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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1131-1141
RAPID COMMUNICATION
Ex Vivo Expansion of Genetically Marked Rhesus Peripheral Blood
Progenitor Cells Results in Diminished Long-Term Repopulating Ability
By
J.F. Tisdale,
Y. Hanazono,
S.E. Sellers,
B.A. Agricola,
M.E. Metzger,
R.E. Donahue, and
C.E. Dunbar
From the Hematology Branch, National Heart, Lung, and Blood
Institute, Bethesda, MD.
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ABSTRACT |
The possibility of primitive hematopoietic cell ex vivo expansion is
of interest for both gene therapy and transplantation applications. The
engraftment of autologous rhesus peripheral blood (PB) progenitors
expanded 10 to 14 days were tracked in vivo using genetic marking. Stem
cell factor (SCF)/granulocyte colony-stimulating factor
(G-CSF)-mobilized and CD34-enriched PB cells were divided
into two equal aliquots and transduced with one of two retroviral
vectors carrying the neomycin-resistance gene (neo) for 4 days
in the presence of interleukin-3 (IL-3), IL-6, and SCF in the first 5 animals, IL-3/IL-6/SCF/Flt-3 ligand (FLT) in 2 subsequent
animals, or IL-3/IL-6/SCF/FLT plus an autologous stromal monolayer
(STR) in the final 2. At the end of transduction period, one aliquot
(nonexpanded) from each animal was frozen, whereas the other was
expanded under the same conditions but without vector for a total of 14 days before freezing. After total body irradiation, both the
nonexpanded and expanded transduced cells were reinfused. Despite 5- to
13-fold higher cell and colony-forming unit (CFU) doses
from the expanded fraction of marked cells, there was greater short-
and long-term marking from the nonexpanded cells in all animals. In
animals receiving cells transduced and expanded in the presence of
IL-3/IL-6/SCF/FLT, engraftment by the marked expanded cells was further
diminished. This discrepancy was even more pronounced in the animals
who received cells transduced and expanded in the presence of FLT and
autologous stroma, with no marking detectable from the expanded cells.
Despite lack of evidence for expansion of engrafting cells, we found
that the addition of FLT and especially STR during the initial brief
transduction period increased engraftment with marked cells into a
clinically relevant range. Levels of marked progeny cells originating
from the nonexpanded aliqouts were significantly higher than that seen in previous 4 animals receiving cells transduced in the presence of
IL-3/IL-6/SCF, with levels of 10% to 20% confirmed by Southern blotting from the nonexpanded IL-3/IL-6/SCF/FLT/STR graft compared with
0.01% in the original IL-3/IL-6/SCF cohort. These results suggest
that, although expansion of PB progenitors is feasible ex vivo, their
contribution towards both short- and long-term engraftment is markedly
impaired. However, a brief transduction in the presence of specific
cytokines and stromal support allows engraftment with an encouraging
number of retrovirally modified cells.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
PRIMITIVE HEMATOPOIETIC stem cells (HSCs)
are particularly suitable as targets for genetic manipulation based on
the ease of their collection and the potential for their manipulation
ex vivo. The use of integrating vectors, such as retroviruses, permits the passage of transferred genetic material to all progeny cells, potentially allowing definitive treatment of a wide variety of congenital and acquired diseases, including hemoglobinopathies, immunodeficiencies, metabolic storage disorders, and cancer. However, early preclinical and clinical studies using retroviral vectors to
transduce human and nonhuman primate hematopoietic progenitor and stem
cells have resulted in levels of engraftment with genetically altered
cells too low to hope for clinical benefit in most
disorders.1
Ex vivo expansion of retrovirally transduced HSCs could theoretically
increase the overall number of gene-corrected cells infused and
therefore improve competition against endogenous nontransduced stem
cells. More importantly, even if transduction of stem cells is
inefficient, expansion of these cells after positive selection for a
retrovirally encoded marker gene such as a cell surface protein might
result in a safe transplantation dose of almost pure genetically
modified cells. This approach has shown promise in
vitro.2,3 In murine models, transplantation of positively selected cells has resulted in high-level engraftment with
vector-containing cells, but ex vivo expansion postselection has not
been explored.4,5 Given the requirement for cell division
with current vector systems, strategies effective in expanding
engrafting cells ex vivo might also improve transduction
efficiency.6
The use of ex vivo-expanded hematopoietic cells to improve the safety
and efficacy of autologous and allogeneic transplantation is another
application that has generated intense interest and experimentation
over the past 5 years.7,8 Ex vivo expanded or activated
progenitor and stem cells might result in more rapid recovery after
autologous transplantation, allow cells from a single short apheresis
procedure or outpatient bone marrow (BM) harvest to support a
transplantation procedure, increase applicability of cord blood
transplantation to larger recipients, and allow more effective tumor
cell purging if expanded cell populations could reliably provide
long-term or even short-term engraftment.
In mice, ex vivo culture of marrow cells for 7 days in the presence of
multiple hematopoietic growth factors accelerates hematopoietic recovery and increases radioprotection.9 Successful
secondary transplantation of cells from the primary recipients suggests that the long-term proliferative potential of murine stem cells is not
lost during ex vivo culture.10 However, when cultured cells
are competed against fresh cells, a significant engraftment defect was
observed.11,12 Many groups have reported ex vivo expansion
of human BM or peripheral blood (PB) cells using combinations of
hematopoietic growth factors or stromal support systems, with up to
several log expansion of both total cell number and colony-forming units (CFU).13-15 Flow cytometric analysis at
the end of the culture period has shown the majority of the
amplification occurring via terminal differentiation. However,
quantitation of more primitive stem cell surrogates such as long-term
culture-initiating cells (LTCIC) suggest that these cells can
be maintained or expanded under some conditions.16,17
There have been several human clinical trials designed to document the
safety and feasibility of infusing expanded progenitor cells in the
setting of high-dose chemotherapy. Rapid hematopoietic recovery has
been reported after administration of PB CD34-enriched cells expanded
for 10 days. Long-term repopulating ability could not be definitively
assessed in this study, because fully myeloablative conditioning
treatment was not used.18 More worrisome was the report
that several patients did not engraft after receiving expanded PB cells
in the setting of full
myeloablation.19
To more clearly assess the engrafting ability of ex vivo-expanded
hematopoietic cells in a model with relevance to human gene therapy and
transplantation, we used retroviral gene marking to track the effect of
ex vivo expansion on the engraftment of mobilized rhesus monkey PB
cells. Our studies indicate that ex vivo expansion of transduced
CD34-enriched PB cells in the presence of interleukin-3 (IL-3), IL-6,
and stem cell factor (SCF), with or without flt 3 ligand (FLT) or
autologous stromal cells (STR), does not increase short-term or
long-term engraftment as compared with transduced but nonexpanded cells
and diminishes or at best maintains long-term repopulating ability. Our
data does not support any contribution to engraftment, even
transiently, by committed progenitors such as colony-forming
unit-granulocyte-macrophage (CFU-GM). However, a brief ex
vivo culture in the presence of flt3 ligand and stromal cells in
addition to IL-3, IL-6, and SCF resulted in transduction of
repopulating cells at levels of 10% to 20%, a clinically relevant range.
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MATERIALS AND METHODS |
PB progenitor cell mobilization and harvesting.
Young rhesus macaques (Macaca mulatta) were housed and handled in
accordance with the guidelines set forth by the Committee on Care and
Use of Laboratory Animals of the Institute of Laboratory Animal
Resources, National Research Council (DHHS Publication No. NIH 85-23),
and the protocol was approved by the Animal Care and Use Committee of
the National Heart and Lung and Blood Institute. The animals received
recombinant pegylated human SCF (200 µg/kg; Amgen, Thousand Oaks, CA)
and recombinant human granulocyte colony-stimulating factor (G-CSF; 10 µg/kg; Amgen) subcutaneously daily for 5 days, followed by apheresis
of 2.5 times the blood volume on day 6.20 The mononuclear
fraction was purified by density gradient centrifugation over
lymphocyte separation media (LSM; Organon Teknika, Durham, NC).
Enrichment for primitive progenitor and stem cells was performed using
the Ceprate LC CD34 immunoabsorption column as directed (Cellpro,
Bothell, WA). The degree of progenitor enrichment was calculated from
CFU-GM assays performed before and after column purification.
Ex vivo transduction and expansion.
The producer cell lines G1Na and LNL6 were grown to confluence in
Dulbecco's modified Eagle's medium (DMEM; Mediatech, Herndan, VA)
supplemented with 10% fetal calf serum (FCS; GIBCO/BRL, Gaithersburg MD).21,22 The biologic titer of these supernatants assayed by transfer of G418-resistance to NIH 3T3 cells ranged from 1 to 5 × 106 cfu/mL. Undiluted vector supernatant was
collected and passed through a 0.4-µm filter before transduction of
target CD34-enriched cells. For each animal, CD34-enriched PB cells
were divided into two equal fractions and each was transduced under
identical conditions with one of the two vectors. Transductions were
performed as previously described for a total of 96 hours at a starting
concentration of 1 × 105 cells/mL with daily
replacement of vector supernatant and cytokines.23 All
transduction cultures were supplemented with 4 µg/mL protamine sulfate (Sigma, St Louis, MO), 20 ng/mL recombinant human
IL-3 (Sandoz, East Hanover, NJ), 50 ng/mL recombinant human IL-6
(Sandoz), and 100 ng/mL recombinant human SCF (Amgen). For 2 animals,
100 ng/mL recombinant human FLT (Research Diagnostics, Flanders, NJ) was also used in the transduction and expansion cultures. An additional 2 animals had their cells cocultured on a preformed irradiated autologous marrow stromal layer (STR) along with all four cytokines. This stromal layer was produced by culturing mononuclear cells from a
10-mL BM aspirate for 2 days in DMEM plus 10% FCS and then removing
nonadherent cells by vigorous washing with phosphate-buffered saline
(PBS) and continuing to culture the adherent cells until a confluent
monolayer formed (~14 days of culture). Before adding CD34-enriched
PB cells, the stromal flask was irradiated with 1,500 cGy.
At the conclusion of the 96-hour transduction period, all the cells
transduced with one vector (the nonexpanded fraction) from each animal
were harvested by vigorous washing with PBS, followed by trypsinization
to remove any adherent cells, and cryopreserved in DMEM, 50%
autologous serum, and 10% dimethyl sulfoxide (DMSO; Sigma) using a
controlled rate freezer. The other fraction (expanded) from each animal
was replated in fresh DMEM, 10% FCS, and cytokines and returned to
culture in the same flask, without further exposure to viral
supernatant, under the identical culture conditions used during the
transduction. Media and cytokines were replenished at day 7 of the
culture, and the total volume of the culture was doubled at that time.
On day 10 or 14, the expanded cells were harvested and cryopreserved as
described above.
Autologous transplantation.
The animals received 650 cGy total body  -irradiation daily for 2 days. The following day, both the nonexpanded and expanded fractions
were thawed in a 37°C water bath and infused. Recombinant human
G-CSF (Amgen) was administered at a dose of 5 µg/kg/d as a continuous
infusion until the white blood cell count (WBC) reached 6,000/µL.
Standard supportive care was administered
posttransplantation.24
Analysis of transduction and expansion.
Total cell number and colony-forming units (CFU-GM, CFU-G, CFU-M, and
burst-forming unit-erythroid [BFU-E]) were determined for the mononuclear cells precolumn immunoabsorption, for the CD34-enriched cells postcolumn, at the end of the 96-hour transduction period for both fractions, at the end of the expansion period for the
expanded fraction, and postthaw for both nonexpanded and expanded
fractions. CFU assays were performed using methylcellulose media
(StemCell Technologies, Vancouver, British Columbia, Canada) supplemented with 5 U/mL recombinant human erythropoietin (Epo; Amgen),
10 ng/mL IL-3 (Sandoz), 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Sandoz), and 100 ng/mL SCF (Amgen).
At day 14, colonies containing greater than 50 cells were counted, and
24 individual colonies from each fraction were plucked into 50 µL of
distilled water, digested with 20 mg/mL proteinase K at 55°C for 1 hour followed by 99°C for 10 minutes, and assessed for vector
neo sequences by polymerase chain reaction (PCR) as described
below. Simultaneous PCR for  -actin sequences was performed on each
plucked colony and the percentage of transduction was calculated by
dividing the number of CFU positive for the neo gene by the
number of CFU positive for -actin, as previously described.21,23
PB and BM samples were obtained at the time of recovery of the
neutrophil count to greater than 1,000/µL, and at time intervals of 1 month, 3 months, 6 months, 9 months, and 1 year posttransplantation. PB
and BM mononuclear cells were purified by density centrifugation over
LSM. The sedimented pellet containing granulocytes was layered over LSM
a second time, and the granulocyte pellet was shown to be greater than
95% pure on cytospin analysis. DNA was extracted as directed using the
QIAamp Blood Kit (Qiagen, Santa Clarita, CA). For isolation of T and B
cells, PB cells were stained with anti-CD2-fluorescein isothiocyanate
(FITC)/CD19-phycoerythrin (PE) (Immunotech, Marseille, France) and
sorted using a Coulter Epics Elite instrument (Coulter, Hialeah,
FL). Sorted populations had purities of greater than 95%.
PCR analyses for neo and -actin sequences were performed
using primers and conditions as previously described.23
Negative controls consisted of normal rhesus PB samples extracted
concurrently with each set of test samples and a reagent control. A
positive control dilution series was prepared by making serial
dilutions of DNA from the producer cell line G1Na (containing 2 integrated copies per cell) into normal rhesus PB DNA. Additionally,
DNA from the LNL6 producer line was mixed with DNA from the G1Na
producer cell line in ratios of 0/100, 20/80, 50/50, 80/20, and 100/0
and diluted into control rhesus BM DNA to an combined percentage of 2%. All neo and -actin PCR reactions were optimized to
yield linear amplifications in the range of the intensity of the in vivo samples. The neo PCR products were run on a 25-cm 8%
polyacrylamide gel (National Diagnostics, Atlanta, GA) to allow
separation of the 16-bp difference between the G1Na and LNL6
radiolabeled amplification products. Phosphorimager technology was used
to measure the intensity of each band (Molecular Dynamics, Sunnyvale,
CA). Neo band intensity was normalized for amplifiable DNA
content based on the -actin signal, and the overall contribution of
each vector to in vivo marking was calculated by plotting the signal
intensities of each band to those of a standard curve generated from
the positive control dilutions.
For Southern blotting, 10 µg of genomic DNA was cut with Kpn
I (Boehringer Mannheim, Indianapolis, IN) for 8 hours at 37°C and
electrophoresed on a 0.8% agarose gel. The DNA was transferred to a
nylon membrane and subsequently hybridized with a radiolabeled 611-bp
neo probe and analyzed by autoradiography.
Statistical analysis.
The paired two-tailed Student's t-test, ANOVA, and regression
analysis were performed using Microsoft Excel 5.0 (Microsoft, Seattle,
WA).
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RESULTS |
Experimental design.
Figure 1 summarizes the experimental design used to test
the competitive repopulating ability of genetically marked nonexpanded CD34-enriched PB cells compared with genetically marked expanded cells.
SCF/G-CSF-mobilized PB cells from each rhesus monkey were CD34-enriched and then divided into two equal fractions. Each fraction
was transduced with one of two retroviral vectors (G1Na or LNL6)
containing the bacterial neomycin resistance phosphotransferase gene
(neo). At the completion of the 96-hour transduction period, one fraction was frozen and the other was cultured under the same culture conditions but without vector supernatant for a total of 10 to
14 days. The vector (LNL6 v G1Na) used to transduce the nonexpanded versus the expanded fraction was alternated in sequential monkeys to control for possible differences in each vector's gene transfer efficiency into primitive hematopoietic cells despite equivalent in vitro biologic titers as assessed on 3T3 cells and CFU-GM. Table 1 summarizes the transduction/expansion
culture conditions used for each animal.
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| Fig 1.
Experimental design. Rhesus macaques underwent
mobilization with G-CSF/SCF followed by apheresis of 2.5 times their
blood volume. The apheresis product was enriched for primitive
progenitors by CD34 selection and split into two equal fractions for a
96-hour transduction with either of two retroviral vectors: G1 (G1Na) or LN (LNL6). One aliquot was frozen at the end of transduction, whereas the other was returned to the original culture conditions without further exposure to retrovirus for a total of 10 to 14 days
before freezing. Culture condition A: IL-3, IL-6, SCF; culture condition B: IL-3, IL-6, SCF, Flt-3 ligand; culture condition C: IL-3,
IL-6, SCF, Flt-3 ligand, and an autologous stromal monolayer. After 650 cGy TBI on 2 consecutive days, both the transduced (nonexpanded) and
the transduced and expanded aliquots were thawed and simultaneously reinfused.
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Total cell and CFU expansion and transduction.
CD34 column immunoabsorption of the mobilized PB mononuclear cells
resulted in a 204- ± 64.5-fold enrichment of CFU. As shown in
Fig 2, total cell numbers increased 3.85- ± 1.2-fold
during the 96-hour transduction period and then a total of 42.3-±
18-fold during the expansion period. There was no statistically
significant difference (P = .09, ANOVA) in the total cellular
expansion between the three transduction/culture conditions, although
there was a trend towards less expansion in the stromal cocultures
(IL-3/IL-6/SCF, 47.4-fold; IL-3/IL-6/SCF/FLT, 54.4-fold; and
IL-3/IL-6/SCF/FLT/STR, 19.8-fold).
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| Fig 2.
Total cell numbers during transduction and expansion
periods. For each animal, the total cell number present in the cultures at the following time points are shown: starting CD34 enriched fraction
(Starting), the end of transduction for the nonexpanded fraction (End
Transduction-NE), the end of transduction for subsequently expanded
fraction (End Transduction-Exp), and the end of expansion (End
Expansion). The culture conditions are shown below: IL-3, interleukin
3; IL-6, interleukin-6; SCF, stem cell factor; FLT, flt3 ligand; STR,
autologous stromal monolayer.
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The expansion of committed progenitors (CFU-GM, CFU-G, CFU-M, and
BFU-E) is shown in Fig 3. CFU increased
1.8- ± 0.8-fold during the 96-hour transduction and a total of
11.8- ± 10.4-fold in the expanded fraction. There was no
statistically significant difference (P = .55, ANOVA) in the
total CFU expansion between the three culture conditions
(IL-3/IL-6/SCF, 14.5-fold; IL-3/IL-6/SCF/FLT, 14.3-fold; and
IL-3/IL-6/SCF/FLT/STR, 4.5-fold). The number of CFU in both nonexpanded
and expanded fractions after thawing were not significantly different
from prefreeze CFU content, and viability was greater than 95% (data
not shown).
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| Fig 3.
CFU during transduction and expansion periods. For each
animal, the total CFU (CFU-GM, CFU-M, CFU-G, and BFU-E) present in the
cultures at the following time points are shown: starting CD34 enriched
fraction (Starting), the end of transduction for the nonexpanded
fraction (End Transduction-NE), the end of transduction for
subsequently expanded fraction (End Transduction-Exp), and the end of
expansion (End Expansion). The culture conditions are shown below:
IL-3, interleukin-3; IL-6, interleukin-6; SCF, stem cell factor; FLT,
flt3 ligand; STR, autologous stromal monolayer.
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Figure 4 shows the percentage of
neo-marked CFU-GM present in the two fractions from each animal
at the end of the 4-day transduction period, as well as in the expanded
fraction at the end of the 10- to 14-day expansion period. Both
aliquots (nonexpanded and to be expanded) were transduced with
equivalent efficiencies as assessed by the percentage of
neo-marked CFU-GM at the end of the transduction period
(51.0% ± 23.5% v 48.4% ± 29.2%, P = .73, two-tailed t-test). In the first 4 animals,
transductions and expansions were performed in the presence of IL-3,
IL-6, and SCF, and the mean transduction efficiency with either vector
was 39.2% ± 22.2%. In the 2 animals whose cells were transduced
and expanded with the addition of FLT, the mean transduction efficiency
was 47.9% ± 28.7%. In the final 2 animals whose cells were
transduced and expanded with the addition of both FLT and STR, the mean
transduction efficiency was 72.4% ± 18.4%. There was a trend
towards a higher in vitro transduction efficiency for cultures
including FLT (P = .099, ANOVA). Importantly, the percentage of
neo-positive CFU from the end of the expansion period was in
every case almost identical to the percentage of neo-positive
CFU present in the same fraction aliquot before expansion (48.4% ± 29.2% end transduction v 45.3% ± 26.3% on the same
fractions at the end of expansion; P = .64, paired two-tailed
t-test), indicating no selective loss of successfully
transduced CFU during the expansion period, and similar levels of
transduction of cells fated to become CFU during the expansion period.
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| Fig 4.
Marking efficieny of CFU in nonexpanded and expanded
fractions. In vitro transduction efficiency of CFU was assessed at the end of transduction for both fractions and the end of expansion for the
expanded fraction. The efficiency was calculated by from the percentage
of individual plucked CFU positive for neo sequences by PCR.
For each animal, the percentage of CFU positive for the neo
gene is shown at the end of transduction for the nonexpanded fraction (End Transduction-NE), at the end of transduction for the
fraction subsequently expanded (End Transduction-Exp), as well as
colonies plated at the end of the expansion period for the expanded
fraction (End Expansion).
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Engraftment kinetics.
Engraftment was prompt in the majority of the animals, with a median of
7 days (range, 5 to 19 days) to reach a neutrophil count of 500/µL
(Table 1). The time to engraftment correlated well with the starting
CFU dose per kilogram placed into culture from each animal (r = .941, linear regression analysis), but not with the end expansion CFU
dose (r = .440). Engraftment was more rapid than in historical
animals transplanted by our program using CD34-enriched BM grafts
(neutrophil recovery days 18 to 24) or combined mobilized PB and primed
BM CD34-enriched grafts (days 10 to 14).25,26 However, the
seemingly more rapid engraftment did not appear to be due to the use of
ex vivo expanded cells, because more recently a series of animals
receiving equivalent doses of transduced but not expanded
SCF/G-CSF-mobilized PB cells have also engrafted as early as days 5 to
7 (Y. Hanazono and J. Tisdale, data not shown). The very rapid
engraftment is most likely due to progressively better apheresis and
CD34-enrichment techniques, resulting in higher total primitive cell
dosages, and agrees with murine data relating engraftment time to
primitive cell dose.27
In vivo detection of transduced cells.
The use of the two vectors differing by 16 bp to transduce the
nonexpanded versus expanded fractions of cells from each animal allowed
simple quantitation of the relative contribution of each fraction to in
vivo marking (example shown in Fig 5).
Figure 6A and B summarizes the in vivo
marking data from all 8 animals observed long-term, with levels of
marked cells derived from the nonexpanded and expanded fractions shown
and the ratio of marking in cells derived from the nonexpanded to
expanded fractions calculated for each time point. In the first cohort
of 4 animals, transductions and expansions were performed in the
presence of IL-3/IL-6/SCF (Fig 6A). Low-level long-term marking was
detected, with only 1/10,000 cells or 0.01% containing the vector,
assuming a single copy of vector per marked cell. In almost every cell
type analyzed for each time point, the ratio of marked cells derived
from the nonexpanded to the expanded fraction was greater than 1, reflecting a greater contribution towards in vivo marking in progeny
cells derived from the nonexpanded marked fraction. The level of marked cells originating from the expanded aliquots did not reflect the in
vitro transduction efficiency of CFU, despite the 10-fold or greater
numbers of total cells, total CFU, and marked CFU infused from the
expanded aliquots. On the day of engraftment, the level of marked cells
originating from the expanded aliquot was no higher than the level of
marked cells originating from the nonexpanded aliquot, suggesting that
marked CFU were not contributing to early hematopoietic reconstitution.
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| Fig 5.
PCR analysis of posttransplantation samples on animal
L161. L161 received cells that were transduced (G1Na) and transduced (LNL6) and expanded in the presence of IL-3, IL-6, SCF, and Flt-3 ligand. PB mononuclear cells (PBMNCs), granulocytes (PB GRANS), and BM
mononuclear cells (BMMNCs) were analyzed by PCR for the neo
gene. The 16-bp difference between the vectors in the amplified neo
region allows simultaneous assessment of marking in progeny from
the nonexpanded (G1Na) and the expanded (LNL6) infused fractions. Concurrent amplification of -actin was performed on each sample to
quantitate the amount of amplifiable template DNA. Serial dilutions and
mixtures of DNA from the producer cell lines containing a known copy
number of the neo gene into control rhesus PB DNA at the
indicated percentages were used to quantitate the percentage of marked
cells posttransplant. RQ1255 represents a sample simultaneously processed and extracted from a negative control animal.
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| Fig 6.
Percentage of marking calculated by semiquantitative PCR
originating from both the nonexpanded and expanded population of marked
cells posttransplant for all 8 animals. For each animal, the upper
panel represents the percentage of marking originating from transduced
but nonexpanded cells (NE), while the lower panel represents the
percentage of marking from the expanded cells (EXP). Marking was
assessed in PB mononuclear cells (PB monos), PB granulocytes (PB
Grans), and BM mononuclear cells (BM monos) at 2 weeks, 2 months, 3 months, 6 months, 9 months, and 12 months. In each panel, the ratio of
marking from the nonexpanded cells to the marking from the expanded
cells is given for each fraction analyzed at each time point in the
upper portion of each panel. A ratio greater than 1 signifies higher
marking from the nonexpanded cells, and  denotes no detectable
marking from the expanded cells, at least a 3 log difference given the
PCR sensitivity.
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In an attempt to improve engraftment by expanded cells, FLT with or
without an autologous stromal monolayer (STR) was included during both
transduction and expansion, based on multiple reports of the unique
properties of FLT in stimulating division of very primitive
hematopoietic cells.28-31 In the 2 animals receiving cells
marked and expanded in IL-3/IL-6/SCF/FLT, the relative defect in
engraftment of marked cells originating from the expanded fraction was
even greater (Figs 5 and 6B). This was partly due to improved marking
from the nonexpanded fraction compared with nonexpanded cells
transduced without FLT (average levels of 1% compared with 0.01%),
with no improvement in long-term marking from the expanded cells. In 1 animal (J039), no marking from the expanded fraction was detectable
despite 83.3% marked CFU infused. In the 2 animals receiving cells
transduced and expanded in the presence of FLT and STR in addition to
IL-3/IL-6 and SCF (RC402 and 94E048), no marking from the expanded cell
population could be detected (sensitivity of the PCR assay 0.001%).
However, marking from the nonexpanded fraction was further improved,
with initial levels of greater than 10% estimated by PCR (Figs 5 and
6B).
Southern blotting confirmed the PCR-quantitated marking levels in
animal RC402, with posttransplantation mononuclear cells and
granulocyte samples having levels of 12.5% to 50% marked cells, all
originating from the nonexpanded cell fraction (LNL6), and no
detectable marking from the expanded cell fraction (G1Na)
(Fig 7). Digestion of the
same DNA samples with an enzyme that cuts once within the proviral DNA
for insertion site analysis resulted in no detectable bands, suggesting
that the marking did not originate from a single transduced clone and
was at least oligoclonal (data not shown). Sorted populations of PB B
cells, T cells, and granulocytes collected just before death from
radiation enteritis 3 months posttransplantation showed equivalent
marking in each cell fraction of approximately 5%. Southern blotting
of DNA from postmortem tissues showed bands corresponding to the
nonexpanded graft in the spleen, axillary lymph node, and BM at levels
of 3% to 12%. Five of 16 CFU (31.3%) from a postmortem BM sample
were positive for the neo gene by PCR.
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| Fig 7.
Southern blot analysis of genetic marking in animal
RC402. Ten micrograms of genomic DNA was digested with Kpn I,
which cuts within the viral LTRs. In this animal, the nonexpanded
fraction was transduced with LNL6 and the expanded fraction with G1Na
in the presence of IL-3, IL-6, SCF, Flt-3 ligand, and an autologous stromal monolayer. PB mononuclear cells (PB MNCs) and BM mononuclear cells (BM MNCs) were analyzed, along with positive control dilutions of
G1Na producer cell line DNA mixed with normal rhesus PB DNA at the
percentages shown. The band present in these samples (3.0 kb) match the
predicted size for LNL6 (3.0 kb).
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DISCUSSION |
Ex vivo expansion of hematopoietic progenitor cells remains an area of
active interest in the field of stem cell transplantation and HSC gene
transfer, yet the long-term fate of ex vivo-expanded cells remains
unresolved.7,8 Using a relevant large animal model and
genetic marking, we were able to track the fate of the progeny of ex
vivo-expanded hematopoietic cells transduced and expanded under a
variety of conditions.32 The experimental design allowed
simultaneous tracking of cells derived from both nonexpanded and
expanded marked cells within the same animal after complete myeloablation, serving as an in vivo competitive repopulation assay and
allowing assessment of the relative engrafting ability of marked cells
under nonexpanded and expanded conditions. In vitro measures of
expansion yielded results similar to those reported by others, with
significant expansion of both total cell number and CFU.7
There was no selective loss of marked CFU during the expansion period,
and animals received a log or greater marked expanded as compared with
nonexpanded cells. Despite this difference, in vivo marking did not
reflect the relative number of marked nonexpanded versus expanded CFU
infused at any time posttransplantation, even on the day of
engraftment. There was no evidence of expansion of marked long-term
repopulating cells; at best, the cells were maintained and, in fact,
there was a significant defect in long-term marked engraftment
originating from the expanded cell fraction compared with the
nonexpanded fraction under some culture conditions.
Time to engraftment, although rapid, correlated with the number of
primitive cells present in the original CD34-enriched collection and
not with that from prolonged ex vivo expansion. PCR analysis of
granulocytes, even on the day of recovery, showed an equal or greater
signal originating from the nonexpanded graft at levels not reflecting
the high percentage of marked CFU infused, directly confirming data
from the murine model that lineage-committed CFU do not contribute to
engraftment and that numbers of ex vivo-cultured CFU do not predict
engraftment.27,33 In human transplantation, the dose of
unmanipulated CD34+ cells or the number of CFU-GM may
predict time to engraftment and other important clinical outcomes, but
it may be ill advised to assume that these surrogate stem cell assays
are meaningful after ex vivo manipulation.34-36 We did not
quantitate LTCIC or attempt FACS analysis of primitive cell phenotypes,
but culture conditions similar to those shown to expand the numbers of
LTCIC or CD34+/CD38 cells in culture did
not in our primate model translate into improved in vivo engrafting
ability.17,31
One animal initially entered on this study did not mobilize well and
had a very low number of CD34-enriched cells available for transduction
and expansion in the presence of IL-3/IL-6/SCF. Despite a 21-fold
increase in total cell number and 26-fold increase in CFU over the
expansion period, bringing these values into a safe range for rapid
engraftment using fresh cells (1.6 × 106 CFU/kg),
only a transient increase in neutrophil count was seen without any
evidence of platelet recovery, and the animal had to be killed at day
38 for failure to engraft. A similar phenomena has recently been
reported in a clinical trial in which ex vivo-expanded cells were
infused without unmanipulated cells in a small number of patients (4)
after myeloablative conditioning; lack of sustained hematopoietic
recovery prompted infusion of the unmanipulated cryopreserved back-up
cells.19 Problems with excessive cell loss due to clumping
in 1 patient, heavy pretreatment in a second (including a prior
autograft), and fungal contamination of the culture in the third left
only 1 patient evaluable; therefore, this study serves as a caution but
not definitive proof that ex vivo expansion is detrimental to
engraftment of hematopoietic progenitors. Taken together with our
results, this clinical trial underscores the need for more predictive
and rapid assays of human hematopoiesis and the need for carefully
designed large animal studies before large-scale human clinical
applications of HSC ex vivo expansion technology.
There are several caveats to the interpretation of our data. First, we
are only able to follow cells long-term that have been successfully
transduced, which in the first cohort of animals, especially,
represented a very small fraction of those cells contributing to
hematopoiesis. There may be specific characteristics of transducible primitive cells that preclude ex vivo expansion. For instance, these
cells must have cycled early during the culture period to be
transduced, and perhaps cycling at this time resulted in phenotypic changes precluding homing or other necessary steps in engraftment, even
if the cells avoided apoptosis and terminal differentiation. One group
has suggested that the engraftment defect seen in a murine model using
ex vivo-cultured cells results from an inability of cycling cells to
engraft.11,37 There is also evidence that only certain
subsets of murine repopulating cells express receptors for amphotropic
retroviral vectors, and extrapolation of this data might suggest that
cells expressing the receptor can not be expanded ex
vivo.38 Thus, our only definitive conclusion can be that
retovirally marked engrafting cells were not expanded ex vivo. This has
important implications for gene therapy protocols, but not necessarily
for other applications in clinical transplantation.
Second, we have evaluated only G-CSF/SCF-mobilized, CD34-enriched PB
cell grafts. Although these results may also apply to other cell
sources such as BM or cord blood, this should be tested. Some
encouraging in vitro and xenograft data have suggested that cord blood
stem cells may have unique expansion capacities.39-41 The
ability to expand cord blood-engrafting cells would also have the most
immediate clinical application, because the success and widespread use
of allogeneic cord blood transplantation in adult recipients is
currently limited by low cell doses and slow
engraftment.42,43 Third, only three culture conditions were
tested. Other cytokine combinations, stromal support systems, or even
the use of bioreactor technology might have improved expansion of
engrafting cells.15,44 Other culture components aimed at
preventing apoptosis or terminal differentiation, such as
anti-transforming growth factor- antibodies, or new
cytokines with activity on primitive cells, such as thrombopoietin, should also be tested.45-47 In one study, expansion of
LTCIC was achieved only when relatively high doses of hematopoietic
growth factors were used, yet these high doses were not required to
expand total cell number or CFU.17 A relatively prolonged
ex vivo culture period was used (10 to 14 days). Shorter culture
periods may produce more modest increases in cell number or CFU, but
allow retention or even expansion of true engrafting cells. Data from
the NOD/SCID xenograft model suggests that 4 days of ex vivo expansion
allows good engraftment of human cells, but longer periods are very
detrimental.48
Although ex vivo expansion in our study was detrimental to marking in
vivo for all three culture conditions studied, marking from the
nonexpanded but transduced fraction was markedly improved by
manipulations that were designed to enhance expansion. In the second
cohort of animals receiving cells transduced in the presence of FLT,
stable long-term marking of 1% was seen, compared with a large number
of animals in this and other studies with levels of only 0.01%
long-term when transductions were performed in the presence of
IL-3/IL-6/SCF alone. With the addition of FLT and autologous stroma,
clinically relevant levels of greater than 10% in vivo gene transfer
were obtained. These results are encouraging for the eventual
successful application of hematopoietic stem cell gene transfer and
need to be confirmed and studied in more detail in a larger cohort of
nonhuman primates and ultimately in humans.
| |
FOOTNOTES |
Submitted April 14, 1998;
accepted May 31, 1998.
Address reprint requests to J.F. Tisdale, MD, Bldg 10/Room
7C208, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD
20892-1652; e-mail: tisdalej{at}gwgate.nhlbi.nih.gov.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
This is a US government work. There are no restrictions in its use.
| |
ACKNOWLEDGMENT |
The authors thank Malcolm Brenner, Arthur Nienhuis, and Neal Young for
helpful discussions; Martha Kirby for FACS; Barrington Thompson and
Earl West for their assistance in caring for the animals; and Amgen for
providing cytokines.
| |
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P. Kurre, J. Morris, B. Thomasson, D. B. Kohn, and H.-P. Kiem
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C. S. McCauslin, J. Wine, L. Cheng, K. D. Klarmann, F. Candotti, P. A. Clausen, S. E. Spence, and J. R. Keller
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D. S. An, S. K. P. Kung, A. Bonifacino, R. P. Wersto, M. E. Metzger, B. A. Agricola, S. H. Mao, I. S. Y. Chen, and R. E. Donahue
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S. E. Sellers, J. F. Tisdale, B. A. Agricola, M. E. Metzger, R. E. Donahue, C. E. Dunbar, and B. P. Sorrentino
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S. Barrette, J. L. Douglas, N. E. Seidel, and D. M. Bodine
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P. F. Kelly, J. Vandergriff, A. Nathwani, A. W. Nienhuis, and E. F. Vanin
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D. A. Persons and A. W. Nienhuis
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B. Schiedlmeier, K. Kuhlcke, H. G. Eckert, C. Baum, W. J. Zeller, and S. Fruehauf
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D. S. An, R. P. Wersto, B. A. Agricola, M. E. Metzger, S. Lu, R. G. Amado, I. S. Y. Chen, and R. E. Donahue
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R. E. Donahue, R. P. Wersto, J. A. Allay, B. A. Agricola, M. E. Metzger, A. W. Nienhuis, D. A. Persons, and B. P. Sorrentino
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M. Goerner, B. Bruno, P. A. McSweeney, G. Buron, R. Storb, and H.-P. Kiem
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Abstract
Introduction
Abstract