high levels
of the marker gene in FTOC.
As the CD34++Lin UCB cells expressed GFP
after transduction, we wondered if the transduction protocol conserved
T-cell precursor potential of these cells. To test this, precultured
CD34++Lin cord blood cells were
transferred without prior selection for marker gene expression to SCID
mouse fetal thymic lobes to initiate organ cultures. After 14 days of
FTOC, less than 5,000 human thymocytes were recovered (n = 2). As shown
in Fig 4, half of the human cells stained
positive for HLA-DR. About one in three of these cells were
CD4+ (Fig 4) but CD14 and
CD3 (not shown) and thus represent the dendritic
cells. In the experiment shown, half of the dendritic cells expressed
the GFP marker. After 30 to 38 days of culture, on the average 5 × 104 human thymocytes (range, 1.5 to 8 × 104; n = 8) were recovered per lobe. Flow cytometry showed
generation of T cells expressing low, intermediate, and high levels of
CD3 (Fig 5), and some cells expressing high
levels of CD3 were negative for CD1. Of this most mature subset, half
of the cells expressed a -TCR (data not shown). As shown by other
groups, this indicates that UCB
CD34++Lin cells retain the ability to
generate T cells after several days of cytokine-supplemented suspension
culture5,12,18,19 or cryopreservation.5
Likewise, FTOC started with CD34++Lin
cord blood cells, precultured in complete medium without cytokines, were successful and phenotypically comparable (data not shown).
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| Fig 4.
Transduced CD34++Lin UCB
cells generate dendritic cells expressing high levels of GFP in FTOC.
Flow cytometric analysis of thymocytes recovered from FTOC initiated
with transduced CD34++Lin UCB cells that
were not selected for GFP expression (in this experiment, 30%
GFP+ cells at initiation of FTOC) and used to initiate
FTOC. After 14 days of culture, thymocytes were recovered from FTOC and
prestained with antimouse CD45 Cychrome (see the Materials and
Methods). Subsequently, aliquots were stained with IgG1 PE and IgG2a
BIO/SA-TC (not shown) or CD4 PE and HLA-DR BIO/SA-TC and analyzed by
flow cytometry. The left dot plot shows the forward scatter (FSC)
versus TC + Cychrome staining of all cells recovered. R1 is set to
include less than 1% of the human cells in isotypic control antibody
staining and contains the HLA-DR+ human cells. Above R1,
the cluster of Cychrome+ murine cells is seen, which are
at 14 days of FTOC still more numerous than human cells. The right dot
plot shows CD4 staining versus GFP expression gated on
HLA-DR+ human cells (R1). The values in the crosses
indicate the percentage of cells present in the corresponding quadrant.
Quadrants were set to include 99% of cells stained, with isotypic
control antibody in lower quadrants, gated on all human cells. The data
shown are representative of two independent experiments.
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| Fig 5.
Transduced CD34++Lin UCB
cells generate T cells expressing high levels of GFP in FTOC. Flow
cytometric analysis of thymocytes recovered from FTOC initiated with
transduced CD34++Lin UCB cells that were
not selected for GFP expression (in this experiment, 40%
GFP+ cells at initiation of FTOC) before initiation of
FTOC. After 35 days of culture, thymocytes recovered from FTOC were
stained with IgG1 PE (not shown), IgG2a PE, CD1 PE, CD3 PE, CD4 PE,
CD8 PE and analyzed by flow cytometry. Dot plots show forward
scatter (FSC) and staining versus GFP expression of live, human cells recovered. The values in the crosses indicate the percentage of cells
present in the corresponding quadrant. Quadrants were set to include
99.5% of the cells stained, with isotypic control antibody in the
lower quadrants. The data shown are representative of six independent
experiments.
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In FTOC initiated with transduced
CD34++Lin cord blood cells, part of the
human cells expressed high levels of the GFP marker gene at days 30 to
35 of FTOC (Fig 5). The ratio of percentage of cells expressing the
marker gene in the thymocyte progeny cells over that in the
CD34++Lin cells that initiated the FTOC
varied from experiment to experiment (average, 0.5; range, 0.1 to 1.2;
n = 8). Analysis of coexpression of GFP with CD1, CD3, CD4, and CD8
showed that the expression of the marker gene did not hamper T-cell
development of transduced cells (Fig 5). Most cultures also contained
NK cells (Fig 6E) and -T cells (data
not shown) expressing the marker gene. The level of GFP expression in
the thymocytes was very high, up to more than 1,000 times the
autofluorescence level. Murine cells recovered from FTOC (mouse
CD45-Cychrome+) were always FL1,
indicating the absence of replication competent virus in the FTOC. We
concluded that transduced CD34++Lin cord
blood cells could generate mature T, NK, and dendritic cells expressing
high levels of the marker gene in FTOC.
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| Fig 6.
GFP+ transduced
CD34++Lin UCB cells do not downregulate
GFP expression during thymic development in FTOC. Histogram (A) shows the GFP expression of transduced
CD34++Lin UCB 48 hours postinfection (in
this experiment, 49% GFP+ cells; N = number of cells).
The cells were purified in a GFP+ fraction (sort gate R1)
and a GFP fraction (sort gate R2) by cell sorting. The
unseparated population (indicated by R3) served as a control. Dot plots
show flow cytometric analysis of thymocytes recovered after 21 days of
FTOC and stained with CD1 PE for FTOC initiated with the
GFP+ fraction (B), the GFP fraction (C),
and the unseparated population (D). Flow cytometric analysis of
thymocytes stained with CD56 PE, gated on CD3 TC cells,
after 21 days of FTOC initiated with the unseparated population (E) is
also shown. Dot plots show staining versus GFP expression of live,
human cells recovered. The values in the crosses indicate the
percentage of cells present in the corresponding quadrant. Quadrants
were set to include 99.5% of the cells stained, with isotypic control
antibody in lower quadrants. The data shown are representative of two
experiments.
|
|
Transduced human CD34++Lin cord blood
cells, sorted for GFP expression, generate only thymocytes expressing
high levels of the marker gene in FTOC.
As described above, unselected transduced
CD34++Lin UCB cells generate a mixed
thymocyte population expressing or not expressing GFP after FTOC (Figs
4 and 5). On the average, the original transduction percentages of
CD34++Lin cells was two times that
observed in their thymocyte progeny. This phenomenon could be due to
either downregulation of GFP expression early after introduction in the
thymic lobe or to a difference in progenitor potential between
transduced and nontransduced cells. To dissect these phenomena, we
performed experiments with transduced CD34++Lin cells that were sorted for
presence or absence of GFP expression. As shown in Fig 6A, sort gates
were set to purify transduced CD34++Lin
cells expressing (R1) or not expressing (R2) GFP. Also, the total population (indicated by R3) was used in comparison. An equal number of
sorted cells (>99% pure) was cultured in FTOC. After 21 days of
culture, the thymocyte progeny of transduced
CD34++Lin cells expressing GFP was
homogeneously GFP+. No single human
CD1+GFP cell (Fig 6B) or
CD4+GFP cell (data not shown) was
detected. The level of GFP expression in the thymocytes was very high
(GFP++), up to more than 1,000 times the autofluorescence
level. Interestingly, in the thymocyte progeny of transduced
CD34++Lin cells not expressing GFP, a
minor fraction expressed moderate levels of GFP (GFP+; Fig
6C). FTOC initiated with GFP transduced
CD34++Lin cells yielded as many
thymocytes as FTOC initiated with GFP+ cells, at least
after 21 days of culture. This is not clear from the dot plots, because
many GFP++ cells are clustered at the last FL1 channel and
project on the frame of the plot (Fig 6D).
As observed earlier, cultures initiated with transduced
CD34++Lin that were not selected for GFP
expression generated a mixed progeny of GFP,
GFP+, and GFP++ cells (Fig 5D). In this FTOC,
most cells do not yet express CD3 after 21 days of culture. The
CD3CD56+ population is then still
prominent, with both a GFP+ and GFP
subset (Fig 6E).
In summary, the analysis of FTOC with sorted transduced cells suggest
that the MoMuLV LTR is not switched off, but in contrast upregulated
during development of transduced
CD34++Lin UCB cells into thymocytes,
which is in line with other reports.12,18
Both CD38 and CD38+
CD34++Lin cord blood cells generate
mature T cells expressing high levels of the marker gene in FTOC.
From our experiments we concluded that
CD34++Lin UCB cells expressed GFP after
transduction and generated mature T cells in FTOC expressing the marker
gene. As shown in Fig 1, these CD34++Lin
UCB cells are heterogeneous in CD38 expression. Because UCB HSC reside
in the CD34+CD38 cell
fraction,15,19 we analyzed whether this subset of the CD34++Lin UCB cells was transduced and
could generate T cells in the FTOC. As shown in
Fig 7, transduction was about half as
efficient in CD34++CD38Lin as
compared with CD34++CD38+Lin
UCB cells. Also, on the average, 3 times more (range, 1.5 to 6; n = 5)
cells were recovered from CD38+ compared with
CD38 CD34++Lin UCB
cells after the 3 days of culture in the transduction protocol, ie,
posttransduction only as many cells as the input number were recovered
per well from
CD34++CD38Lin UCB
cells.
View larger version (42K):
[in this window]
[in a new window]
| Fig 7.
Both CD38+ and CD38
CD34++Lin UCB cells generate T cells
expressing high levels of GFP in FTOC. Before FTOC indicates flow
cytometric analysis of the progeny of CD38+ and
CD38 CD34++Lin UCB cells
after culture for 3 days in medium with SCF (100 ng/mL) and IL-3 (50 U/mL) supplemented with retroviral supernatant after 1 day of culture.
Dot plots shows CD34 PE staining versus GFP expression gated on live
cells. The values in the crosses indicate the percentage of cells
present in the corresponding quadrant. Quadrants were set arbitrarily.
FTOC day 30 indicates flow cytometric analysis of thymocytes recovered
from FTOC initiated with transduced CD38+ and
CD38 CD34++Lin UCB cells
shown before FTOC that were not selected for GFP expression before
initiation of FTOC. After 30 days of culture, thymocytes recovered from
FTOC were stained with IgG1 PE (not shown), CD1 PE, CD4 TC, and CD3 PE
and analyzed by flow cytometry. Dot plots show staining versus GFP
expression of live, human cells recovered. The values in the crosses
indicate the percentage of cells present in the corresponding quadrant.
Quadrants were set to include 99.5% of the cells stained, with
isotypic control antibody in the lower quadrants. The data shown are
representative of three independent experiments.
|
|
As described above for total CD34++Lin
UCB cells, the progeny of precultured, transduced
CD38 and CD38+ subsets of
CD34++Lin UCB cells was transferred
without prior selection for marker gene expression to FTOC. After 30 days of FTOC, gene-marked thymocytes were recovered from cultures
initiated with both subsets (Fig 7). For the markers tested, no
phenotypical differences were observed between the thymocyte progenies
from transduced CD38 versus CD38+
CD34++Lin UCB. However, relatively more
GFP+ cells in cultures initiated with
CD34++CD38Lin cells
stained negative for the surface markers tested, as compared with FTOC
initiated with
CD34++CD38+Lin UCB cells.
We conclude from these data that both the CD38 and
CD38+ subsets of CD34++Lin
UCB cells can be transduced and can generate T cells in FTOC after
transduction.
| |
DISCUSSION |
In this report, we show that highly purified
CD34++Lin UCB cells can generate
dendritic, NK, and mature T cells in an FTOC. This ability is
maintained after transduction of IL-3/SCF-stimulated CD34++Lin UCB cells with retrovirus
encoding GFP, the thymocyte progeny expressing high levels of the
marker gene. Both the CD38 and CD38+
subsets of CD34++Lin UCB cells can be
transduced and can generate T cells in FTOC posttransduction. We thus
present an in vitro assay for thymic T-cell development from transduced
HSC/HPC.
Phenotypical analysis of the purified UCB
CD34++CD1CD3CD4CD7CD8CD19gpA
cells shows no expression of other lineage markers such as CD2, CD5,
CD7, CD16, and CD33. In contrast, the cells are homogeneously positive
for CD44 (not shown) and HLA-DR (Fig 1). Their immature phenotype is
underscored by the expression of c-kit30 and the low or
absent expression of CD38.15,30
Also, the absence of transduction without cytokine
stimulation10 sustains the notion that these cells are
immature. Part of the cells stained positive for CD45RA. A low
expression of CD10 (on the average 5% of
CD34++Lin UCB cells; our unpublished
observations) was detected only on some of these
CD34+CD45RA+Lin cells. A
previous study14 showed that
CD34++CD38+CD45RA+CD10+Lin
cells from adult bone marrow are nonprimitive progenitors for lymphoid
and dendritic cells. Further experiments will teach us what the
contribution of this and other subsets within the
CD34++Lin UCB cells is to the
colonization of the thymic lobes in FTOC. However, our data (Fig 7)
indicate that
CD34++CD38Lin UCB can
also generate T cells in the FTOC, indicating that more primitive cells
than the aforementioned population can contribute to the thymocyte
progeny in FTOC. The development into T and dendritic cells
described in this report (Fig 2) is very similar to that as we
described for CD34++Lin fetal liver
cells.21,22 The development of T cells in this system
allows a kinetic analysis and study of T-cell development pathways.22 The T cells develop from a
CD3CD4CD8stage,
via CD3CD4+CD8 and
CD3/dim CD4+CD8+ cells into
CD3++CD4+ or CD3++CD8+
mature T cells, as reported for human T-cell development observed in
SCID-hu mice and human thymus organ culture (reviewed by Shortman and
Wu1). Several reports31,32 show the generation
of CD3++CD4+ or
CD3++CD8+ cells from CD34+ bone
marrow cells in vitro by culture on thymic stroma in the absence of the
three-dimensional thymic microenvironment. However, it is unclear
whether these models reflect T-cell development as observed in the
thymus. In the experiments of Freedman et al,31 no clear
CD4+CD8+ double-positive intermediate was
observed. Interestingly, a recent report shows the existence of an
extra thymic pathway of CD4+ T-cell development from
peripheral blood progenitors that also do not develop by a
CD4+CD8+ double-positive
intermediate.33 Such an intermediate is
present in the data reported by Rosenzweig et al,32 who use
cultures of human HSC/HPC on primate thymic stroma. However, this model is less readily available and less studied than FTOC.
Staal et al34 reported the development of CD34+
thymocytes, transduced by coculture with  -CRIP packaging cells
producing MFG-LacZ retrovirus, into more mature thymocytes using FTOC.
Using FACS detection of -galactosidase expression, they show
staining of part of the CD4+CD8 and
CD4+CD8+ thymocytes after 3 weeks of culture.
In our hands, transduction with retrovirus generated with the MFG-LacZ
vector in -CRIP packaging cell line suffered from major drawbacks
(our unpublished results). Similar to the findings of
Staal et al,34 we obtained a uniform peak shift in
FITC-fluorescence of CD34++ cells after coculture with
-CRIP/MFG-LacZ cells. However, in the majority of cells, this was
due to enzyme rather than gene-transfer of and did not mirror stable
transduction. Mixing of cells showed that, in our hands, enzyme was
transferred from nontransduced to transduced cells, hampering or
eliminating discrimination between both populations. This problem was
not observed by using -galactosidase engineered to contain nuclear
localization sequences (Dr C. Bagnis, Institut Paoli Calmettes,
Marseille, France; personal communication, June 1997), possibly by
preventing enzyme leakage. Overestimation of transduction due to
protein rather than gene-transfer has also been reported with
retrovirus-encoding cell surface markers.35 With the
retroviral supernatant that was used in the present report, Jurkat
cells were transduced (data not shown). In these experiments, the first
GFP-expressing cells were detected only after more than 8 hours
posttransduction, and GFP fluorescence intensity was only maximal after
48 hours. These observations argue against detectable passive protein
transfer and suggest active protein synthesis. By using mixing
experiments with homogeneous populations of GFP+ and
GFP cells, transfer of GFP to nontransduced cells
was not detected. Finally, cloning of transduced Jurkat cells showed
stable expression after gene-transfer. In our experiments with
CD34++Lin UCB cells, very few
GFP+ cells were detected, unless the cells were stimulated
with cytokines (Fig 3). This suggests that UCB CD34++ cells
have to be put into cell cycle, before gene-transfer can occur, as is
believed to be necessary for nonlentiviral retroviral vectors.5,10 We therefore conclude that FACS GFP detection is a reliable marker of productive gene-transfer in our experiments. As
with cell surface markers,12,18,36 GFP expression allows the identification and separation of effectively transduced cells soon
after infection. In this way, competition between nontransduced and
transduced cells in subsequent assays can be excluded. By sorting
transduced CD34++Lin cells for GFP
expression, we showed that the MoMuLV LTR is not at all downregulated
in the development of T cells from transduced UCB HSC/HPC (Fig 6). This
observation is in line with in vivo observations of other
groups.11,12,18 Interestingly, GFP
transduced CD34++Lin cells generated
some GFP+ thymocytes. Although not detected, we never can
exclude contamination during sorting. However, because unselected
transduced CD34++Lin generate both
GFP+ (FL1  4,000) and GFP++ (FL1 >4,000)
thymocytes (Fig 5D) and GFP+ transduced
CD34++Lin cells generate mainly
GFP++ thymocytes (Fig 5B), we favor the idea that at least
part of the GFP+ thymocytes are the progeny of effectively
transduced CD34++Lin cells not
expressing GFP at the moment of analysis. This would indicate an
upregulation rather than downregulation of the MoMuLV LTR during T-cell
development. The increase of average GFP fluorescence (250 stronger
than autofluorescence in transduced UCB CD34++ cells to
more than 1,000 times stronger than autofluorescence in thymocytes)
sustains this conclusion. Also, protein accumulation is suggested by
the fact that GFP fluorescence intensity in transduced UCB
CD34++ cells is higher 48 hours compared with 24 hours
posttransduction (data not shown). Because GFP is an intracellular,
spontaneously fluorescent protein, no antibody staining is needed to
detect its expression. This easy detection has the advantage that no background fluorescence is present in nontransduced cells. However, in
contrast to cell surface markers, transduced cells can only be purified
by cell sorting and cannot be phenotyped with FITC-labeled antibodies.
In some of our experiments, we used the bulk of
CD34++Lin cells exposed to virus,
containing both cells expressing or not the transgene, to start FTOC
(Figs 4 and 5). We observed the development of both transduced and
nontransduced dendritic, NK, and T cells. The markers CD56, CD1, and
CD4 appeared with the same kinetics in GFP+ and
GFP cells. However, CD3 and CD8 expression was less
in the GFP+ compared with the GFP
populations after 35 days of FTOC. Moreover, in one experiment with
transduced CD34++Lin cells sorted for
GFP expression before FTOC, we recovered fewer thymocytes after 14 days
of FTOC in cultures initiated with GFP+ cells, compared
with cultures initiated with GFP cells (data not
shown). This difference was not evident after 21 and 30 days. Further
experiments will teach us if these observations might indicate a slower
development of mature GFP+ compared with that of
GFP T cells. A toxic effect of GFP expression in the
transduced CD34++ cells or thymocytes cannot be excluded.
However, although reduced proliferation was observed in yeast cells
expressing S65T GFP,37 no reports show a toxic effect of
S65T GFP or of EGFP expressed in mammalian cells. Still, both
GFP+ and GFP mature T cells were present
after 35 days of FTOC. This indicates that neither expression of
retrovirally encoded GFP nor exposure to the retroviral supernatant
blocks the development of CD34++Lin
cells in the FTOC.
Several diseases that affect the hematopoietic system, such as ADA
deficiency,8,10 Gaucher disease,4 chronic
granulomatous disease,9 and acquired immunodeficiency
syndrome,12 are considered curable by gene therapy if the
transduced gene is expressed in mature T cells. However, T-cell
progenitor potential of transduced HSC is largely unknown in most gene
therapy protocols. In clinical trials reported so far,6,8
the in vivo fraction of cells transduced in myeloid and lymphoid
lineages is low compared with that in in vitro assays, such as methyl
cellulose cultures, started with an aliquot of the same transduced HSC.
Also, in in vivo models such as SCID-hu mice intrathymically injected
with transduced cord blood, the level of gene marking in the thymocytes
is considerably lower than that of the input HSC/HPC
(80-fold11 to 5-fold18 reduction). A similar
(~40-fold) reduction20 in gene marking levels was
observed in human T cells found in the bone marrow of bnx mice
repopulated with transduced CD34+ cells from normal human
bone marrow or mobilized peripheral blood. The reduced marking of
generated T cells in vivo compared with that of the input HSC/HPC
population is probably due to the fact that true HSC, responsible for
long-term repopulation of the acceptor, are less frequently or not
transduced. In our experiments with the bulk of
CD34++Lin cells exposed to virus,
containing both cells expressing or not expressing the transgene, an
average of twofold reduction in gene marking of the thymocyte progeny
was observed. We showed that this phenomenon was not due to
downregulation of GFP expression, because the thymocyte progeny of
GFP+ transduced CD34++Lin
cells was homogeneously GFP+ (Fig 6). We therefore
currently consider the possibility that transduced
CD34++Lin cells have, on the population
level, a reduced T-cell progenitor potential compared with their
nontransduced counterparts. This might be an indirect consequence of a
more limited survival. More experiments are needed to evaluate the
impact of cytokines, cell cycle induction and differentiation kinetics
on the observed differences in progenitor potential. Also, we are
setting up inverse PCR experiments20 to assay
(oligo)clonality of the transduced T, NK, and dendritic cells generated
in the FTOC.
The CD34++Lin cord blood cells used in
some of our experiments were CD38 and thus encompass
both the most primitive HSC.15 These cells could be
transduced, albeit at lower efficiency compared with CD34++CD38+Lin cells (Fig
7). Our preliminary data show that gene-transfer to CD34++CD38Lin UCB
cells is higher if the virus is added only after 2 days instead of 1 day of cytokine stimulation, whereas this difference was not clear for
CD34++CD38+Lin UCB cells
(data not shown). This probably reflects differences in cell cycle
entry between the two subsets. After transduction, both the
CD38+ and the CD38 subset generate T
cells expressing high levels of the marker gene in the FTOC (Fig 7). We
observed that relatively more GFP+ cells in cultures
initiated with
CD34++CD38Lin cells
stained negative for the surface markers tested, as compared with FTOC
initiated with
CD34++CD38+Lin UCB cells.
Future experiments will inquire if this phenomenon indicates
non-T-cell development in the FTOC of transduced
CD34++CD38Lin UCB
cells. Our observations consistently show (7 independent experiments
with different donors) T-cell development from freshly sorted
CD34++CD38Lin UCB
cells in FTOC, in contrast to the data of Blom et al.27 These investigators report that
CD34++CD38 UCB cells from some but not
all donors fail to generate CD3+CD4+ cells in
FTOC. However, because these investigators isolate CD34 FITC++CD38 PE cells for FTOC using
2-deoxyguanosine-treated RAG-1/ fetal thymic
lobes, we cannot directly compare our results. Differences in
fluorochrome of the antibody used can affect the expression level from
which a surface marker is considered to be expressed or not. To resolve
this issue, the two staining and isolation protocols should be compared
in parallel.
Besides failure of transduction of HSC, one can infer that a
transduction protocol affects T-lymphoid progenitor capacity of
transduced cells that become nonlymphoid HPC rather than stay HSC. We
have indeed observed that certain cytokine mixes, while resulting in
comparable transduction efficiencies as those reported here, completely
abrogate T-cell development in vitro of transduced HSC/HPC (manuscript
in preparation).
In conclusion, we show in this report that retrovirally transduced
human CD34++Lin and
CD34++CD38Lin UCB
cells can generate a thymocyte progeny expressing the transgene in
vitro. GFP, under control of the MoMuLV LTR promotor, was shown to be a
suitable marker gene. The FTOC model we use allows an array of cytokine
combinations and concentrations to be compared in parallel in their
effect on transduction and T-cell development of transduced cells. The
in vitro results could prove valuable in establishing a transduction
protocol for in vivo studies and gene therapy. As in the SCID-hu model,
developmental kinetics can be studied. This allows us to study the
effect of genes of interest, such as (anti-)human immunodeficiency
virus genes, on the development of transduced HSC/HPC.
| |
FOOTNOTES |
Submitted June 20, 1997;
accepted September 8, 1997.
Supported by grants from the Gezamelijk Overlegde Actie (GOA) and the
Fund for Scientific Research-Flanders (Belgium). B.V. is a research
assistant of the Fund for Scientific Research-Flanders (Belgium).
Address reprint requests to Bruno Verhasselt, MD, Department of
Clinical Chemistry, Microbiology and Immunology, University of Ghent,
University Hospital of Ghent, 4 Blok A, De Pintelaan 185, B-9000 Ghent,
Belgium.
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.
| |
ACKNOWLEDGMENT |
The authors thank Christian De Boever for artwork, Achiel Moerman and
Veronique Debacker for animal care, and the Department of Obstetrics
(Prof M. Dhont) of the University Hospital of Ghent for the supply of
human tissue. We also gratefully acknowledge the support of Dr H. Spits
and collaborators (The Netherlands Cancer Institute, Amsterdam, The
Netherlands) in introducing us in retroviral vector construction. We
are greatly indebted to Dr R.C. Mulligan (Howard Hughes Medical
Institute, Boston, MA) for making the MFG vector backbone and -CRIP
packaging cell line available to us and to Dr G.P. Nolan (Stanford
University School of Medicine, Stanford, CA) for the generous gift of
the Phoenix-A packaging cell line. Finally, we thank our colleagues Drs
B. Vandekerckhove and D. Vanhecke for critical reading of the
manuscript.
| |
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Abstract
Introduction
Abstract