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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 80-86
Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem
and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice:
Implications for Niemann-Pick Disease Gene Therapy and the Development
of Improved Stem Cell Gene Transfer Procedures
By
Shai Erlich,
Silvia R.P. Miranda,
Jan W.M. Visser,
Arie Dagan,
Shimon Gatt, and
Edward H. Schuchman
From the Department of Human Genetics and Institute for Gene Therapy
and Molecular Medicine, Mount Sinai School of Medicine, New York, NY;
the Laboratory of Stem Cell Biology, Lindsley F. Kimball Research
Institute, New York Blood Center, New York, NY; and the Department of
Biochemistry, Hebrew University Hadassah School of Medicine,
Jerusalem, Israel.
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ABSTRACT |
The general utility of a novel, fluorescence-based procedure for
assessing gene transfer and expression has been demonstrated using
hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic
cells were isolated from the bone marrow or fetal livers of acid
sphingomyelinase-deficient mice, and retrovirally transduced with
amphotropic or ecotropic vectors encoding a normal acid
sphingomyelinase (ASM) cDNA. Anti-c-Kit antibodies were then used to
label stem- and progenitor-enriched cell populations, and the Bodipy
fluorescence was analyzed in each group after incubation with a
Bodipy-conjugated sphingomyelin. Only cells expressing the functional
ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing
their Bodipy fluorescence as compared with nontransduced cells. The
usefulness of this procedure for the in vitro assessment of gene
transfer into hematopoietic stem cells was evaluated, as well as its
ability to provide an enrichment of transduced stem cells in vivo. To
show the value of this method for in vitro analysis, the effects of
retroviral transduction using ecotropic versus amphotropic vectors,
various growth factor combinations, and adult bone marrow versus fetal
liver stem cells were assessed. The results of these studies confirmed
the fact that ecotropic vectors were much more efficient at transducing
murine stem cells than amphotropic vectors, and that among the three
most commonly used growth factors (stem cell factor [SCF] and
interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant
effect on the transduction of stem cells, whereas IL-6 had the most
significant effect on progenitor cells. In addition, it was determined
that fetal liver stem cells were only approximately twofold more
"transducible" than stem cells from adult bone marrow.
Transplantation of Bodipy-selected bone marrow cells into lethally
irradiated mice showed that the number of spleen colony-forming units
that were positive for the retroviral vector (as determined by
polymerase chain reaction) was 76%, as compared with 32% in animals
that were transplanted with cells that were nonselected. The methods
described within this manuscript are particularly useful for evaluating
hematopoietic stem cell gene transfer in vivo because the marker gene
used in the procedure (ASM) encodes a naturally occurring mammalian
enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells
before transplantation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIETIC STEM cells (HSCs) are an
important target for gene therapy.1-4 However, until now
they have remained refractable to most gene transfer techniques because
of their low numbers and lack of proliferation. Thus, before HSCs can
be widely used in a clinical setting, new vectors must be developed and
improved methods of HSC enrichment and transformation must be obtained.
As these new gene transfer techniques are developed, ways to assess
their usefulness will be needed.
Towards this end, we have developed a new method to quantitatively
determine gene transfer and expression in hematopoietic stem and
progenitor cells. The technique takes advantage of the fact that cells
expressing a functional acid sphingomyelinase (ASM; sphingomyelin
phosphodiesterase) can metabolize fluorescent sphingomyelin
derivatives, whereas those that lack ASM activity (eg, from acid
sphingomyelinase-deficient [ASMKO] mice5 or human
patients with the genetic disorder Niemann-Pick disease [NPD]6) cannot. Thus, after labeling with fluorescent
sphingomyelin, normal cells (expressing ASM) and NPD cells (lacking ASM
activity) can be readily discriminated by fluorescence microscopy or
flow cytometry.7,8 The same is true for NPD cells and such
cells that have been enzymatically corrected by gene transfer.
This manuscript shows the utility of this method using hematopoietic
stem and progenitor cells obtained from the bone marrow or fetal livers
of ASMKO mice. The advantages of this technique include the facts that
(1) the target cells do not need to be proliferating (such as HSCs),
and any gene transfer system can be analyzed after inserting ASM into
the vector; (2) it is fluorescence based and highly sensitive,
permiting analysis of rare cell populations; and (3) it can be easily
used for long-term in vivo analysis because it requires only 2 days to
complete, does not involve extensive cell manipulation, and the
transplanted cells do not express a foreign protein with potentially
adverse effects.
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MATERIALS AND METHODS |
Cell preparations.
To obtain adult nucleated bone marrow cells, the tibia and femurs of
12- to 16-week-old C57BL/SV129 normal and ASMKO mice were flushed with
buffered Hanks solution (10 mmol/L Hepes, pH 7.5). Single-cell
suspensions were obtained by passing the cells through a 0.4-micron
mesh (Becton Dickinson Labware, Franklin Lakes, NJ). Low-density bone
marrow cells (<1.085 g/cm3) were isolated by
discontinuous density gradient centrifugation using Nycoprep (Nycomed
Pharma AS, Oslo, Norway),9 and washed with buffered Hanks
solution containing 5% heat-inactivated fetal calf serum (HSA). To
obtain fetal liver cells, the livers of day 14.5 fetuses were isolated
in buffered Hanks solution. Single-cell suspensions were prepared by
gently pipetting the tissue up and down through the bore of a 5-mL
pipette. The cells were then washed with HSA.
Lineage depletion.
To obtain Lin cells, a modification of the method of
Bertoncello et al10 was used. Isolated adult bone marrow
and fetal liver cells were incubated for 30 minutes on ice with the
biotinylated antibodies anti-TER-119, anti-CD45R/B220, and anti-Ly-6G
(Pharmingen, San Diego, CA). The concentration of each antibody was 1 µg/106 cells. The cells were than washed with HSA,
resuspended in buffered Hanks solution, and incubated with
streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin;
Dynal, Lake Success, NY) at 4°C for 30 minutes at a 10:1 bead:cell
ratio. Magnetic force was then applied for 1 minute and the supernatant
was collected. The cell pellet was washed with buffered Hanks solution
three times using the same procedure, and the supernatants were
combined.
Anti-c-Kit labeling.
Cultured or freshly collected cells were washed once with HSA and
counted. The cells were than incubated with phycoerythrin (PE)-conjugated anti-CD117 (c-Kit) antibodies (Pharmingen) at a
concentration of 1 µg/106 cells for 30 minutes on ice.
After labeling, the cells were washed once with HSA and resuspended in
buffered Hanks solution. Control labeling was performed with a rat
IgG2b, kappa isotype (Pharmingen).
Synthesis of fluorescent sphingomyelin.
Sphingomyelin to which the fluorescent probe Bodipy was covalently
linked via a 12-carbon spacer (Bodipy dodecanoyl sphingosyl phosphocholine; B12SPM) was synthesized as previously described for
lissamine rhodamine sphingomyelin,7 except that Bodipy dodecanoic acid (Molecular Probes Inc, Eugene, OR) was condensed with
sphingosyl phosphocholine. To incorporate B12SPM into liposomes, B12SPM
was mixed with phosphatidyl choline (PC; Sigma, St Louis, MO) at a
molar ratio of 1:4. The solvent was evaporated and the mixture was
resuspended in buffered Hanks solution followed by a 1-minute
sonication.
"Pulse-chase" labeling with B12SPM.
B12SPM/PC liposomes (final concentration 0.5 to 1 nmol/mL) were
incubated at 37°C for 4 hours with Lin cells
that had been suspended in buffered Hanks solution. Labeling was
terminated by centrifuging the cells (400g) and washing the pellets once with HSA. Fresh medium was then added and the cells were
further incubated for 48 hours in standard culture media containing
Iscove's Modified Dulbecco's Medium (IMDM; GIBCO-BRL, Gaithersburg,
MD), 10% HSA (GIBCO-BRL), and antibiotics, but no B12SPM/PC liposomes.
Retroviral transduction.
To achieve retroviral transduction, adult bone marrow and fetal liver
cells were cocultured for 48 hours with amphotropic or ecotropic
retroviral-producing cells containing an ASM/MFG retroviral vector.8 Control untreated cells were cocultured with producer cells alone. Cocultures were performed in 0.4-mm Transwell dishes (Corning Costar, Cambridge, MA) containing the packaging cells in the upper compartment. Stem cell factor (SCF; 50 ng/mL), interleukin-3 (IL-3; 20 ng/mL), and IL-6 (10 ng/mL) (Genzyme,
Cambridge, MA) were added to the media unless indicated otherwise.
Fluorescence-activated cell sorter (FACS) analysis and expansion of
sorted cells.
Cells were analyzed using a FACScan instrument (Becton Dickinson
Immunocytometry Systems, San Jose, CA) and the WinMDI program. Cells
were sorted using a FACStar flow cytometer (Becton Dickinson Immunocytometry Systems). Sorted cells were resuspended in expansion media containing IMDM, 10% HSA, SCF (50 ng/mL), IL-6 (10 ng/mL), IL-3
(20 ng/mL), and antibiotics, and then grown at 37°C for 10 days.
Polymerase chain reaction (PCR) analysis.
A modification of our previously described procedure was
used.8 Murine and human ASM sequences were amplified using
one common sense primer, 5 -TGCTGAGGATCGAGGAGACAA-3 (P1)
constructed from human and murine ASM exon 3, and two species-specific
antisense primers, 5-GGGTAGAGTGACAGAAGATTGA-3 (P2) and
5-GGCACAAGAGTAGCCAGACG-3 (P3), constructed from murine
ASM intron 3 and human ASM exon 6, respectively. Primer pair P1 and P2
amplified a 211-bp genomic murine ASM product, whereas primer pair P1
and P3 amplified a 554-bp product from the human ASM/MFG sequence. Each
amplification reaction (100 µL final volume) contained 200 pmol of
primer P1, 100 pmol each of primers P2 and P3, 300 ng of genomic DNA,
1× PCR buffer (Promega, Madison, WI), 1.5 mmol/L
MgCl2, 5 U of Taq polymerase (Promega) and 200 mmol/L each
of dNTPs. A standard curve was generated using DNA mixtures as
described in Yeyati et al.8 After amplification (30 cycles,
each consisting of 1 minute at 93°C, 1 minute at 61°C, 1 minute
at 72°C), PCR products were electrophoresed on 1.5% agarose gels
and stained with ethidium bromide. The intensity of the bands was
determined using the National Institutes of Health Imager software
package (NIH, Bethesda, MD). By comparing the intensity of
the two amplified bands, the number of transduced cells in the sorted
populations could be estimated. This calculation was based on the
assumption that each transduced cell contained one proviral genome. It
should be noted that the murine-specific ASM band is present in all
samples and serves as an internal control, and that while the ASMKO
mice have no ASM activity, the murine ASM genomic sequences are still
present.
Enzyme assays.
Fresh or cultured adult bone marrow cells were obtained, washed once
with HSA, and incubated on ice for 15 minutes in 0.2% Triton
X-100. Total protein was determined by the method of Stein et al.11 The standard 15-µL ASM assay mixture consisted
of 10 µL of protein source and 2 nmol of B12SPM suspended in 0.1 mol/L sodium acetate buffer, pH 5.2, containing 0.6% Triton X-100 and 5 mmol/L EDTA. After incubating the assay mixture at 37°C (up to 3 hours), the samples were loaded onto thin layer chromatography plates
(TLC LK6 D Silica gel; Whatman, Clifton, NJ) and resolved using
chloroform/methanol (95:5 vol/vol). After resolution, the band
containing the fluorescently labeled ceramide (the product of B12SPM
hydrolysis) was scraped from the plates, extracted in chloroform/methanol/water (1:2:1 vol/vol) for 15 minutes at 55°C, and quantified in a spectrofluorometer (fluorescence spectrophotometer 204-A, Perkin-Elmer, Norwalk, CT). The instrument settings
were excitation 505 nm and emission 530 nm.
Spleen colony-forming unit (CFU-S) assays.
Adult nucleated bone marrow cells were obtained from ASMKO mice that
had been pretreated with 5-fluorouracil (5-FU) (150 mg/Kg) 2 days before harvesting, retrovirally transduced with the ecotropic vector, and "pulse-chase"-labeled with B12SPM as described above, and then sorted by FACS. Cells representing the least 25% fluorescent in FL-1 (B12low) were collected and 4 × 104 were injected into the tail veins of lethally
irradiated (800 cGy) adult ASMKO mice. For comparison, the same number
of nonselected, transduced cells were injected into another set of
animals. After 14 days the mice were killed, and the spleens were
removed and then fixed in a 70% solution of formalin:acetic
acid:ethanol (1:1:20 vol/vol/vol) for 3 days.
To prepare DNA from the CFU-S colonies, a modification of the method of
Frank et al12 was used. The CFU-S colonies were separated
and washed individually three times overnight in 1× Tris-EDTA
(TE) (pH 8.2) at 4°C, and then minced and incubated overnight again at 37°C in a solution containing 50 mmol/L Tris (pH
8.2) and 200 ng/µL Proteinase K (Boehringer Mannheim, Mannheim, Germany). The microcentrifuge tubes containing the digested materials were then immersed in boiling water for 8 minutes, and the extracted DNA was placed on ice. For PCR, the DNA solutions were diluted 1:100
and 40 µL was used. The PCR was performed as described above except
that only primers P1 and P3 were used and the number of cycles was 40. A positive colony was defined as a colony in which the human ASM
(hASM) transgene-specific PCR product was found in at
least three independent amplification reactions.
| |
RESULTS |
Identification of stem- and progenitor-enriched cell populations.
Bone marrow was obtained from normal and ASMKO adult mice and analyzed
by FACS. After pre-enrichment by Nycodenz (Nycomed Pharma, Oslo,
Norway) density gradient centrifugation and lineage depletion,9,10 two main populations of nucleated cells were identified, designated FSChigh and FSClow
(Fig 1A). Labeling of these cells with
antibodies against c-Kit showed that both groups contained
c-Kit+ cells (Fig 1B), and that the number of lineage
depleted (Lin)/c-Kit+ cells was
approximately 2% of the total nucleated cell population. c-Kit was
used as a marker because previous work had shown that the
Lin/FSChigh/c-Kit+
population is highly enriched for progenitor cells, whereas the Lin/FSClow/c-Kit+ population
is highly enriched for stem cells.13,14
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| Fig 1.
Analysis of Lin adult bone marrow cells.
The light scatter properties of the cells were measured (A), and two
populations were identified, designated FSClow and
FSChigh. These populations were analyzed individually for
the presence of c-Kit on the cell surface using PE-conjugated
anti-c-Kit antibodies (B). The shaded area indicates control cells,
whereas the open area indicates cells labeled with anti-c-Kit
antibodies. Note that FL-2 measures PE.
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"Pulse-chase" labeling of normal, ASMKO, and transduced cells.
Lin cells from normal or ASMKO mice were incubated
for 4 hours with B12SPM, and then grown at 37°C for 48 hours in
standard culture media without B12SPM. Analysis of the stem- and
progenitor-enriched populations for B12 fluorescence is shown in
Fig 2A and B. Comparison of the Bodipy
fluorescence (FL-1) of normal and ASMKO adult bone marrow cells after
labeling with B12SPM showed that in both the Lin/FSClow/c-Kit+ (stem
cell-enriched) and
Lin/FSChigh/c-Kit+
(progenitor cell-enriched) populations, the normal cells were less
fluorescent than those from ASMKO animals. These results showed that
stem and progenitor cells could internalize B12SPM and that both types
of normal cells expressed a functional ASM.
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| Fig 2.
Analysis of B12SPM labeling of hematopoietic stem- and
progenitor-enriched cell populations. (A) and (C) depict the
Lin/FSClow/c-Kit+ stem
cell-enriched population, whereas (B) and (D) depict the
Lin/FSChigh/c-Kit+ progenitor
cell-enriched population. (A) and (B) shaded area, ASMKO cells; open
area, normal cells. (C) and (D) shaded area, nontransduced ASMKO cells;
open area, transduced ASMKO cells. Note that FL-1 measures B12SPM. The
experiment was repeated three times, and the representative data from
one experiment are shown.
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The same experiment was then repeated on ASMKO cells that had been
retrovirally transduced with an amphotropic MFG retroviral vector
expressing human ASM. The results of this experiment are shown in Fig
2C and D. A significant shift to lower fluorescence was observed in the
Lin/FSChigh/c-Kit+ cells
(progenitor cell-enriched), whereas only a very minor shift was
observed in the
Lin/FSClow/c-Kit+ (stem
cell-enriched) population. These results confirmed previous studies15,16 showing that murine progenitor cells (but not stem cells) could be efficiently transduced by amphotropic vectors.
PCR and enzyme analysis of sorted cells.
To further investigate this finding, retrovirally transduced
Lin/c-Kit+ ASMKO adult bone marrow cells
were labeled with B12SPM and subjected to FACS sorting. Cells with the
highest (B12high) and lowest (B12low) FL-1 were
sorted from the stem- and progenitor-enriched groups (Lin/FSClow/c-Kit+ and
Lin/FSChigh/c-Kit+,
respectively), grown in expansion cultures, and subjected to semiquantitative PCR analysis and ASM activity assays
(Fig 3). Note that within the sorted
B12low group of
Lin/FSClow/c-Kit+ cells, a
small number were transduced (1 in 230, based on the PCR assay),
whereas in the B12low group of
Lin/FSChigh/c-Kit+ cells,
significantly more were transduced (1 in 8). By comparison, without
selection for FL-1, among the
Lin/FSClow/c-Kit+ cells no
transduced cells could be identified, whereas among the
Lin/FSChigh/c-Kit+ cells the
frequency of transduced cells was approximately 1 in 20 (not shown).
These data confirmed the fact that the stem cell-enriched population
from adult bone marrow was very difficult to transduce with amphotropic
vectors compared with the progenitor-enriched population, but also
showed that the Bodipy selection technique provided a significant
enrichment of transduced cells. Indeed, by using this method,
transduced cells from both groups could be identified and collected.
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| Fig 3.
Sorting and analysis of retrovirally transduced ASMKO
bone marrow cells. PCR analysis and ASM enzyme assays were conducted in
triplicate on sorted B12low and B12high cells
from the Lin/FSClow/c-Kit+
population (left; stem cell-enriched) and
Lin/FSChigh/c-Kit+ population
(right; progenitor cell-enriched) as described in the text. ND, not
detected. The experiment was repeated three times, and the
representative data from one experiment are shown.
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Analysis of fetal liver stem and progenitor cells.
We next used this system to assess the transduction of fetal liver
c-Kit+ cells by the amphotropic vector
(Fig 4). Lin fetal liver
cells were isolated, labeled with PE-conjugated anti-c-Kit antibodies,
and analyzed on FACS. The light scatter properties of the cells were
measured, and in contrast to adult bone marrow, only one population of
Lin cells was identified. This population was
analyzed for the presence of the c-Kit molecule (FL-2) on the cell
surface, and it was determined that the number of c-Kit+
cells was approximately 5% of the total nucleated fetal liver cells.
The Lin ASMKO fetal liver cells were either
retrovirally transduced with the ASM/MFG amphotropic vector or
untreated, labeled with B12SPM, and the FL-1 of the two groups was
compared. Only a small shift to lower fluorescence was observed in the
retrovirally transduced population, corresponding to a low transduction
efficiency of approximately 1 in 50 to 1 in 100 cells.
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| Fig 4.
Analysis of retroviraly transduced Lin
fetal liver cells. The light scatter properties of the cells were
measured (A), and the major population was identified and gated for
further analysis (shown in box). This population was then analyzed for
the presence of c-Kit on the cell surface ([B] shaded area, control
cells; open area; cells labeled with anti-c-Kit antibodies). Note that
FL-2 measures PE. The c-Kit+ cells were further analyzed
for B12 fluorescence ([C] shaded area, control cells; open area,
transduced cells). Note that FL-1 measures B12SPM. The experiment was
repeated twice, and the representative data from one experiment are
shown.
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Comparison of the retroviral transduction efficiencies using
amphotropic versus ecotropic viruses.
Because the transduction of stem cell-enriched bone marrow or fetal
liver cells using the amphotropic vector was low, we next sought to
determine if this could be improved using an ecotropic vector. Several
recent reports have shown increased transduction of murine
hematopoietic stem cells using ecotropic versus amphotrophic retroviral
vectors.15,16 As shown in Fig
5, by using the B12SPM selection technique the transduction
efficiencies of
Lin/FSClow/c-Kit+ stem cells
with the amphotropic versus ecotropic ASM/MFG vectors could be directly
compared. Of note, the ecotropic vector led to a more than 50-fold
increase in transduction over that found with the amphotropic vector.
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| Fig 5.
Comparison of the transduction efficiencies using
amphotropic versus ecotropic retroviral vectors. (A) and (C) depict the
Lin/FSClow/c-Kit+ stem
cell-enriched population, whereas (B) and (D) depict the
Lin/FSChigh/c-Kit+ progenitor
cell-enriched population. The shaded areas represent nontransduced
ASMKO cells, whereas the open areas depict transduced cells. Note that
FL-1 measures B12SPM. The experiment was repeated three times, and the
representative data from one experiment are shown.
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Effects of individual growth factors on the transduction of murine
stem and progenitor cells.
To evaluate the specific contribution of growth factors to the
transduction of hematopoietic cells, various combinations of SCF, IL-3,
and IL-6 were used during the retroviral transduction of B12SPM-labeled
cells with the ecotropic vector (Fig 6).
This was followed by FACS analysis to determine the B12 fluorescence of
the cells. In the
Lin/FSClow/c-Kit+ population
(stem cell-enriched), SCF had the biggest effect on the transduction
efficiency, whereas among the
Lin/FSChigh/c-Kit+
population (progenitor-enriched), the largest contribution was provided
by IL-6. In both groups of cells, the combination of all three growth
factors led to the highest transduction efficiencies.
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| Fig 6.
Effects of growth factors on transduction efficiences
using ecotropic vectors. The standard retroviral transduction methods
and concentrations of each growth factor are described in the Materials
and Methods. FL-1 measures B12SPM fluorescence. The bars indicate ±1
standard deviation using data derived from three independent
experiments.
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CFU-S analysis of sorted cells.
To ensure that cells selected with these techniques were enriched for
hematopoietic stem cells, ASMKO bone marrow cells were transduced with
the ecotropic ASM/MFG vector in the prescence of SCF, IL-3, and IL-6;
B12low cells were collected by flow cytometry; and then
transplanted into lethally irradiated adult ASMKO mice. Fourteen days
later the spleens were obtained and the number of CFUs-S positive for the retroviral vector was determined by PCR. As shown in
Table 1, in the absence of selection, 32%
of the CFU-S colonies were vector positive, whereas among animals
transplanted with B12SPM-selected cells, 76% of the colonies were
vector positive. Thus, these results showed that a significant
enrichment of transduced repopulating stem cells can be obtained using
this procedure.
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DISCUSSION |
A novel, fluorescence-based method is presented for the analysis of
gene transfer into hematopoietic stem and progenitor cells. In our view
there are two applications of this technology, one specific for the
treatment of NPD, and the other more general. With regards to NPD, the
data clearly show that by using these techniques, enriched populations
of retrovirally transduced NPD hematopoietic stem and progenitor cells
can be identified and collected for subsequent transplantation into
patients. Such cells are currently being transplanted into ASMKO mice
so that their engraftment potential and clinical usefulness can be
compared with transduced hematopoietic cells that have not undergone
the selection procedure. It is hypothesized that in a competitive repopulation setting, particularly in young animals that have not been
lethally ablated, such selected cells may be clinically advantageous
because the likelihood of engrafting a transduced, long-term
repopulating stem cell will be greater due to the fluorescent enrichment procedure. Indeed, the results of the CFU-S experiment suggest that this may be the case. If successful, similar methods can
be applied to CD34+ cells obtained from human NPD patients.
Although obtaining sufficient numbers of such
B12low/CD34+ cells for human transplantation
may be problematic at the present time, the development of high-speed
flow cytometers17-19 promises to overcome this limitation.
We also propose that this simple analytical system can be broadly used
in a variety of gene therapy and basic research settings. The target
cells do not need to be proliferating to take up B12SPM (S.E. and
E.H.S., unpublished data), making these techniques
amenable to HSCs and other quiescent cells. We have shown the
usefulness of this method by evaluating transduction with retroviral
vectors; however, other gene transfer systems can be easily studied by inserting the ASM cDNA as a marker. This would include viral- or
nonviral-based systems. Importantly, the system relies on expression of
the transferred gene as its final endpoint, and is fluorescence based,
making it highly sensitive. Also of note, the corrected cells can be
isolated from the noncorrected ones using a FACS, even when they
represent 1% or less of the total cell population, and the properties
of the two groups can be directly compared in vitro or in vivo, as long
as ASMKO mice are used as a source of target cells.
Several findings were presented in this paper regarding the
transduction of murine hematopoietic stem and progenitor cells with
retroviral vectors to show the usefulness of these techniques. First,
we confirmed the fact that amphotropic vectors could not transduce
murine hematopoietic stem cells efficiently as compared with ecotropic
vectors.15,16 Indeed, the transduction frequency of the
stem cell-enriched population was increased more than 50-fold using
ecotropic versus amphotropic vectors. Of note, we also found that among
the three most commonly used growth factors, SCF contributed the most
to stem cell transduction, whereas IL-6 contributed most to progenitor
cell transduction. Again, these results confirmed previous
reports20 and showed the usefulness of these techniques. Finally, we documented that c-Kit+ fetal cells, which are
approximately twofold more prevelant than c-Kit+ bone
marrow cells, were transduced with amphotropic vectors at an
intermediate frequency (approximately 1 in 100 in the Bodipy-selected populations) when compared with bone marrow stem or progenitor cells.
Clearly, new vector systems, such as the Lentivirus-based retroviral
vectors21,22 as well as new growth factor cocktails and
packaging cell lines,23,24 can be readily analyzed by these methods. Before the development of these and similar fluorescence-based techniques (see below), data such as these could only be obtained by
transduction with vectors containing marker genes such as those encoding neomycin resistance or  -galactosidase activity, followed by
labor-intensive and indirect colony-forming assays. Direct isolation of
the transduced cells would have been impossible.
Several alternative fluorescence-based selection systems have been
developed in recent years to enrich for transduced hematopoietic stem
cells.25-27 Among these, the use of the green fluorescence protein (GFP) has attracted considerable interest.28
However, one important difference between our system and those which
use GFP is that in our system the fluorescent marker (B12SPM) is not encoded by the gene transfer vector. Identification of the transduced cells relies on enzymatic expression of ASM, leading to degradation of
the fluorescent molecule and a subsequent loss of fluorescence. Indeed,
the target cells are only exposed to B12SPM for a short period of time,
resulting in little or no toxicity. In the GFP system, the GFP gene is
inserted into the vector of interest and the cells are selected for an
increase in fluorescence. Stably transduced cells will continue to
produce GFP and remain fluorescent, possibly leading to increased
toxicity or abnormal metabolism if the transduced cells are followed
for long periods of time in vivo.
Thus, we believe that the selection approach described in this
manuscript will be of interest to a wide range of scientists, and is
particularly advantageous for the development of hematopoietic stem
cell gene therapy for NPD and the analysis of HSC gene transfer in
general.
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ACKNOWLEDGMENT |
We thank Dr Kevin Kelly for his expert assistance in the preparation of
the fetal liver cells.
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FOOTNOTES |
Submitted November 6, 1997;
accepted September 2, 1998.
Supported by research grants from the National Institutes of Health (HD
28607 and HD 32654), March of Dimes Birth Defects Foundation (1-2224),
a grant (RR 0071) from the National Center for Research Resources for
the Mount Sinai General Clinical Research Center, and a grant
(93-00015) from the US-Israel Binational Science Foundation.
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 Edward Schuchman, PhD, Department of Human
Genetics, Box 1498, Mount Sinai School of Medicine, 1425 Madison Ave,
Room 14-20A, New York, NY 10029; e-mail: Schuchman{at}msvax.mssm.edu.
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REFERENCES |
1.
Brenner MK:
Gene transfer into human hematopoietic progenitor cells: A review of current clinical protocols.
J Hematother
2:7, 1993[Medline]
[Order article via Infotrieve]
2.
Brenner MK, Cunningham JM, Sorrentino BP, Heslop HE:
Gene transfer into human hemopoietic progenitor cells.
Br Med Bull
51:167, 1995[Abstract/Free Full Text]
3.
Huss R:
Applications of hematopoietic stem cells and gene transfer.
Infus Ther Transfus Med
23:147, 1996
4.
Kaye EM:
Therapeutic approaches to lysosomal storage diseases.
Curr Opin Pediatr
7:650, 1995[Medline]
[Order article via Infotrieve]
5.
Horinouchi K, Erlich S, Perl DP, Ferlinz K, Bisgaier CL, Sandhoff K, Desnick RJ, Stewart CL, Schuchman EH:
Acid sphingomyelinase deficient mice: A model of types A and B Niemann-Pick disease.
Nat Genet
10:288, 1995[Medline]
[Order article via Infotrieve]
6.
Schuchman EH, Desnick RJ:
Niemann-Pick disease types A and B: Acid sphingomyelinase deficiencies, in
Scriver CR,
Beaudet AL,
Sly WS,
Valle D
(eds):
The Metabolic and Molecular Bases of Inherited Disease, vol 2. New York, NY, McGraw-Hill, 1995, p 2601.
7.
Dinur T, Schuchman EH, Fibach E, Dagan A, Suchi M, Desnick RJ, Gatt S:
Toward gene therapy for Niemann-Pick disease (NPD): Separation of retrovirally corrected and noncorrected NPD fibroblasts using a novel fluorescent sphingomyelin.
Hum Gene Ther
3:633, 1992[Medline]
[Order article via Infotrieve]
8.
Yeyati PL, Agmon V, Fillat C, Dinur T, Dagan A, Desnick RJ, Gatt S, Schuchman EH:
Fluorescence-based selection of retrovirally transduced cells in the absence of a marker gene: Direct selection of transduced type B Niemann-Pick disease cells and evidence for bystander correction.
Hum Gene Ther
6:975, 1995[Medline]
[Order article via Infotrieve]
9.
Bertoncello I, Bartelmez SH, Bradley TR, Hodgson GS:
Increased Qa-m7 antigen expression is characteristic of primitive hemopoietic progenitors in regenerating marrow.
J Immunol
139:1096, 1987[Abstract]
10.
Bertoncello I, Bradley TR, Watt SM:
An improved negative immunomagnetic selection strategy for the purification of primitive hemopoietic cells from normal bone marrow.
Exp Hematol
19:95, 1991[Medline]
[Order article via Infotrieve]
11.
Stein S, Bohlen P, Stone J, Dairman W, Udenfriend S:
Amino acid analysis with fluorescamine at the picomole level.
Arch Biochem Biophys
155:202, 1973[Medline]
[Order article via Infotrieve]
12.
Frank T, Svoboda-Newman S, Hsi ED:
Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR.
Diagn Mol Pathol
5:220, 1996[Medline]
[Order article via Infotrieve]
13.
Okada S, Nakauchi H, Nagayoshi K, Nishikawa S, Nishikawa S, Miura Y, Suda T:
Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule.
Blood
78:1706, 1991[Abstract/Free Full Text]
14.
Ikuta K, Weissman IL:
Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation.
Proc Natl Acad Sci USA
89:1502, 1992[Abstract/Free Full Text]
15.
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]
16.
Orlic D, Girard LJ, Anderson SM, Do BK, Seidel NE, Hordan CT, Bodine DM:
Transduction efficiency of cell lines and hematopoietic stem cells correlates with retrovirus receptor mRNA levels.
Stem Cells
15:23, 1997 (suppl)
17.
Keij JF, van Rotterdam A, Groenewegen AC, Stokdijk W, Visser JW:
Coincidence in high-speed flow cytometry: Models and measurements.
Cytometry
12:398, 1991[Medline]
[Order article via Infotrieve]
18.
Tanke HJ, van der Keur M:
Selection of defined cell types by flow-cytometric cell sorting.
Trends Biotechnol
11:55, 1993[Medline]
[Order article via Infotrieve]
19.
Leary JF, Schmidt DF, Gram JG, McLaughlin SR, Dalla Torre C, Burde S:
High-speed flow cytometric analysis and sorting of human fetal cells from maternal blood for molecular characterization.
Ann NY Acad Sci
731:138, 1994[Medline]
[Order article via Infotrieve]
20.
Wognum AW, De Jong MO, Wagemaker G:
Differential expression of receptors for hemopoietic growth factors on subsets of CD34+ hemopoietic cells.
Leuk Lymphoma
24:11, 1996[Medline]
[Order article via Infotrieve]
21.
Poeschla E, Corbeau P, Wong-Staal F:
Development of HIV vectors for anti-HIV gene therapy.
Proc Natl Acad Sci USA
93:11395, 1996[Abstract/Free Full Text]
22.
Lever AM:
HIV and other lentivirus-based vectors (editorial).
Gene Ther
3:470, 1996[Medline]
[Order article via Infotrieve]
23.
Miller AD, Chen F:
Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry.
J Virol
70:5564, 1996[Abstract/Free Full Text]
24.
Schnierle BS, Stitz J, Bosch V, Nocken F, Merget-Millitzer H, Engelstadter M, Kurth R, Groner B, Cichutek K:
Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells.
Proc Natl Acad Sci USA
94:8640, 1997[Abstract/Free Full Text]
25.
Fehse B, Uhde A, Fehse N, Eckert HG, Clausen J, Ruger R, Koch S, Ostertag W, Zander AR, Stockschlader M:
Selective immunoaffinity-based enrichment of CD34+ cells transduced with retroviral vectors containing an intracytoplasmiatically truncated version of the human low-affinity nerve growth factor receptor (deltaNGFR) gene.
Hum Gene Ther
8:1815, 1997[Medline]
[Order article via Infotrieve]
26.
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]
27.
Pawliuk R, Kay R, Lansdorp P, Humphries RK:
Selection of retrovirally transduced hematopoietic cells using CD24 as a marker of gene transfer.
Blood
84:2868, 1994[Abstract/Free Full Text]
28.
Persons DA, Allay JA, Allay ER, Smeyne RJ, Ashmun RA, Sorrentino BP, Nienhuis AW:
Retroviral-mediated transfer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo.
Blood
90:1777, 1997[Abstract/Free Full Text]
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Abstract
Introduction