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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3487-3493
Efficient Retroviral-Mediated Gene Transfer to Human Cord Blood Stem
Cells With In Vivo Repopulating Potential
By
E. Conneally,
C.J. Eaves, and
R.K. Humphries
From the Terry Fox Laboratory; BC Cancer Agency; and the Departments
of Pathology and Laboratory Medicine, Medical Genetics, and Medicine of
the University of British Columbia, Vancouver, BC, Canada.
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ABSTRACT |
Recent studies have shown efficient gene transfer to primitive
progenitors in human cord blood (CB) when the cells are incubated in
retrovirus-containing supernatants on fibronectin-coated dishes. We
have now used this approach to achieve efficient gene transfer to human
CB cells with the capacity to regenerate lymphoid and myeloid progeny
in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34+ cell-enriched populations
were first cultured for 3 days in serum-free medium containing
interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor,
Flt3-ligand, and Steel factor followed by two 24-hour incubations with
a MSCV-NEO virus-containing medium obtained under either
serum-free or serum-replete conditions. The presence of serum during
the latter 2 days made no consistent difference to the total number of
cells, colony-forming cells (CFC), or long-term culture-initiating
cells (LTC-IC) recovered at the end of the 5-day culture period, and
the cells infected under either condition regenerated similar numbers
of human CD34+ (myeloid) CFC and human
CD19+ (B lymphoid) cells for up to 20 weeks in NOD/SCID
recipients. However, the presence of serum increased the viral titer in
the producer cell-conditioned medium and this was correlated with a
twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% ± 3%
and 17% ± 8%, G418-resistant in vivo repopulating cells and LTC-IC
were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% ± 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively),
whereas values for LTC-IC and NOD/SCID repopulating cells were highly
and significantly correlated (r2 = 0.85). These findings
provide further evidence of a close relationship between human LTC-IC
and NOD/SCID repopulating cells (assessed using a  6-week CFC output
endpoint) and indicate the predictive value of gene transfer
measurements to such LTC-IC for the design of clinical gene therapy
protocols.
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INTRODUCTION |
TRANSDUCTION OF PLURIPOTENT hematopoietic
stem cells using recombinant retroviruses forms the basis of most
current strategies for the correction of single gene defects. Efficient
transfer of genes into murine hematopoietic stem cells with long-term
in vivo repopulating ability can now be routinely achieved using this
approach.1-4 Encouraging results have also been obtained with human progenitors detectable in vitro as colony-forming cells (CFC) and their more primitive precursors identified as long-term culture-initiating cells (LTC-IC).5-9 More recent findings
indicate the possibility of gene transfer to human hematopoietic cells capable of engrafting immune-deficient mice.10-12 However,
the application of this technology to clinical transplants has,
overall, yielded disappointing results with a few notable exceptions.
The latter include results obtained using bone marrow cells from
children undergoing hematopoietic recovery13 and a
preliminary report of improved gene transfer under conditions that may
favor maintenance of proliferating hematopoietic stem cells in
vitro.14,15
Our approach has focused on the identification of factors that rapidly
stimulate the proliferation of human cell populations that include
transplantable progenitors without loss of their original functional
potential. Recently, we showed that LTC-IC (defined using a 6-week CFC
output endpoint16) and cells able to regenerate human
lymphomyelopoiesis in sublethally irradiated nonobese diabetic
(NOD)/severe combined immunodeficiency (SCID) mice (referred to as
competitive repopulating units [CRU]) are similarly amplified in
short-term cultures of CD34+CD38lo human cord
blood (CB) cells stimulated by high concentrations of Flt3-ligand (FL),
Steel factor (SF), interleukin-3 (IL-3), IL-6, and granulocyte
colony-stimulating factor (G-CSF).17 In addition, we found
that LTC-IC and CRU in freshly isolated CB cells are similarly
distributed between the CD38+ and CD38- subsets
of the CD34+ CB population. These findings suggested a
close relationship between the cells identified by these two assays and
encouraged us to continue to use the LTC-IC assay to identify
conditions for optimizing retroviral-mediated gene transfer to CRU.
This allowed the development of a supernatant infection protocol that gives reproducibly high levels of retroviral-mediated gene transfer to
human CB CRU ( 30%), which is significantly correlated with the
levels of gene transfer obtained for coinfected 6-week LTC-IC, but not
for coinfected CFC.
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MATERIALS AND METHODS |
Human cells.
Samples of CB from normal, full-term infants delivered by cesarean
section were collected in heparin according to protocols approved by
the University of British Columbia Clinical Screening Committee for
Research Involving Human Subjects. This included obtaining informed
consent from the mother before delivery. A light density (<1.077
g/mL) cell fraction was first isolated by centrifugation of the CB
cells on ficoll-hypaque (Pharmacia, Uppsala, Sweden). These cells were
then either used directly or were further fractionated on a
StemSepÔ column (StemCell Technologies Inc, Vancouver, BC) to
isolate a CD34+ cell-enriched population (by removal of
cells expressing surface antigens characteristic of various mature
hematopoietic cells18), according to the manufacturer's
instructions. In some experiments, highly purified (>99.9%)
CD34+ or CD34+CD38lo cells were
isolated from these lin- cells by fluorescence-activated cell sorting (FACS), as described in detail elsewhere.17 In the remainder, the enriched CD34+ cells were used without
further purification. Surplus human bone marrow (BM) cells were
obtained with informed consent from normal adult donors of allogeneic
BM transplants or were cadaveric samples obtained from the Northwest
Tissue Centre (Seattle, WA). To generate marrow fibroblasts for the
infection experiments, fresh BM cells were first cultured for at least
5 weeks in Iscove's medium with 20% fetal calf serum (FCS; StemCell)
and then subcultured repeatedly until a pure fibroblast monolayer was
obtained.
Human cytokines.
Highly purified recombinant IL-3 and granulocyte-macrophage CSF
(GM-CSF) were gifts from Novartis (formerly Sandoz, Basel, Switzerland). IL-6 and SF were purified from media conditioned by COS
cells that had been transiently transfected in the Terry Fox Laboratory
with the corresponding human cDNAs. FL was a gift from Immunex Corp
(Seattle, WA) and purified human erythropoietin (Ep) and G-CSF were
kindly provided by StemCell.
Retroviral vector.
An MSCV-NEO virus19 constructed using the MSCV 2.1 vector
(kindly provided by Dr R. Hawley, University of Toronto, Toronto, Canada) was used to establish a GP-env AM12 MSCV-NEO producer cell line
as described.18 The titer of these producer cells was
107 colony-forming units/mL as assessed by the transfer of
G418 resistance to NIH-3T3 cells.20 The producer cells were
shown to be free of helper virus, as indicated by the inability to
recover infectious virus from MSCV-NEO-infected NIH-3T3 cells (capable
of transferring G418 resistance to a culture of naive NIH-3T3 cells).
Supernatants were collected from confluent cultures of MSCV-NEO
virus-producing cells after incubation of these overnight with fresh
Iscove's medium containing 20% FCS or bovine serum albumin, insulin,
and transferrin (BIT, StemCell) as indicated. The medium was then obtained, filtered through 0.4-mm filters, and stored frozen at  196°C, or was used directly.
CFC and LTC-IC assays.
Methylcellulose assays (all reagents from StemCell) were performed
essentially as previously described.16 After infection, some cells were plated in methylcellulose both with and without G418
(1.6 mg/mL, dry weight, GIBCO-BRL, Burlington, Canada). At these
concentrations of G418, no colony growth from uninfected cells was seen
in control groups (mock-infected cells) included in every experiment.
LTC-IC assays were performed also as described16 with
maintenance of the cultures at 37°C with weekly half-medium changes
for 6 weeks, at the end of which the nonadherent and adherent fractions
were obtained, pooled, and plated in methylcellulose with and without
G418 as indicated.
Assessment of mice transplanted with human cells.
NOD/LtSZ-scid/scid mice21 bred in the animal
facility at our institution were housed in microisolator cages and
given autoclaved food and water, acidified just before and after total
body irradiation (350 cGy). Human CB cells plus 106
irradiated (1,500 cGy) normal human BM cells as carrier cells were then
injected intravenously. Mice were maintained at least 6 weeks after
transplantation, at which time they were killed and the cellular
contents of both femurs and both tibias flushed out with HFN (Hanks'
buffered salt solution containing 2% fetal calf serum and 0.1% sodium
azide) and a single cell suspension obtained from each mouse. Aliquots
were stained as previously described17 with
anti-CD45-fluorescein isothiocyanate (FITC; HLe 1; Becton Dickinson,
Mountain View, CA) and anti-CD71-FITC (OKT9),22 anti-CD19-phycoerythrin (PE; Leu12; Becton
Dickinson) and anti-CD34-CY5 (8G12),23 or FITC-conjugated
and PE-conjugated mouse Ig as negative controls. Normal mouse BM cells
showed less than 0.1% nonspecific staining with these antibodies. In
animals containing both human lymphoid (CD19+) and human
CD34+ populations, the human CD34+ cells were
sorted and plated in CFC assays with and without G418 as described
above.
Polymerase chain reaction (PCR) analysis.
Colonies generated in CFC assays were plucked and analyzed individually
using the PCR and Southern blotting with a NEOr probe to
amplify and identify incorporated NEO-specific sequences as previously
described.24
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RESULTS |
Validation of the supernatant infection protocol.
In an initial series of experiments, the efficiency of infecting human
CB CFC and LTC-IC when the target cells were incubated with MSCV-NEO
virus-containing supernatants under various culture conditions was
compared with the levels of gene transfer obtained by cocultivation
with MSCV-NEO viral-producer cells. The conditions chosen were based on
previously reported findings that coincubation of the target cells on
fibronectin25,26 or fibroblasts5,27 could
improve the efficiency of gene transfer to primitive human hematopoietic cells. Either light density (106/mL) or
lin- (36% ± 6% CD34+, 105/mL)
CB cells were first incubated in the absence of virus for 48 hours in
Iscove's medium with 20% FCS and 20 ng/mL IL-3, 10 ng/mL IL-6, and 50 ng/mL SF. Aliquots of these prestimulated cells were then incubated in
the same culture volume for an additional 48 hours either in cell-free
virus-containing medium supplemented with the same cytokines in petri
dishes or in dishes that had been precoated with human full-length
fibronectin (Sigma, St Louis, MO) at a concentration of 5 µg/cm2, or on top of a monolayer of irradiated (1,500 cGy) allogeneic human marrow-derived fibroblasts, or in fresh medium
containing the same cytokines on top of a monolayer of irradiated (150 cGy) producer cells, as indicated. Polybrene was added to all media to
give a final concentration of 4 µg/mL. The cytokine-supplemented viral supernatants (and control media) were replaced halfway through the 48-hour infection period, at the end of which all nonadherent cells
were obtained, washed, and assessed for G418-resistant CFC and LTC-IC.
The results are summarized in Table 1.
Supernatant infection on fibronectin-coated plates gave similarly high
levels of gene transfer to LTC-IC, as were obtained by cocultivation (44% v 39%) and both conditions also gave a high level of
gene transfer to CFC. Supernatant infection in the absence of either fibronectin or human marrow fibroblasts produced very low levels of
gene transfer to any type of progenitor. The presence of human fibroblasts improved gene transfer efficiencies to CFC, but the gene
transfer efficiencies and recoveries of LTC-IC were reduced to levels
that precluded their assessment.
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Table 1.
Comparison of Transduction Efficiencies (% G418-Resistant Progenitors) Obtained Using Supernatant Infection Alone,
Supernatant With Fibronectin, Supernatant With Stromal Support or
Cocultivation
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Retention of CRU activity during infection.
A series of six experiments were then undertaken to determine how the
maintenance of CRU activity might be influenced by incubation of the
cells with a retroviral supernatant generated in medium containing 20%
FCS or medium supplemented with a defined serum substitute (BIT;
StemCell). At the time of starting these experiments, we had just
determined that FL in addition to SF, IL-3, and IL-6 (or G-CSF) is
important for achieving optimal expansion of LTC-IC and CFC in
short-term cultures of normal adult human BM,28 and that
this combination of cytokines would also support some expansion of CB
LTC-IC and CRU (fourfold and twofold, respectively), in 5- to 8-day
cultures.17 Therefore, the cytokines selected for use in
this next set of gene transfer experiments were changed from the
previous combination to FL and SF (100 ng/mL each) plus IL-3, IL-6, and
G-CSF (20 ng/mL each). To avoid the toxicity that polybrene had been
found to have on primitive cells29 (which we confirmed),
the polybrene was replaced with 5 µg/mL of protamine sulphate. In
addition, the period of prestimulation was extended from 48 to 72 hours. This latter change was based on our observations of single
CD34+CD38- CB cells, which showed that under
the conditions used, all viable cells would divide within 5 days, but
not before.17 Four of the experiments were set up with
lin- CB cells (at 105 cells/mL), one with
FACS-purified CD34+ CB cells (at 105 cells/mL),
and one with FACS-purified CD34+CD38lo CB cells
(at 104 cells/mL). The rest of the protocol was the same as
had been found to be optimal in the previous experiments, ie, the cells were prestimulated in cytokine-supplemented, serum-free medium followed
by 48 hours of infection on fibronectin-coated petri dishes with
replacement of the cytokine-supplemented viral supernatants (prepared
either in medium plus 20% FCS or serum-free plus BIT) after the first
24 hours. At the end of the second 24 hours of infection, the cells
were obtained and assayed for CFC, LTC-IC, and for their ability to
generate lymphoid and myeloid progeny after their transplantation into
sublethally irradiated NOD/SCID mice. The input cell type and numbers
and the number of resulting positive mice for human CFC and
CD19+ cells is shown in Table
2. Figure 1 shows a representative dot plot
of the relative numbers of total human cells and human
CD34+ cells present in the marrow of one of the mice
transplanted with human cells from these experiments. As shown in
Table 3, the presence or absence of serum
in the cultures from which the cells transplanted were obtained made no
consistent difference to any of the endpoints of human engraftment
assessed in mice up to 15 weeks posttransplant. In addition, there was
also no difference in the total numbers of cells, CFC, or LTC-IC
recovered from the two types of infection cultures (ie, viral
supernatants prepared in serum-free or serum-replete medium, data not
shown). The results from both procedures were therefore pooled to
derive mean (± standard error of mean [SEM]) yields of each
progenitor cell type at the end of the 5-day infection culture period
(per 105 input CD34+ cells) as follows: 3.4 ± 1.1 × 106 total cells, 1.4 ± 0.4 × 106 CFC, and 790 ± 290 LTC-IC (the results from the
experiment that was performed with CD34+CD38lo
cells was excluded from this analysis).
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| Fig 1.
Phenotypic analysis of BM cells derived from a NOD/SCID
mouse transplanted 20 weeks previously with the infected progeny of 3.5 × 104 FACS-purified CD34+ human CB cells.
In (A), the cells were stained with irrelevant isotype-matched mouse
IgG labelled with FITC and PE and the gates shown set to exclude 99.9%
of these cells. In (B), the cells were stained with a combination of
anti-CD45/71-FITC and anti-CD19-PE. In (C), the cells were stained
with anti-CD34-PE.
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Table 3.
Output of Human Myeloid CFC and CD19+
Cells From NOD/SCID Mice Engrafted With Cells Obtained From
Cultures of CB Cells That Contained SFM for the First 3 Days and
SFM or FCS Replete Medium for the Final 2 Days
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Gene transfer to human CB progenitors.
To assess gene transfer efficiencies in these latter experiments, the
proportion of G418-resistant CFC, or progeny CFC derived from LTC-IC or
(in vivo) from the CRU injected into the NOD/SCID mice was determined.
The results, shown in Fig 2, show average gene transfer efficiencies that range from a maximum of 68%
(burst-forming unit-erythroid [BFU-E] exposed to
FCS-containing supernatants) to a low of 8% (CRU exposed to
BIT-containing supernatants). However, for each progenitor type, there
was an approximately twofold to threefold higher proportion of
G418-resistant cells when these were infected with FCS-containing
supernatants, despite the fact that the total number of progenitors
present had not been affected. Assessment of the viral titer of the
supernatants prepared with FCS and BIT showed a threefold difference (6 × 106 in FCS v 2 × 106 in
BIT, n = 2). Thus, the most likely cause of the reduced gene transfer
obtained with the BIT-containing supernatants was simply their reduced
content of virus.
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| Fig 2.
Comparison of gene transfer efficiencies to different
types of human CB progenitors infected under serum-free versus
serum-replete conditions and assessed by measurement of
G418-resistance. Values represent the mean ± SEM.
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The results shown in Fig 2 also indicate a similar efficiency of gene
transfer to human CB CRU and LTC-IC under the best conditions (17% ± 3% and 17% ± 8%, respectively). When gene transfer
efficiencies to CRU, LTC-IC, and CFU-GM in individual experiments were
compared, the results for LTC-IC and CRU were significantly correlated
(r2 = 0.85, P < .01), whereas there was no
correlation between the corresponding gene transfer efficiencies to
LTC-IC or CRU and CFU-GM (r2 = 0.16 and 0.17, respectively, P > .05) (Fig 3).
Results for BFU-E and CFU-GEMM were similar to those shown for CFU-GM,
although the numbers of these were lower and hence the data less
reliable (data not shown).
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| Fig 3.
Correlation analysis of gene transfer efficiency to CRU
and LTC-IC (A) and to CRU and CFU-GM (B). Results shown include data from protocols in which either FCS (solid symbols) or BIT-containing (open symbols) viral supernatants were used. The average number of
colonies counted to calculate the efficiency of gene transfer to CFU-GM
ranged from 77 to 404 (mean = 154) and from 41 to 350 (mean = 123)
in the presence and absence of G418, respectively. For assessment of
LTC-IC, the numbers of colonies counted ranged from 12 to 115 (mean = 57) and from 1 to 85 (mean = 20) in the presence and absence of G418,
respectively.
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Although assessment of G418-resistant colony formation provides a
convenient method of quantitating gene transfer efficiency using
NEO-containing retroviruses, such estimates typically underestimate the
frequency of infected cells due to a variety of mechanisms that may
block or reduce expression of the integrated retroviral cDNA. In
addition, some cell types cannot be monitored this way. Therefore, to
further characterize the progeny of the infected CB cells that
engrafted the NOD/SCID mice, some of the human colonies generated in
vitro from the human CD34+ cells isolated from their
marrows (6 to 20 weeks posttransplant) were plucked and assessed
individually for the presence of the NEO gene by PCR. In addition, in
one experiment, highly purified human CD19+ (B lineage)
cells sorted from three mice were similarly analyzed. The human
lymphoid and human myeloid cells from all mice analyzed showed
integration of the NEO gene. Results from a representative experiment
are shown in Fig 4.
Table 4 shows a detailed comparison of the
estimates of gene transfer to the CRU obtained from the infected CB
cultures based on the G418 resistance of versus the presence of NEO
sequences in human CFC obtained from mice 6 to 20 weeks after they had
been injected with the infected human cells. Values determined by PCR
analysis were consistently approximately twofold to threefold higher,
suggesting that the actual efficiency of gene transfer to the
transplanted CRU was correspondingly higher (ie, 32% ± 10%).
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| Fig 4.
PCR detection of NEO sequences in cells obtained from
NOD/SCID recipients engrafted with infected human CB cells (Exp. 3 in Table 4). The upper panel shows results for total BM and isolated human
CD34+ and CD19+ populations. The lower
panel shows results for individual colonies derived from the sorted
human CD34+ cells plated with and without G418.
Densitometric analysis of the amount of DNA in each lane relative to
PCR of the actin gene done on the same colonies were as follows: lanes
1 to 16: 0.8, 0.7, 0.7, 0.4, 0.3, 0.5, 1, 0.4, 0.4, 0.9, 0.7, 0.6, 0.4, 0.4, 0.7, 0.7. Because the NEO signal in lane 1 is weaker than expected relative to the amount of actin present, we have not called this colony
positive.
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Table 4.
Comparisons of Gene Transfer Efficiencies Measured by
Assessing G418-Resistance and PCR Detection of the NEO Gene in
Individual Colonies for Human CFC Obtained From NOD/SCID Mice
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DISCUSSION |
The studies described in this report identify conditions that allow
human in vivo repopulating cells to be reproducibly infected by
recombinant retroviruses at high efficiency ( 30%) using a protocol
that should be readily adaptable to clinical applications. This
infection procedure builds on a series of previous important observations including the identification of a cytokine cocktail that
stimulates the expansion in vitro of human CB in vivo repopulating cells17 and recognition of the ability of
fibronectin-coated dishes to enhance gene transfer efficiencies using
cell-free viral supernatants.25,26 The toxicity that
polybrene has for primitive human hematopoietic cells29 was
circumvented by using protamine sulphate as a substitute. We also chose
to focus on human CB as a source of the hematopoietic stem cells to be
infected. This was based on previous work suggesting that these might
be more susceptible to retroviral infection,8,9 and that
they also regenerate larger numbers of lymphoid and myeloid progeny in
NOD/SCID mice and for more prolonged periods compared with the
repopulating cells present in normal adult human BM.30 The
adoption of a 5-day infection protocol (3 days of prestimulation plus 2 days of infection) was based on studies indicating that even under conditions of optimized cytokine stimulation, some human
CD34+CD38lo cells require this duration of
cytokine exposure before they will divide.17,31,32
To document gene transfer to human CB cells with in vivo repopulating
activity, sublethally irradiated NOD/SCID mice were injected with the
5-day progeny of relatively large numbers of input CD34+
cells, sufficient to obtain greater than 1% engraftment of human cells
in greater than 80% of the recipients (20 of 23). Evidence of the
expression and/or presence of the NEO gene in the human CFC and
CD19+ (B lineage) cells isolated from the bone marrow of
the engrafted mice 6 to 20 weeks posttransplant was used to infer the
presence of infected human CRU in the cells originally injected (ie, in vivo repopulating human stem cells with lymphomyeloid differentiation potential). We have previously shown that greater than 90% of NOD/SCID
mice transplanted with limiting numbers of human CB cells produce both
lymphoid and myeloid progeny, indicative of the origin of both of these
populations from a common stem cell.17 Thus, although
direct evidence of clonal populations containing both retrovirally
marked lymphoid and myeloid elements was not obtained in the present
experiments, our previous findings would suggest that the genetically
marked CFC and pre-B cells detected were generated from infected human
CB CRU. It should be noted that we made a conscious effort to
transplant nonlimiting numbers of infected CRU into the NOD/SCID mice
to minimize variability between recipients in the proportion of
regenerated human cells that would be genetically marked. This was then
tested by comparing the proportions of G418-resistant and NEO
sequence-positive human CFC demonstrable in individual mice injected
with aliquots of the same infected CB cell population. The level of
gene transfer achieved was sufficient to mark a readily detectable
proportion of the human CFC present in the 80% of mice where CFC were
regenerated, and the proportion of marked CFC was highly consistent
between all mice in a given set, regardless of the method used to
identify the marked cells. If the mice had been injected with only one
or two CRU, a larger proportion of mice in each experiment would have
been expected to not contain any human cells, and all of the human
cells in the engrafted mice would have been either marked or not, a
situation which, interestingly, fits the findings reported by
Larochelle et al.11
Efficient gene transfer to human in vivo repopulating cells present in
adult marrow10,33 and fetal liver12 has also
been reported recently by other groups. In two of these studies,
beige-nude-xid (bnx) mice were used as recipients. These mice allow
human myeloid and T-cell progeny to be generated, but not B-lineage
cells and overall seem to support much lower levels of human
hematopoiesis than NOD/SCID mice. Nevertheless, high level gene
transfer to human marrow cells able to engraft bnx mice was reported
for cells infected in the presence of stroma, and FL could partially
overcome this requirement for stromal cells, which our present studies confirm. In the studies of Yurasov et al,12 who used human
fetal liver cell targets, greater infectivity would be expected from their likely increased proliferative activity.34 Our
findings thus extend those recently reported by others highlighting the importance of using an infection protocol that optimizes stem cell
recovery, as well as infection efficiency. Moreover, the present
studies show that these requirements can be met under conditions that
are suitable for clinical application. In the future, the possibility
of adding other strategies to selectively isolate retrovirally-infected
human stem cells,18 as has been achieved with murine stem
cells35,36 or other types of human cells,37
should, with the gene transfer efficiencies now achievable, allow
useful numbers of viable 100% gene-modified human stem cell populations to be obtained.
Many groups have shown that LTC-IC from different sources can be
subdivided into biologically distinct subtypes according to the
longevity of their CFC-producing ability.38-40 In fact, this principle was first used to discriminate LTC-IC as a population distinct from CFC.38,41 It has also allowed murine cells
with short- versus long-term in vivo repopulating abilities to be
distinguished.42-45 Thus, some functional heterogeneity
among human cells detectable either as LTC-IC or as CRU likely exists,
consistent with their heterogeneity in CD38 expression.17
However, because human CB CRU are identified at a frequency that is
several hundred-fold lower than the frequency of CB LTC-IC, independent
of their phenotype,17 it is difficult to establish the
precise relationship of CRU and LTC-IC, particularly in the absence of
any independent information concerning the relative efficiencies of the
procedures used to detect them. Nevertheless, the highly significant
correlation shown here between the efficiency of gene transfer to
LTC-IC and CRU in human CB (both assessed using a 6-week endpoint)
provides further evidence of a close relationship between these two
populations and also serves to emphasize the predictive value of
measuring gene transfer to such LTC-IC as a prelude to more ambitious
and labor-intensive in vivo experiments.
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FOOTNOTES |
Submitted August 4, 1997;
accepted December 15, 1997.
Supported by Grant No. HL55435 from the National Institutes of Health
(Bethesda, MD), the National Cancer Institute of Canada (NCIC)
(Toronto, Ontario, Canada) with funds from the Terry Fox Run, the
Medical Research Council of Canada (Ottawa, Ontario, Canada), and
Novartis (Basel, Switzerland). E.C. held a Terry Fox Physician
Scientist Fellowship and C.J.E. is a Terry Fox Cancer Research
Scientist of the NCIC.
Address reprint requests to R.K. Humphries, MD, PhD, Terry Fox
Laboratory, 601 W 10th Ave, Vancouver, BC V5Z 1L3, Canada.
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.
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ACKNOWLEDGMENT |
The authors thank Dr R. Hawley (University of Toronto); Dr P. Lansdorp
(Terry Fox Laboratory, Vancouver, BC); and StemCell and Novartis for
valuable gifts of vectors, antibodies, and other reagents. The expert
technical help of Margaret Hale, Gayle Thornbury, Jessyca Maltman, and
Maya Sinclaire, and the secretarial assistance of Bernadine Fox are
also acknowledged.
| |
REFERENCES |
1.
Williams DA,
Lemischka IR,
Nathan DG,
Mulligan RC:
Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse.
Nature
310:476,
1984[Medline]
[Order article via Infotrieve]
2.
Szilvassy SJ,
Fraser CC,
Eaves CJ,
Lansdorp PM,
Eaves AC,
Humphries RK:
Retrovirus-mediated gene transfer to purified hemopoietic stem cells with long-term lympho-myelopoietic repopulating ability.
Proc Natl Acad Sci USA
86:8798,
1989[Abstract/Free Full Text]
3.
Sorrentino BP,
Brandt SJ,
Bodine D,
Gottesman M,
Pastan I,
Cline A,
Nienhuis AW:
Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1.
Science
257:99,
1992[Abstract/Free Full Text]
4.
Einerhand MPW,
Bakx TA,
Kukler A,
Valerio D:
Factors affecting the transduction of pluripotent hematopoietic stem cells: Long-term expression of a human adenosine deaminase gene in mice.
Blood
81:254,
1993[Abstract/Free Full Text]
5.
Moore KA,
Deisseroth AB,
Reading CL,
Williams DE,
Belmont JW:
Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells.
Blood
79:1393,
1992[Abstract/Free Full Text]
6.
Hughes PFD,
Thacker JD,
Hogge D,
Sutherland HJ,
Thomas TE,
Lansdorp PM,
Eaves CJ,
Humphries RK:
Retroviral gene transfer to primitive normal and leukemic hematopoietic cells using clinically applicable procedures.
J Clin Invest
89:1817,
1992
7.
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]
8.
Moritz T,
Keller DC,
Williams DA:
Human cord blood cells as targets for gene transfer: Potential use in genetic therapies of severe combined immunodeficiency disease.
J Exp Med
178:529,
1993[Abstract/Free Full Text]
9.
Lu L,
Xiao M,
Clapp DW,
Li Z-H,
Broxmeyer HE:
High efficiency retroviral mediated gene transduction into single isolated immature and replatable CD343+ hematopoietic stem/progenitor cells from human umbilical cord blood.
J Exp Med
178:2089,
1993[Abstract/Free Full Text]
10.
Nolta JA,
Dao MA,
Wells S,
Smogorzewska M,
Kohn DB:
Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.
Proc Natl Acad Sci USA
93:2414,
1996[Abstract/Free Full Text]
11.
Larochelle A,
Vormoor J,
Hanenberg H,
Wang JCY,
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.
Nature Med
2:1329,
1996[Medline]
[Order article via Infotrieve]
12.
Yurasov S,
Kollmann TR,
Kim A,
Raker CA,
Hachamovitch M,
Wong-Staal F,
Goldstein H:
Severe combined immunodeficiency mice engrafted with human T cells, B cells, and myeloid cells after transplantation with human fetal bone marrow or liver cells and implanted with human fetal thymus: A model for studying human gene therapy.
Blood
89:1800,
1997[Abstract/Free Full Text]
13.
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]
14.
Stewart AK,
Dube ID,
Kamel-Reid S,
Keating A:
Clinical protocol: A phase I study of autologous bone marrow transplantation with stem cell gene marking in multiple myeloma.
Hum Gene Ther
6:107,
1995[Medline]
[Order article via Infotrieve]
15. (abstr, suppl 1)
Stewart AK,
Nanji S,
Ruedy C,
Singaraja R,
Nayar R,
Peck B,
Sutherland DR,
McGarry G,
Dube ID:
Engraftment of gene marked long term marrow culture (LTMC) cells in myeloma patients.
Blood
88:270a,
1996
16.
Hogge DE,
Lansdorp PM,
Reid D,
Gerhard B,
Eaves CJ:
Enhanced detection, maintenance and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human Steel factor, interleukin-3 and granulocyte colony-stimulating factor.
Blood
88:3765,
1996[Abstract/Free Full Text]
17.
Conneally E,
Cashman J,
Petzer A,
Eaves C:
Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice.
Proc Natl Acad Sci USA
94:9836,
1997[Abstract/Free Full Text]
18.
Conneally E,
Bardy P,
Eaves CJ,
Thomas T,
Chappel S,
Shpall EJ,
Humphries RK:
Rapid and efficient selection of human hematopoietic cells expressing murine heat-stable antigen as an indicator of retroviral-mediated gene transfer.
Blood
87:456,
1996[Abstract/Free Full Text]
19.
Hawley RG,
Lieu FHL,
Fong AZC,
Hawley TS:
Versatile retroviral vectors for potential use in gene therapy.
Gene Ther
1:136,
1994[Medline]
[Order article via Infotrieve]
20.
Cone RD,
Mulligan RC:
High-efficiency gene transfer into mammalian cells: Generation of helper-free recombinant retrovirus with broad mammalian host range.
Proc Natl Acad Sci USA
81:6349,
1984[Abstract/Free Full Text]
21.
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.
Immunology
154:180,
1995
22.
Sutherland R,
Delia D,
Schneider C,
Newman R,
Kemshead J,
Greaves M:
Ubiquitous cell-surface glycoprotein on tumor cells is proliferation-associated receptor for transferrin.
Proc Natl Acad Sci USA
78:4515,
1981[Abstract/Free Full Text]
23. Lansdorp PM, Dougherty GJ, Humphries RK: CD34 epitopes, in Knapp
W, Dorken B, Gilks WR, Rieber EP, Schmidt RE, Stein H, von dem Borne
AEG (eds): Leucocyte Typing IV. White Cell Differentiation Antigens.
New York, NY, Oxford, 1989, p 826
24.
Hughes PFD,
Eaves CJ,
Hogge DE,
Humphries RK:
High-efficiency gene transfer to human hematopoietic cells maintained in long-term marrow culture.
Blood
74:1915,
1989[Abstract/Free Full Text]
25.
Moritz T,
Patel VP,
Williams DA:
Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors.
J Clin Invest
93:1451,
1994
26.
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.
Nature Med
2:876,
1996[Medline]
[Order article via Infotrieve]
27.
Nolta JA,
Smogorzewska EM,
Kohn DB:
Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells.
Blood
86:101,
1995[Abstract/Free Full Text]
28.
Petzer AL,
Zandstra PW,
Piret JM,
Eaves CJ:
Differential cytokine effects on primitive (CD34+CD38-) human hematopoietic cells: Novel responses to flt3-ligand and thrombopoietin.
J Exp Med
183:2551,
1996[Abstract/Free Full Text]
29.
Flasshove M,
Banerjee D,
Mineishi S,
Li ML,
Bertino JR,
Moore MAS:
Ex vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer.
Blood
85:566,
1995[Abstract/Free Full Text]
30.
Cashman J,
Bockhold K,
Hogge DE,
Eaves AC,
Eaves CJ:
Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haematopoietic cells in NOD/SCID mice transplanted with human cord blood.
Br J Haematol
98:1026,
1997[Medline]
[Order article via Infotrieve]
31.
Petzer AL,
Hogge DE,
Lansdorp PM,
Reid DS,
Eaves CJ:
Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.
Proc Natl Acad Sci USA
93:1470,
1996[Abstract/Free Full Text]
32.
Jordan CT,
Yamasaki G,
Minamoto D:
High-resolution cell cycle analysis of defined phenotypic subsets within primitive human hematopoietic cell populations.
Exp Hematol
24:1347,
1996[Medline]
[Order article via Infotrieve]
33.
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]
34.
Fleming WH,
Alpern EJ,
Uchida N,
Ikuta K,
Spangrude GJ,
Weissman IL:
Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells.
J Cell Biol
122:897,
1993[Abstract/Free Full Text]
35.
Richardson C,
Bank A:
Preselection of transduced murine hematopoietic stem cell populations leads to increased long-term stability and expression of the human multiple drug resistance gene.
Blood
86:2579,
1995[Abstract/Free Full Text]
36.
Pawliuk R,
Eaves CJ,
Humphries RK:
Sustained high-level reconstitution of the hematopoietic system by preselected hematopoietic cells expressing a transduced cell-surface antigen.
Hum Gene Ther
8:1595,
1997[Medline]
[Order article via Infotrieve]
37.
Mavilio F,
Ferrari G,
Rossini S,
Nobili N,
Bonini C,
Casorati G,
Traversari C,
Bordignon C:
Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer.
Blood
83:1988,
1994[Abstract/Free Full Text]
38.
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]
39.
Prosper F,
Stroncek D,
Verfaillie CM:
Phenotypic and functional characterization of long-term culture-initiating cells present in peripheral blood progenitor collections of normal donors treated with granulocyte colony-stimulating factor.
Blood
88:2033,
1996[Abstract/Free Full Text]
40.
Hao QL,
Thiemann FT,
Petersen D,
Smogorzewska EM,
Crooks GM:
Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population.
Blood
88:3306,
1996[Abstract/Free Full Text]
41.
Sutherland HJ,
Eaves CJ,
Eaves AC,
Dragowska W,
Lansdorp PM:
Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro.
Blood
74:1563,
1989[Abstract/Free Full Text]
42.
Magli MC,
Iscove NN,
Odartchenko N:
Transient nature of early haematopoietic spleen colonies.
Nature
295:527,
1982[Medline]
[Order article via Infotrieve]
43.
Ploemacher RE,
Brons RHC:
Separation of CFU-S from primitive cells responsible for reconstitution of the bone marrow hemopoietic stem cell compartment following irradiation: Evidence for a pre-CFU-S cell.
Exp Hematol
17:263,
1989[Medline]
[Order article via Infotrieve]
44.
Jordan CT,
Lemischka IR:
Clonal and systemic analysis of long-term hematopoiesis in the mouse.
Genes Dev
4:220,
1990[Abstract/Free Full Text]
45.
Miller CL,
Rebel VI,
Helgason CD,
Lansdorp PM,
Eaves CJ:
Impaired steel factor responsiveness differentially affects the detection and longterm maintenance of fetal liver hematopoietic stem cells in vivo.
Blood
89:1214,
1997[Abstract/Free Full Text]
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[Abstract]
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|
|
|
|
|
|
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[Full Text]
[PDF]
|
|
|
|
|
|
|
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|
|
|
|
|
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|
|
|
|
|
|
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[Abstract]
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|
|
|
|
|
|
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|
|
|
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|
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[Abstract]
[Full Text]
[PDF]
|
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|
|
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J. Exp. Med.,
May 17, 1999;
189(10):
1601 - 1610.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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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
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73(5):
3960 - 3967.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
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Differential Kinetics of Primitive Hematopoietic Cells Assayed In Vitro and In Vivo During Serum-Free Suspension Culture of CD34+ Blood Progenitor Cells
Stem Cells,
May 1, 1999;
17(3):
152 - 161.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
V. I. Rebel, M. Tanaka, J.-S. Lee, S. Hartnett, M. Pulsipher, D. G. Nathan, R. C. Mulligan, and C. A. Sieff
One-Day Ex Vivo Culture Allows Effective Gene Transfer Into Human Nonobese Diabetic/Severe Combined Immune-Deficient Repopulating Cells Using High-Titer Vesicular Stomatitis Virus G Protein Pseudotyped Retrovirus
Blood,
April 1, 1999;
93(7):
2217 - 2224.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Guenechea, J.C. Segovia, B. Albella, M. Lamana, M. Ramirez, C. Regidor, M.N. Fernandez, and J.A. Bueren
Delayed Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice Transplanted With Ex Vivo-Expanded Human CD34+ Cord Blood Cells
Blood,
February 1, 1999;
93(3):
1097 - 1105.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Miyoshi, K. A. Smith, D. E. Mosier, I. M. Verma, and B. E. Torbett
Transduction of Human CD34+ Cells That Mediate Long-Term Engraftment of NOD/SCID Mice by HIV Vectors
Science,
January 29, 1999;
283(5402):
682 - 686.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. D. Cashman and C. J. Eaves
Human Growth Factor-Enhanced Regeneration of Transplantable Human Hematopoietic Stem Cells in Nonobese Diabetic/Severe Combined Immunodeficient Mice
Blood,
January 15, 1999;
93(2):
481 - 487.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. B. van Hennik, M. M.A. Verstegen, M. F.A. Bierhuizen, A. Limon, A. W. Wognum, J. A. Cancelas, J. Barquinero, R. E. Ploemacher, and G. Wagemaker
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
Blood,
December 1, 1998;
92(11):
4013 - 4022.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. E. Frankel, M. Lilly, R. Kreitman, D. Hogge, M. Beran, M. H. Freedman, P. D. Emanuel, C. McLain, P. Hall, E. Tagge, et al.
Diphtheria Toxin Fused to Granulocyte-Macrophage Colony-Stimulating Factor Is Toxic to Blasts From Patients With Juvenile Myelomonocytic Leukemia and Chronic Myelomonocytic Leukemia
Blood,
December 1, 1998;
92(11):
4279 - 4286.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract