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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4013-4022
RAPID COMMUNICATION
Highly Efficient Transduction of the Green Fluorescent Protein Gene in
Human Umbilical Cord Blood Stem Cells Capable of Cobblestone Formation
in Long-Term Cultures and Multilineage Engraftment of Immunodeficient
Mice
By
Paula B. van Hennik,
Monique M.A. Verstegen,
Marti F.A. Bierhuizen,
Ana Limón,
Albertus W. Wognum,
José A. Cancelas,
Jordi Barquinero,
Rob E. Ploemacher, and
Gerard Wagemaker
From the Institute of Hematology, Erasmus University Rotterdam, The
Netherlands; and the Department of Cryobiology and Cell Therapy,
Institut de Recerca Oncologica, Barcelona, Spain.
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ABSTRACT |
Purified CD34+ and
CD34+CD38 human umbilical cord blood (UCB)
cells were transduced with the recombinant variant of Moloney murine
leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP
expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer
cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell
line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope
proteins. Using a 2-day growth factor prestimulation, followed by a
2-day, fibronectin fragment CH-296-supported transduction, CD34+ and CD34+CD38 UCB
subsets were efficiently transduced using either vector. The use of the
SF-EGFP/PG13 retroviral packaging cell combination consistently
resulted in twofold higher levels of EGFP-expressing cells than the
MFG-EGFP/Am12 combination. Transplantation of 105 input
equivalent transduced CD34+ or 5 × 103
input equivalent CD34+CD38 UCB cells in
nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages
of 8% and 5%, respectively, which showed that the in vivo
repopulating ability of the cells had been retained. In addition, mice
engrafted after transplantation of transduced CD34+ cells
using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP
with median values of 2% and 23% of human CD45+ cells,
respectively, which showed that the NOD/SCID repopulating cells were
successfully transduced. EGFP+ cells were found in all
human hematopoietic lineages produced in NOD/SCID mice including human
progenitors with in vitro clonogenic ability. EGFP-expressing cells
were also detected in the human cobblestone area-forming cell (CAFC)
assay at 2 to 6 weeks of culture on the murine stromal cell line
FBMD-1. During the transduction procedure the absolute numbers of CAFC
week 6 increased 5- to 10-fold. The transduction efficiency of this
progenitor cell subset was similar to the fraction of
EGFP+ human cells in the bone marrow of the NOD/SCID mice
transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced
CD34+ cells, ie, 6% and 27%, respectively. The study
thus shows that purified CD34+ and highly purified
CD34+CD38 UCB cells can be transduced
efficiently with preservation of repopulating ability. The SF-EGFP/PG13
vector/packaging cell combination was much more effective in
transducing repopulating cells than the MFG-EGFP/Am12 combination.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
EFFICIENT PROCEDURES for gene transfer
into human immature hematopoietic cells with repopulating capacities
after transplantation may in principle open new avenues for the
treatment of a variety of hereditary and acquired diseases.
Retroviral-mediated gene transfer to such cells, which is attractive by
its simplicity and efficiency, has, however met with considerable
difficulty, which is only partly understood.1,2 The
availability of a rapid selectable marker, such as the green
fluorescent protein (GFP), is thought to be of pivotal importance to
study major variables influencing the efficiency of gene transfer, as
well as to track the progeny of transduced cells after transplantation.
In the present study we evaluated the use of the enhanced (E)
recombinant variant of GFP to label immature human umbilical cord blood
cells, using outgrowth in nonobese diabetic/severe combined
immunodeficient (NOD/SCID) mice1,3,4 as well as cobblestone
area-forming cells (CAFC)5,6 as assays for immature cells
with considerable hematopoietic reconstitution capacity.
The CAFC assay and the long-term culture-initiating cell (LTC-IC) assay
allow for frequency analysis of cells capable of long-term repopulation
in vitro.5,7 Murine studies have shown that the CAFC scored
at week 2 are related to colony-forming unit-spleen (CFU-S) day 12, while CAFC week 5 strongly correlate with
long-term repopulating cells in vivo.6,8 In human
hemopoiesis the rare population with the primitive phenotype of
CD34+CD38 is highly enriched for CAFC
week 6. The primitive nature of CAFC week 6 is further illustrated by
enrichment after incubation with 5-fluorouracil (5-FU), a drug
cytotoxic for proliferating cells. The CAFC week 2, however, are absent
in the CD34+CD38 population and more
than 1 log reduced after 5-FU treatment. Based on these results, the
CAFC week 6 have been proposed to be representative for cells with
long-term repopulating ability in vivo in the human
situation.9 On this basis, this assay is considered
suitable to assess the effect of manipulation of human hematopoietic
progenitor cell populations, such as by gene-transfer protocols.10,11
The efficiency of gene transfer to stem cells is limited by the
inability of most retroviral vectors to integrate DNA into the cellular
genome of quiescent cells.12-15 Stimulation of stem-cell cycling with hematopoietic growth factors (HGF) such as interleukin-3 (IL-3), IL-6, stem cell factor (SCF), or Flt3-L16 before
and during virus exposure would seem to be essential to promote
transduction,17,18 but may result in loss of repopulating
ability of transduced cells as a result of
differentiation.16,19 In addition, colocalization of target
cells and virus on dishes coated with the recombinant fibronectin-fragment CH-296 has been shown to further increase gene
transfer efficiency.20,21
For transduction of human hematopoietic cells, murine retroviruses
based on the Moloney murine leukemia virus (MoMLV) are most commonly
used. However, expression of functional receptors for the MoMLV
envelope protein is presumably low, and pseudotyping the vector with
the GaLV envelope protein resulted in higher transduction efficiencies
in hematopoietic progenitor cells,22-24 which has been
attributed to a higher expression of functional pseudotyped GaLV
receptor (Pit-1) by the immature hematopoietic cells22,24 than the amphotropic retroviral receptor (Pit-2).24-28 A
study in which CD34+ cells were transduced by the
GaLV-pseudotyped retroviral vector showed that CD34+ cells
were efficiently transduced (21% to 33% transduction) as determined
by culture in a colony-forming cell assay.2 It is not known
to what extent the relative transduction inefficiency of the MoMLV type
viruses is caused by a low Pit-2 expression on immature stem cells or
by inefficient activation and provirus integration in quiescent cells.
Transplantation of CD34+ or
CD34+CD38 transduced cells in
immunodeficient beige/nude/xid (bnx) mice showed that 8 of 10 mice
transplanted with CD34+ transduced cells contained the
retrovirally transduced bacterial neomycin phosphotransferase
resistance (neo), gene whereas only 2 of 14 mice that had received
CD34+CD38 cells contained low levels of
transduced cells.2 The ability to engraft the bone marrow
(BM) of NOD/SCID mice and provide multilineage outgrowth, which resides
exclusively in the CD34+CD38-
population,3 has been described as unsuccessful, in
contrast to the LTC-IC or CAFC week 6, which were transduced with
efficiencies ranging between 10% and 70%.1 These
differences led to the suggestion that NOD/SCID repopulating cells are
distinct from the LTC-IC or CAFC week 6.1 However, recent
data obtained with vectors that contained the neo-gene show that
transplantation of retrovirally transduced CD34+ UCB cells
in NOD/SCID mice result in transduced human hematopoiesis in the
NOD/SCID BM with transduction levels similar to those obtained for
LTC-IC.29
Use of the GFP gene from the jellyfish Aequorea victoria as a
retrovirally transduced marker allows rapid identification of transduced cells by fluorescence microscopy, flow cytometry, or culture
in real time without additional staining steps in contrast to other
genetic markers such as the neo-gene30-32 and the bacterial  -galactosidase gene (LacZ).33-36 As wild-type
GFP produces a weak (but stable) green fluorescence
signal, several GFP variants, such as EGFP, have been created which are
better suited for detection of expression by fluorescence microscopy
and flow cytometry.37,38 Studies with murine cells have
shown that cells with the ability of in vivo reconstitution can be
transduced with EGFP.39 Our ongoing studies show that high
expression levels of EGFP could be detected in mouse BM, peripheral
blood, spleen, and thymus for a current observation period of 6 months
after transplantation and were retained in secondary recipient mice,
indicating that long-term repopulating stem cells can be successfully
transduced. Human cell lines and purified CD34+ cells were
also transduced using EGFP-containing vectors.28 Therefore,
retroviral vectors containing EGFP genes can be used to transduce a
variety of cells, which can then be easily detected in vitro as well as
in vivo.
To initiate an analysis directed at optimal vectors and transduction
procedures, the MFG-EGFP retroviral vector produced by an amphotropic
packaging cell line and the SF-EGFP vector pseudotyped for the GaLV
envelope protein were used to transduce immature cell subsets in human
umbilical cord blood (UCB). The potential of these vector/packaging
cell combinations for transduction of purified CD34+ and
CD34+CD38 UCB subsets was compared by
assessing the ability of transduced cells to produce EGFP+
cobblestone areas in the CAFC assay and to contribute to multilineage human hematopoiesis in NOD/SCID mice.
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MATERIALS AND METHODS |
Human UCB cells.
UCB samples were obtained from placentas of full-term normal
pregnancies after informed consent in conformity with legal regulations in The Netherlands. Mononucleated cells were isolated by Ficoll density
gradient centrifugation (1.077 g/cm2; Nycomed Pharma AS,
Oslo, Norway), and were cryopreserved in 10% dimethylsulphoxide, 20%
heat-inactivated fetal calf serum (FCS), and 70% Hanks' Balanced Salt
Solution (HBSS; GIBCO, Breda, The Netherlands) at  196°C as
described40 before use. After thawing by stepwise dilution
in HBSS containing 2% FCS, the cells were washed with HBSS containing
1% FCS and used for gene transduction experiments.
Viral vectors and packaging cell lines.
The amphotropic retroviral producer cell line, MFG-EGFP, was obtained
by a 20-hour incubation of GP+envAm12 under standard culture
conditions with supernatants containing ecotropic retrovirus from the
GP+E-86/MFG-EGFP cell line and hexadimethrine bromide at 4 µg/mL
(Sigma, St Louis, MO) as described.38 The pseudotyped retroviral producer cell line PG13/EGFP7 was developed by transducing the PG13 packaging cell line (kindly provided by D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA) with 0.45 µm filtered supernatant from PA317/EGFP cell cultures.28 EGFP
expression was analyzed by flow cytometry and bright single cells were
sorted on 96-well plates by using an EPICS Elite ESP flow cytometer
coupled to an autoclone device (both from Coulter, Miami, FL). Single clones were cultured as previously described.28 The sorted
clones were additionally selected for high virus titer. The viral titer of both the amphotropic and the pseudotyped producer cell line was in
the order of 106 infectious particles per mL as determined
by supernatant titration on cultured murine NIH 3T3 cells and human
HeLa cells, respectively. Absence of replication-competent virus was
verified by failure to transfer GFP expression from a transduced cell
population to a secondary population. Additionally, for the
SF-EGFP/PG13 vector/packaging cell combination pseudotransduction was
tested on HeLa cells and found absent.
Subset purification.
Purification of CD34+ cells was performed by positive
selection using Variomacs Immunomagnetic Separation System as
described41 (CLB, Amsterdam, The Netherlands). The
percentage of CD34+ cells in the unseparated population
(low-density UCB) and in the purified CD34+ and
CD34 fractions was determined by
fluorescence-activated cell sorting (FACS) analysis. For isolation of
CD34+CD38 subsets, purified
CD34+ cells were stained with fluorescein isothiocyanate
(FITC) and R-phycoerythrin (PE) conjugated antibodies against human
CD34 and CD38 (CD34-FITC, CD38-PE; Becton Dickinson, San Jose, CA) for
30 minutes on ice in HBSS, supplemented with 2% (wt/vol) bovine serum
albumin (BSA; Sigma), 0.05% (wt/vol) sodium azide (Merck, Darmstadt,
Germany) and 2% (vol/vol) normal human serum (NHS). After
incubation, the cells were washed twice, resuspended in HBSS and
CD34+CD38 cells, and the window set at
5% of the CD34+ population with the lowest CD38 expression
levels (Fig 1) were sorted using a FACS
Vantage flow cytometer (Becton Dickinson, San Jose, CA).
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| Fig 1.
Flow cytometric profile used to define and sort the
CD34+CD38 cell population (A). The window
R3 was used to define CD34+CD38 cells for
sorting and contains 5% of the CD34+ population (as
defined by window R2) with the lowest CD38 antigen expression.
Re-analysis of the sorted cells is shown in (B).
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Retroviral transduction of UCB subsets.
Supernatants containing recombinant retrovirus were generated by
culturing approximately 80% confluent producer cells for 12 hours in
culture medium consisting of a serum-free enriched version of
Dulbecco's modified Eagle's medium (DMEM; GIBCO, Gaithersburg, MD).3,39,42 Media for all cultures routinely included 100 U/mL of penicillin and 100 µg/mL of streptomycin. The cultures were
maintained at 37°C with 10% CO2 (measured every 15 minutes with read-outs between 9.5% and 10%) in a humidified
atmosphere. The culture supernatant was subsequently procured and
passed through a 0.45-µm filter. To enhance the transfection
efficiency, Falcon 1008 (35-mm) bacteriological culture
dishes (Becton Dickinson, Plymouth, UK) were coated with
the recombinant fibronectin fragment CH-296 (Takara Shuzo, Otsu, Japan)
at a concentration of 10 µg/cm2 as described
previously.21 UCB subsets (CD34+ or
CD34+CD38) were prestimulated for 2 days
in either medium consisting of enriched Dulbecco's medium (GIBCO,
Gaithersburg, MD), or CellGroSCGM (Boehringer Ingelheim, Heidelberg,
Germany). Different combinations of human recombinant HGF were added to
the culture medium; IL-3 (20 ng/mL; Gist-brocades NV, Delft, The
Netherlands), IL-6 (100 ng/mL; Ares-Serono SA, Genève,
Switzerland), thrombopoietin (TPO; 10 ng/mL, kindly provided by
Genentech, South San Francisco, CA), SCF (100 ng/mL), and Flt3-L (50 ng/mL; the latter two kindly provided by Amgen, Thousand Oaks, CA). The
HGF combination of Flt-3L, TPO, IL-6, and SCF was used during the
transduction procedure; in some initial experiments, as indicated in
the legend of the figures and tables, the IL-3, IL-6, SCF combination
was used. Before adding purified cord blood subsets to the
fibronectin-coated dishes, the CH-296 fibronectin fragment was
preincubated with supernatant containing the amphotropic MFG-EGFP or
the pseudotyped SP-EGFP vector for 1 hour at 37°C.20,21
Subsequently, nucleated cells were resuspended in the vector-containing
supernatant supplemented with hematopoietic growth factors and added to
the dishes. Over a period of 2 days, culture supernatant was once
replaced completely by resuspending nonadherent cells into fresh
retrovirus supernatant and HGF. Finally, the cells were obtained and
used for FACS analysis, human granulocyte-macrophage CFU (GM-CFU) and
erythroid burst-forming units (BFU-E) assays, CAFC assay, and
transplantation into NOD/SCID mice.
Flow cytometry.
Cell samples were analyzed using a FACSCalibur flow cytometer (Becton
Dickinson) as previously described.38,39 Immunophenotyping of EGFP-transduced cells was performed by staining with peridinin chlorophyll protein (PercP)-labeled anti-CD45 and cyanin-5-conjugated anti-CD34 (Cy5; Amersham, Buckinghamshire, UK) or PE-conjugated monoclonal antibodies against CD38, CD2, CD4, CD8, CD19, CD20, CD56,
CD33 (Becton Dickinson). Mice were considered engrafted if the
percentage CD45+ cells exceeded 1%.
Transplantation of transduced UCB subsets in immunodeficient mice.
Specific pathogen-free (SPF) NOD/LtSz-scid/scid (NOD/SCID) mice, 6 to 9 weeks of age, were bred and housed under SPF conditions in a laminar
air flow unit and supplied with sterile food and acidified drinking
water containing 100 mg/L ciprofloxacine (Bayer AG, Leverkusen,
Germany) ad libitum. Housing, care, and all animal experimentation were
done in conformity with legal regulations in The Netherlands, which
include approval by a local ethical committee. All mice received total
body irradiation (TBI) at 3.5 Gy, delivered by a 137Cs
source adapted for the irradiation of mice (Gammacell, Atomic Energy of
Canada, Ottawa), 2 to 4 hours before transplantation. The transplants
were suspended in 200 µL HBSS containing 0.1% BSA and injected
intravenously (IV) into a lateral tail vein. Transplanted cell numbers
were 105 CD34+ cells and 5 × 103 CD34+CD38 cells.
Thirty-five days after transplantation the mice were killed by
CO2 inhalation followed by cervical dislocation, both
femurs isolated, and BM cell suspensions prepared by flushing. After counting, the cells were cultured in colony assays and analyzed by flow
cytometry to determine the percentage of human EGFP+ cells
in the mouse BM.
In vitro colony assay.
Purified UCB cells, EGFP-transduced cells, and chimeric mouse BM
samples were assayed for the presence of human GM-CFU and BFU-E by in
vitro colony formation in viscous methylcellulose culture medium as
previously described.3,42-44 The number of colonies was
determined after 14 days of culture in a humidified atmosphere of 10%
CO2 at 37°C. EGFP+ colonies were scored
under excitation by UV light.
Stromal feeders and CAFC assay.
The contact inhibited FBMD-1 murine stromal cell line was used as
described before.5 After 7 to 10 days of culture at
33°C and 10% CO2, the stromal layers had reached
confluence and were overlaid with nontransduced or transduced
CD34+ or CD34+CD38 UCB cells
within the subsequent week. Confluent stromal layers of FBMD-1 cells in
flat-bottom 96-well plates were overlaid with UCB cells in a limiting
dilution setup. Input values of the
CD34+CD38 population and the
CD34+ were 25 nucleated cells and 500 nucleated cells per
well in the first dilution, respectively. Twelve twofold serial
dilutions were used for each sample with 15 replicate wells per
dilution. The cells were cultured at 33°C and 10% CO2
for 6 weeks with weekly half-medium changes. The percentage of wells
with at least one phase-dark hematopoietic clone of at least five cells
(ie, a cobblestone area) beneath the stromal layer was determined
weekly with an inverted microscope. Green fluorescent cobblestone areas
were screened in the same way but with a UV-light excitation source. Frequencies of total and green-fluorescent CAFC were calculated by
using Poisson statistics as described previously.6 During the period of culture, no transfer of the EGFP gene to the stromal underlayer has been observed.
Statistical analysis.
Data are expressed as median (range). Statistical comparisons were
performed according to Mann Whitney U-test. P values <.05, two-tailed, were considered significant.
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RESULTS |
Transduction efficiencies in purified cells with MFG-EGFP and SF-EGFP
vectors.
Purified CD34+ and CD34+CD38
UCB cells (Fig 1) were prestimulated for 2 days and subsequently
transduced with either MFG-EGFP/Am12 or SF-EGFP/PG13 vector/packaging
cell combination, during 2 days of exposure to virus-containing
supernatants in fibronectin fragment-coated bacterial dishes.
Transduction efficiencies obtained by infection using the amphotropic
MFG-EGFP producer cell line were compared to those obtained with the
pseudotyped SF-EGFP cell line. The percentage EGFP+ cells
was assessed by flow cytometry (Fig 2). The
percentage of EGFP+ cells of the purified CD34+
population transduced with the SF-EGFP/PG13 vector/packaging cell
combination (median, 75% EGFP+) was more than twofold
higher compared with MFG-EGFP/Am12-transduced CD34+ cells
(median, 30%) (Table 1). Sorted
CD34+CD38 cells were also transduced at
a higher frequency using the SF-EGFP/PG13 combination (62%) than after
transduction with the MFG-EGFP/Am12 combination (19%). On average,
transduction frequencies were lower in the purified
CD34+CD38- cells than in the CD34+
cell fraction, but only for the MFG-EGFP/Am12-transduced cells the
difference was statistically significant. The level of transduction of
the CD34+CD38 subset within the purified
CD34+ population obtained with the SF-EGFP/PG13
vector/packaging cell combination was more than 2.5-fold higher than
with the MFG-EGFP/Am12 combination. The differences in transduction
efficiency between the two vector/packaging cell combinations in these
cell populations were significant (P < .025).
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| Fig 2.
Flow cytometric analysis of a representative transfection
of purified CD34+ cells with the amphotropic MFG-EGFP
retroviral vector after 2 days of prestimulation and 2 days of
supernatant infection in the presence of IL-3, IL-6, and SCF. This
particular transduction resulted in efficiencies of 30% within the
CD34+ population (A). In (B) CD34+ cells
were gated and the CD38 distribution of the EGFP-transduced cells was
studied. Also, CD34+CD38 cells expressed
the EGFP gene with efficiencies similar to the total
CD34+ population (30% EGFP+).
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Transduction efficiency of CAFC subsets.
The ability of transduced cells to form cobblestone areas was evaluated
in long-term culture supported by FBMD-1 stromal cells. EGFP+ cobblestone areas were identified by fluorescence
microscopy (Fig 3). The absolute numbers of
CAFC at different culture periods increased as a result of the
transduction procedure without significant differences between the
target cells or vector used (Table 2). The
absolute number of CAFC week 2 in the MFG-EGFP/Am12-transduced CD34+ UCB cells increased 5-fold, for the
SF-EGFP/PG13-transduced CD34+ UCB cells the increase was
7-fold. The CAFC week 6 expanded 10-fold and 5-fold, respectively. For
the CD34+CD38 UCB cells, similar results
were obtained, 6-fold and 10-fold of CAFC week 6 after MFG-EGFP/Am12
and SF-EGFP/PG13 transduction, respectively. Consistent with the
immaturity of the CD34+CD38 cell
population, CAFC week 2 could not be detected in the
CD34+CD38 cell fraction before
transduction. These data show that the transduction protocol that has
been used causes a modest expansion of both CAFC week 2 and week 6.
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| Fig 3.
Fluorescence microscopic image of a representative
EGFP+ cobblestone area. The bright green cells are the
mature cells on top of the stromal layer and the dim green cells
represents the EGFP+ cobblestone area.
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Table 2.
Absolute Numbers of CAFC Week 2 and Week 6 and
Percentages of Green Fluorescent Cobblestone Areas After
Transduction of 106 Selected UCB CD34+
Cells or 35 × 103 CD34+CD38
Cells With the Vectors MFG-EGFP or SF-EGFP
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The transduction efficiency of the CAFC week 2 in MFG-EGFP/Am12
transduced CD34+ cells ranged between 23% and 30% with a
median value of 26%, and in SF-EGFP/PG13-transduced CD34+
cells the median value was 60% (46% to 74%) (Table 2). The
transduction efficiency of the CAFC week 6 in MFG-EGFP/Am12-transduced
CD34+ cells ranged between 0% and 11% with a median of
6% EFGP+ cobblestone areas. CAFC week 6 in
SF-EGFP/PG13-transduced CD34+ cells showed transduction as
high as 27%. CAFC week 6 in SF-EGFP/PG13-transduced CD34+CD38 cells showed a similar level
of 25% transduction efficiency. Notably, highly purified
CD34+CD38 cells transduced with the
amphotropic cell line did not produce EGFP+ cobblestone
areas week 6. These experiments clearly showed the superiority of
SF-EGFP/PG13 over MFG-EGFP/Am12 in transducing late appearing CAFC, in
concordance with the results obtained in phenotypically identified
immature CD34+ subsets.
Repopulation of transduced subsets in NOD/SCID mice.
In parallel with analysis of cobblestone formation, the ability of
transduced cells to reconstitute hematopoiesis in vivo was examined by
transplantation of the equivalent of 105 noncultured
CD34+ cells into sublethally irradiated NOD/SCID mice.
After 35 days the level of chimerism and the percentage of
EGFP+ cells in mouse BM were determined by flow cytometry
(Table 3). Similar levels of engraftment
were found in mice transplanted with noncultured or cultured
CD34+ cells. After transplantation of noncultured
CD34+ cells human cells were detected in all mice (n = 11)
(median, 54% [range, 6% to 64%] CD45+
cells). EGFP+ cells were found in 6 of 10 repopulated
chimeric mice transplanted with MFG-EGFP/Am12-transduced
CD34+ cells with a median percentage of EGFP+
cells of 2% (Table 3). CD34+ cells transduced using the
SF-EGFP/PG13 vector produced higher levels of EGFP+ cells
(median, 23%) in the human population in all four mice transplanted.
These data showed that the repopulating cells in the CD34+
population can be transduced effectively and produce EGFP+
progeny in transplanted NOD/SCID mice. In addition, SF-EGFP/PG13 was
much more efficient in transducing the repopulating cells than
MFG-EGFP/Am12.
Transplantation of noncultured CD34+CD38
cells and transduced CD34+CD38 resulted
in chimerism levels of median 10% (range, 6% to 29%) for the
noncultured cells and 5% (range, 1% to 24%) and 6% (range, 4% to
9%) for the MFG-EGFP/Am12- or SF-EGFP/PG13-transduced cells, respectively. In contrast to the results with purified
CD34+ cells, CD34+CD38 cells
transduced with MFG-EGFP/Am12 were not able to repopulate mouse BM with
EGFP-expressing cells, although all four mice engrafted with human
cells (Table 2); this parallels the absence of EGFP expressing CAFC
week 6 in CD34+CD38 cells transduced
with MFG-EGFP/Am12. Only one of three mice engrafted with
SF-EGFP/PG13-transduced CD34+CD38
cells. EGFP+ could only be detected in 3% of the
CD45+ cells produced. This is in contrast to the results
with the CD34+ cells in that apparently most repopulating
cells in the highly purified CD34+CD38
subset were not transduced efficiently or the transduced cells displayed a significant reduction in their engraftment potential compared with the cells that were not transduced during the procedure. Nevertheless, SF-EGFP/PG13 in these experiments was also apparently more efficient than MFG-EGFP/Am12.
Multilineage outgrowth of EGFP-transduced CD34+
cells.
The composition of the EGFP+ human cell population in two
mice was assessed by flow cytometry using a panel of lineage-specific markers (Fig 4). EGFP+ cells of
the myeloid lineage (CD33, range, 31% to 39%; CD11b, range, 20% to
25%; CD4, range, 30% to 45%), T-lymphoid (CD2, range, 20% to 22%),
B-lymphoid (CD20, range, 16% to 23%), and natural killer (NK) cells
(CD56, 1%) were found in mice transplanted with EGFP-transduced
CD34+ cells. Also, immature
EGFP+CD34+ cells were present in the mouse BM
(range, 1.1% to 6.8%) (Fig 5). Transduced
cells and chimeric mice BM were also cultured in standard
methylcellulose medium under conditions that selectively favor the
outgrowth of human monomyeloid and erythroid progenitors3 and fail to stimulate mouse progenitors. In both the graft and the
chimeric mice BM, EGFP+ GM-CFU (15 of 39 in the graft and 3 of 23 in the mouse BM) and BFU-E (23 of 40 in the graft and 5 of 25 in
the mouse BM) were identified by flow cytometry of isolated colonies or
fluorescence microscopy of whole cultures.
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| Fig 4.
Representative immunophenotyping of chimeric NOD/SCID
mouse BM 35 days after transplantation of MFG-EGFP/Am12 transduced,
IL-3-, IL-6-, SCF-stimulated CD34+ UCB cells. BM
(>10% CD45+) was stained with a panel of antibodies
specific against different human blood cell lineages and CD45 as a
marker for human cells. (A) The bright green autofluorescence on the x
axes versus CD45. The window represents all human CD45+
cells. The other dotplots shown are gated cells in this
CD45+ window representing only human cells.
Representative examples are shown for EGFP versus CD34 (B), EGFP versus
CD33 (C), EGFP versus CD11b, (D) EGFP versus CD2 (E), EGFP versus CD4
(F), EGFP versus CD20 (G), and EGFP versus CD56 (H).
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| Fig 5.
Representative chimerism and EGFP expression levels in
chimeric NOD/SCID mouse BM 35 days after transplantation of
nontransduced (A) and transduced (B) CD34+ UCB cells,
relative to the numbers of human (CD45+) cells found.
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| |
DISCUSSION |
The versatile use of EGFP as a selectable marker of retroviral-mediated
gene transfer in hematopoietic cells provides a basis to further
optimize retroviral gene transfer to human repopulating stem cells and
to evaluate the role of hematopoietic growth factors in activation and
expansion of immature hematopoietic cells. This study focused on the
development of optimal conditions for gene transfer to human
CD34+ and CD34+CD38 UCB
cells with the ability to reconstitute hematopoiesis in NOD/SCID mice
and produce cobblestone areas for prolonged periods in stroma-supported long-term cultures.
Comparison of transduction frequencies of immunophenotypically
characterized immature cells and those of SCID repopulating cells and
CAFC may both demonstrate the relationship of these cell types as well
as point to essential differences. In general, there was concordance
between these assays, in that the GaLV-pseudotyped retroviral vector
(SF-EGFP) transduction was much more efficient than the amphotropic
retroviral vector (MFG-EGFP) transduction. Also, transduction
frequencies of the immature CD34+CD38
subset within the CD34+ population related well to those
obtained after transplantation of NOD/SCID mice and CAFC week 6. In
addition, the study showed that repopulating cells in the highly
purified CD34+CD38 cells were resistant
to transduction in the absence of the CD38+ subset,
particularly notable for MFG-EGFP/Am12 as demonstrated by the finding
that the EGFP-transduced CD34+CD38
subset in general failed to produce EGFP+ progeny in
NOD/SCID mice. One mouse transplanted with SF-EGFP/PG13-transduced sorted CD34+CD38 cells displayed 3%
human EGFP+ cells, one order of magnitude less than the
frequency of EGFP+ CAFC week 6 in the same sample.
The more prominent transduction efficiency of the EGFP gene into
purified and highly purified immature UCB cells with the GaLV-pseudotyped SF-EGFP compared to the MFG-EGFP/Am12 retroviral packaging cell combination, is consistent with earlier studies where
transduction of human hematopoietic progenitors was more efficient with
a retroviral vector that uses the GaLV receptor.23-26 The
lower transduction percentage obtained with the amphotropic vector may
thus be primarily attributed to the low or absent expression of the
amphotropic envelope-receptor on the target cells.45,46 This was particularly corroborated by the absence of EGFP expression in
MFG-EGFP/Am12-transduced sorted
CD34+CD38 cells, both in the CAFC week 6 and after transplantation into NOD/SCID mice. Alternatively, UCB cells
may be more efficiently transduced by the SF-EGFP/PG13 vector/packaging
cell combination due to the use of the SFFV/MESV hybrid promoter, which
has been designed to overcome transcriptional inefficiency and
silencing associated with retroviral gene transfer into myeloid
progenitors and hematopoietic stem cells.47 Other variables
that obviously need to be further analyzed include differences in titer
and the ability and efficiency of the vectors to transduce EGFP in
hematopoietic cells. The titers of the two vectors used were
comparable, but tested in different assays. The colocalization of
vector and cells during transduction, using the CH-296 fibronectin
fragment,21 makes it unlikely that differences in titer did
heavily influence the results. This is even more so since preparative
experiments (not shown) with the MFG-EGFP/AM12 retroviral vector showed
that additional charges of the virus supernatant in the transduction protocol did not result in higher transduction frequencies, which indicated that the transduction system is sufficiently saturated with
virus. Also, Hanenberg et al48 concluded that the amount of
retroviral particles present in the supernatant was not a limiting factor for transduction of CD34+ BM cells on CH-296-coated
plates. The higher efficiency of the SF-EGFP/PG13 combination when
compared with the MFG-EGFP/AM12 combination should therefore not be
considered as being caused by supernatant virus titer differences.
The observation that repopulating cells in the CD34+
population can be transduced efficiently and produce transduced
multilineage progeny in transplanted NOD/SCID mice, whereas
repopulating cells in the highly purified
CD34+CD38 subset are either not
transduced effectively or do not develop in vivo, is of considerable
interest for elucidation of mechanisms involved in successful
transduction of immature hematopoietic cells. The transduction
efficiency of the CD34+CD38 tended to be
lower than that of the CD34+ cells,2 and was
significantly so for the MFG-EGFP/Am12 combination, which may be
related to the low or absent expression of the amphotropic receptor.
Because repopulating cells are exclusively present in the small
CD34+CD38 population, and
CD34+CD38+ cells do not effectively engraft,
the low levels of gene expression in the chimeric NOD/SCID BM after
transplantation of transduced CD34+CD38
cells may indicate that the growth factors used during prestimulation and virus infection were not sufficiently effective for activation and
stable virus integration of the NOD/SCID repopulating cells. The much
higher frequency of EGFP expressing cells in the BM of NOD/SCID mice
after transplantation of transduced stem cells from the less pure
CD34+ fraction may indicate that stimuli provided by
accessory CD34+ cells were responsible for the more
efficient transduction of repopulating
CD34+CD38- within the CD34+ cell
fraction. Alternatively, these accessory cells may be needed to
maintain the repopulating ability of stem cells during the transduction
procedure of 4 days, eg, by preventing differentiation, or to promote
the expansion and outgrowth of transduced stem cells after
transplantation. We speculate that these accessory cells are related to
the accessory CD34+CD38+ cells, which are
involved in the maintenance and expansion of CD34+CD38 cells in immunodeficient mice
transplanted with nontransduced human UCB subsets.3 Further
identification of these accessory CD34+ cells and
elucidation of the active principle may therefore be both relevant for
stem cell expansion physiology and for the design of successful gene
transfer strategies for immature hematopoietic cells.
The absolute numbers of CAFC produced after week 2 and week 6 of
culture show a modest increase after transduction with the MFG-EGFP or
SF-EGFP vectors. The frequency of EGFP+CAFC week 6 in
SF-EGFP- or MFG-EGFP-transduced CD34+ UCB cells was
similar to levels of EGFP+CD45+ cells found in
NOD/SCID mice. The reason for the 10-fold discrepancy between the
levels of transduction of the CAFC week 6 and the very low numbers of
EGFP+CD45+ in NOD/SCID BM after transplantation
of the SF-EGFP/PG13-transduced CD34+CD38 population is not clear.
Studies with the murine ADA vector similarly yielded very low numbers
of gene-marked human cells in the NOD/SCID mouse BM, in contrast to
higher numbers of transduced LTC-IC and colony-forming cells (CFC),
which was interpreted as evidence that the latter cell types are
functionally distinct from NOD/SCID repopulating cells.1
However, this distinction might be artificial if effectively transduced
CD34+CD38 require the described
CD34+ accessory cells for in vivo maintenance and expansion
but not for in vitro cobblestone area forming ability.
We conclude that retroviral-mediated EGFP transduction in UCB cells, in
combination with functional assays for repopulating cells, is a rapid
tool to study essential gene transfer variables such as vector tropism
and transduction conditions. In addition, the use of the
GaLV-pseudotyped retroviral vector SF-EGFP resulted in highly efficient
gene transfer in both late CAFC and NOD/SCID repopulating cells, the
latter presently the most immature subset of human
CD34+CD38 cells that can be approached
by a functional assay. These results justify the expectation that the
imminent analysis of variables promoting genetic marking of primitive,
transplantable hematopoietic cells, such as further optimized
transduction conditions and vector constructs, lead to protocols for
clinically relevant levels of therapeutic gene transfer.
| |
ACKNOWLEDGMENT |
The authors thank Dr A.Th. Alberda and staff of the St Franciscus
Hospital (Rotterdam, The Netherlands) for the collection of cord blood
samples used in this study. We thank Alexandra de Koning and Sandra van
Sluijs for excellent technical assistance, Joop Brandenburg for
breeding the immunodeficient mice, and Els van Bodegom for excellent
animal care.
| |
FOOTNOTES |
Submitted August 7, 1998;
accepted September 10, 1998.
P.B.v.H. and M.M.A.V. contributed equally to this manuscript.
Supported in part by grants of the Netherlands Organization for
Scientific Research NWO, the Netherlands Cancer Foundation Koningin
Wilhelmina Fonds, the Royal Netherlands Academy of Arts and Sciences,
contracts of the Commission of the European Communities, and Spanish
CICYT Grant No. SAF96-0130. J.A.C. is a recipient of a postdoctoral
grant from the Areces Fund, Spain.
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 Gerard Wagemaker, PhD, Institute of
Hematology, Room Ee1314, Erasmus University Rotterdam, Dr
Molewaterplein 50, PO Box 1738, 3000 DR Rotterdam, The Netherlands;
e-mail: wagemaker{at}hema.fgg.eur.nl.
| |
REFERENCES |
1.
Larochelle A, Vormoor J, Hanenberg H, Wang JC, Bhatia M, Lapidot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick JE:
Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.
Nat Med
2:1329, 1996[Medline]
[Order article via Infotrieve]
2.
Dao MA, Shah AJ, Crooks GM, Nolta JA:
Engraftment and retroviral marking of CD34+ and CD34+CD38 human hematopoietic progenitors assessed in immune-deficient mice.
Blood
91:1243, 1998[Abstract/Free Full Text]
3.
Verstegen MMA, Hennik van PB, Terpstra W, Bos van den C, Wielenga JJ, Rooijen van N, Ploemacher RE, Wagemaker G, Wognum AW:
Transplantation of human umbilical cord blood cells in macrophage-depleted SCID mice: Evidence for accessory cell involvement in expansion of immature CD34+CD38 cells.
Blood
91:1, 1998[Free Full Text]
4.
Shultz LD, Schweitzer PA, Christianson SW, Gott B, Schweitzer IB, Tennent B, McKenna S, Mobraaten L, Rajan TV, Greiner DL, Leiter EH:
Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.
J Immunol
154:180, 1995[Abstract]
5.
Breems DA, Blokland EA, Neben S, Ploemacher RE:
Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay.
Leukemia
8:1095, 1994[Medline]
[Order article via Infotrieve]
6.
Ploemacher RE, Van der Sluijs JP, Voerman JSA, Brons NHC:
An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse.
Blood
74:2755, 1989[Abstract/Free Full Text]
7.
Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp P:
Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro.
Blood
74:1563, 1989[Abstract/Free Full Text]
8.
Ploemacher RE, Van der Sluijs JP:
In vitro frequency analysis of spleen colony-forming and marrow-repopulating hemopoietic stem cells in the mouse.
J Tissue Cult Meth
13:63, 1991
9.
Breems DA, Van Hennik PB, Kusadasi N, Boudewijn A, Cornelissen JJ, Sonneveld P, Ploemacher RE:
Individual stem cell quality in leukapheresis products is related to the number of mobilized stem cells.
Blood
87:5370, 1996[Abstract/Free Full Text]
10.
Fruehauf S, Breems DA, Knaan-Shanzer S, Brouwer KB, Haas R, Lowenberg B, Nooter K, Ploemacher RE, Valerio D, Boesen JJB:
Frequency analysis of multidrug resistance-1 gene transfer into human primitive hematopoietic stem cells using the cobblestone area forming cell assay and detection of vector-mediated P-glycoprotein expression by rodamine-123.
Hum Gene Ther
7:1219, 1996[Medline]
[Order article via Infotrieve]
11.
Breems DA, Van Driel EM, Hawley RG, Siebel KE, Ploemacher RE:
Stroma-conditioned medium and sufficient prestimulation improve fibronectin fragment-mediated retroviral gene transfer into human primitive mobilized peripheral blood stem cells through effects on their recovery and transduction efficiency.
Leukemia
12:951, 1998[Medline]
[Order article via Infotrieve]
12.
Miller DG, Adam MA, Miller AD:
Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.
Mol Cell Biol
10:4239, 1990[Abstract/Free Full Text]
13.
Eaves CJ, Cashman JD, Kay RJ, Dougherty GJ, Otsuka T, Gaboury LA, Hogge DE, Lansdorp PM, Eaves AC, Humphries RK:
Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer.
Blood
78:110, 1991[Abstract/Free Full Text]
14.
Hao QL, Shah AJ, Thiemann FT, Smogorzewska EM, Crooks GM:
A functional comparison of CD34+CD38 cells in cord blood and bone marrow.
Blood
86:3745, 1995[Abstract/Free Full Text]
15.
Ponchio L, Conneally E, Eaves C:
Quantitation of the quiescent fraction of long-term culture-initiating cells in normal human blood and marrow and the kinetics of their growth factor-stimulated entry into S-phase in vitro.
Blood
86:3314, 1995[Abstract/Free Full Text]
16.
Dao MA, Hannum CH, Kohn DB, Nolta JA:
FLT3 ligand preserves the ability of human CD34+ progenitors to sustain long-term hematopoiesis in immune-deficient mice after ex vivo retroviral-mediated transduction.
Blood
89:446, 1997[Abstract/Free Full Text]
17.
Nolta JA, Crooks GM, Overell RW, Williams DE, Kohn DB:
Retroviral vector-mediated gene transfer into primitive human hematopoietic progenitor cells: Effects of mast cell growth factor (MGF) combined with other cytokines.
Exp Hematol
20:1065, 1992[Medline]
[Order article via Infotrieve]
18.
Nolta JA, Kohn DB:
Comparison of the effects of growth factors on retroviral vector-mediated gene transfer and the proliferative status of human hematopoietic progenitor cells.
Hum Gene Ther
1:257, 1990[Medline]
[Order article via Infotrieve]
19.
Tisdale J, Hanazono Y, Sellers S, Agricola B, Metzger M, Donahue R, Dunbar C:
Ex vivo expansion of genetically marked rhesus peripharal blood progenitor cells results in diminished long-term repopulating ability.
Blood
92:1131, 1998[Abstract/Free Full Text]
20.
Hanenberg H, Xiao XL, Dilloo D, Hashino K, Kato I, Williams DA:
Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells.
Nat Med
2:876, 1996[Medline]
[Order article via Infotrieve]
21.
Moritz T, Dutt P, Xiao X, Carstanjen D, Vik T, Hanenberg H, Williams DA:
Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments.
Blood
88:855, 1996[Abstract/Free Full Text]
22.
Kavanaugh MP, Miller DG, Zhang W, Law W, Kozak SL, Kabat D, Miller AD:
Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.
Proc Natl Acad Sci USA
91:7071, 1994[Abstract/Free Full Text]
23.
Kiem HP, Heyward S, Winkler A, Potter J, Allen JM, Miller AD, Andrews RG:
Gene transfer into marrow repopulating cells: comparison between amphotropic and gibbon ape leukemia virus pseudotyped retroviral vectors in a competitive repopulation assay in baboons.
Blood
90:4638, 1997[Abstract/Free Full Text]
24.
Bauer TJ, Miller AD, Hickstein DD:
Improved transfer of the leukocyte integrin CD18 subunit into hematopoietic cell lines by using retroviral vectors having a gibbon ape leukemia virus envelope.
Blood
86:2379, 1995[Abstract/Free Full Text]
25.
von Kalle C, Kiem HP, Goehle S, Darovsky B, Heimfeld S, Torok SB, Storb R, Schuening FG:
Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector.
Blood
84:2890, 1994[Abstract/Free Full Text]
26.
Bunnell BA, Muul LM, Donahue RE, Blaese RM, Morgan RA:
High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.
Proc Natl Acad Sci USA
92:7739, 1995[Abstract/Free Full Text]
27.
Glimm H, Kiem HP, Darovsky B, Storb R, Wolf J, Diehl V, Mertelsmann R, Von Kalle C:
Efficient gene transfer in primitive CD34+/CD38lo human bone marrow cells reselected after long-term exposure to GALV-pseudotyped retroviral vector.
Hum Gene Ther
8:2079, 1997[Medline]
[Order article via Infotrieve]
28.
Limon A, Briones J, Puig T, Carmona M, Fornas O, Cancelas JA, Nadal M, Garcia J, Rueda F, Barquinero J:
High-titer retroviral vectors containing the enhanced green fluorescent protein gene for efficient expression in hematopoietic cells.
Blood
90:3316, 1997[Abstract/Free Full Text]
29.
Conneally E, Eaves CJ, Humphries RK:
Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential.
Blood
91:3487, 1998[Abstract/Free Full Text]
30.
Brenner MK, Rill DR, Holladay MS, Heslop HE, Moen RC, Buschle M, Krance RA, Santana VM, Anderson WF, Ihle JN:
Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients.
Lancet
342:1134, 1993[Medline]
[Order article via Infotrieve]
31.
Brenner MK, Rill DR, Moen RC, Krance RA, Mirro JJ, Anderson WF, Ihle JN:
Gene-marking to trace origin of relapse after autologous bone-marrow transplantation.
Lancet
341:85, 1993[Medline]
[Order article via Infotrieve]
32.
Brenner MK:
The contribution of marker gene studies to hemopoietic stem cell therapies.
Stem Cells
13:453, 1995[Abstract]
33.
Strair RK, Towle M, Smith BR:
Retroviral mediated gene transfer into bone marrow progenitor cells: use of beta-galactosidase as a selectable marker.
Nucleic Acids Res
18:4759, 1990[Abstract/Free Full Text]
34.
Nolan GP, Fiering S, Nicolas JF, Herzenberg LA:
Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ.
Proc Natl Acad Sci USA
85:2603, 1988[Abstract/Free Full Text]
35.
Staal FJ, Bakker AQ, Verkuijlen M, van OE, Spits H:
Use of bicistronic retroviral vectors encoding the LacZ gene together with a gene of interest: A method to select producer cells and follow transduced target cells.
Cancer Gene Ther
3:345, 1996[Medline]
[Order article via Infotrieve]
36.
Staal FJ, Res PC, Weijer K, Spits H:
Development of retrovirally marked human T progenitor cells into mature thymocytes.
Int Immunol
7:1301, 1995[Abstract/Free Full Text]
37.
Zhang G, Gurtu V, Kain SR:
An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells.
Biochem Biophys Res Commun
227:707, 1996[Medline]
[Order article via Infotrieve]
38.
Bierhuizen MF, Westerman Y, Visser TP, Wognum AW, Wagemaker G:
Green fluorescent protein variants as markers of retroviral-mediated gene transfer in primary hematopoietic cells and cell lines.
Biochem Biophys Res Commun
234:371, 1997[Medline]
[Order article via Infotrieve]
39.
Bierhuizen MF, Westerman Y, Visser TP, Dimjati W, Wognum AW, Wagemaker G:
Enhanced green fluorescent protein as selectable marker of retroviral-mediated gene transfer in immature hematopoietic bone marrow cells.
Blood
90:3304, 1997[Abstract/Free Full Text]
40.
Schaefer UW, Schmidt CG, Dicke KA, van Bekkum DW, Schmitt G:
Cryopreservation of hemopoietic stem cells.
Z Krebsforsch Klin Onkol Cancer Res Clin Oncol
83:285, 1975[Medline]
[Order article via Infotrieve]
41.
Miltenyi S, Muller W, Weichel W, Radbruch A:
High gradient magnetic cell separation with MACS.
Cytometry
11:231, 1990[Medline]
[Order article via Infotrieve]
42.
Wagemaker G, Visser TP:
Erythropoietin-independent regeneration of erythroid progenitor cells following multiple injections of hydroxyurea.
Cell Tissue Kinet
13:505, 1980[Medline]
[Order article via Infotrieve]
43.
Merchav S, Wagemaker G:
Detection of murine bone marrow granulocyte/macrophage progenitor cells (GM-CFU) in serum-free cultures stimulated with purified M-CSF or GM-CSF.
Int J Cell Cloning
2:356, 1984[Abstract]
44.
Guilbert LJ, Iscove NN:
Partial replacement of serum by selenite, transferrin, albumin and lecithin in haemopoietic cell cultures.
Nature
263:594, 1976[Medline]
[Order article via Infotrieve]
45.
Crooks GM, Kohn DB:
Growth factors increase amphotropic retrovirus binding to human CD34+ bone marrow progenitor cells.
Blood
82:3290, 1993[Abstract/Free Full Text]
46.
Orlic D, Girard LJ, Jordan CT, Anderson SM, Cline AP, Bodine DM:
The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.
Proc Natl Acad Sci USA
93:11097, 1996[Abstract/Free Full Text]
47.
Baum C, Hegewisch BS, Eckert HG, Stocking C, Ostertag W:
Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells.
J Virol
69:7541, 1995[Abstract]
48.
Hanenberg H, Hashino K, Konishi H, Hock R, Kato I, Williams D:
Optimization of fibronectin-assisted retroviral genetransfer into human CD34+ hematopoietic cells.
Hum Gene Ther
8:2193, 1997[Medline]
[Order article via Infotrieve]
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October 1, 2000;
96(7):
2432 - 2439.
[Abstract]
[Full Text]
[PDF]
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D. Bryder and S. E. W. Jacobsen
Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro
Blood,
September 1, 2000;
96(5):
1748 - 1755.
[Abstract]
[Full Text]
[PDF]
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P. F. Kelly, J. Vandergriff, A. Nathwani, A. W. Nienhuis, and E. F. Vanin
Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein
Blood,
August 15, 2000;
96(4):
1206 - 1214.
[Abstract]
[Full Text]
[PDF]
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P. W. Zandstra, D. A. Lauffenburger, and C. J. Eaves
A ligand-receptor signaling threshold model of stem cell differentiation control: a biologically conserved mechanism applicable to hematopoiesis
Blood,
August 15, 2000;
96(4):
1215 - 1222.
[Abstract]
[Full Text]
[PDF]
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J. Barquinero, J. C. Segovia, M. Ramirez, A. Limon, G. Guenechea, T. Puig, J. Briones, J. Garcia, and J. A. Bueren
Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice
Blood,
May 15, 2000;
95(10):
3085 - 3093.
[Abstract]
[Full Text]
[PDF]
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B. Schiedlmeier, K. Kuhlcke, H. G. Eckert, C. Baum, W. J. Zeller, and S. Fruehauf
Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice
Blood,
February 15, 2000;
95(4):
1237 - 1248.
[Abstract]
[Full Text]
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D. A. Williams, A. W. Nienhuis, R. G. Hawley, and F. O. Smith
Gene Therapy 2000
Hematology,
January 1, 2000;
2000(1):
376 - 393.
[Abstract]
[Full Text]
[PDF]
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C. Dorrell, O. I. Gan, D. S. Pereira, R. G. Hawley, and J. E. Dick
Expansion of human cord blood CD34+CD38- cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function
Blood,
January 1, 2000;
95(1):
102 - 110.
[Abstract]
[Full Text]
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P. B. van Hennik, A. E. de Koning, and R. E. Ploemacher
Seeding Efficiency of Primitive Human Hematopoietic Cells in Nonobese Diabetic/Severe Combined Immune Deficiency Mice: Implications for Stem Cell Frequency Assessment
Blood,
November 1, 1999;
94(9):
3055 - 3061.
[Abstract]
[Full Text]
[PDF]
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E. C. MacNeill, H. Hanenberg, K. E. Pollok, J. C. M. van der Loo, M. F. A. Bierhuizen, G. Wagemaker, and D. A. Williams
Simultaneous Infection with Retroviruses Pseudotyped with Different Envelope Proteins Bypasses Viral Receptor Interference Associated with Colocalization of gp70 and Target Cells on Fibronectin CH-296
J. Virol.,
May 1, 1999;
73(5):
3960 - 3967.
[Abstract]
[Full Text]
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R. van Os, H. Avraham, N. Banu, P. M. Mauch, J. Whater, Y. Yang, and B. Du
Recombinant Adeno-Associated Virus-Based Vectors Provide Short-Term Rather Than Long-Term Transduction of Primitive Hematopoietic Stem Cells
Stem Cells,
March 1, 1999;
17(2):
117 - 120.
[Abstract]
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Top
Abstract
Introduction