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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1243-1255
Engraftment and Retroviral Marking of CD34+ and
CD34+CD38 Human Hematopoietic Progenitors
Assessed in Immune-Deficient Mice
By
Mo A. Dao,
Ami J. Shah,
Gay M. Crooks, and
Jan A. Nolta
From the Division of Research Immunology/Bone Marrow Transplantation,
Childrens Hospital Los Angeles, Los Angeles, CA, and University of
Southern California School of Medicine, Department of Pediatrics, Los
Angeles, CA.
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ABSTRACT |
Retroviral-mediated transduction of human hematopoietic stem cells
to provide a lifelong supply of corrected progeny remains the most
daunting challenge to the success of human gene therapy. The paucity of
assays to examine transduction of pluripotent human stem cells hampers
progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we
compared the transduction and engraftment of human CD34+
progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38 cells. Comparable extents of
human engraftment and lineage development were obtained from 5 × 105 CD34+ cells and 2,000 CD34+CD38 cells. Retroviral marking of
long-lived progenitors from the CD34+ populations was
readily accomplished, but CD34+CD38 cells
capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days
resulted in low levels of transduction of
CD34+CD38 cells. Flt3 ligand was required
during the 7-day ex vivo culture to maintain the ability of the cells
to sustain long-term engraftment and hematopoiesis in the mice.
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INTRODUCTION |
THE CD34+CD38
CELL population represents a subset of human hematopoietic stem cells
with a primitive and quiescent phenotype that can be isolated from
adult and fetal bone marrow, umbilical cord blood, and fetal liver
tissue.1-5 Human CD34+ cells acquire the cell
surface marker CD38 with differentiation,6,7 and the
CD34+CD38 fraction represents a population
enriched in hematopoietic stem cells not yet committed to specific
lineages.
It has been shown that the reconstituting ability of the
CD34+ cell population lies in the CD38
fraction by using immunoincompetent fetal sheep and NOD/SCID mice as
transplant recipients.8,9 Therefore,
CD34+CD38 cells would be the ideal target
population for gene therapy to repair inborn errors of hematopoiesis
caused by single gene defects, such as Gaucher and Hurler's diseases,
adenosine deaminase deficiency, and others.10 A small
number of CD34+CD38 cells could
theoretically be modified by insertion of a normal copy of the affected
gene and generate corrected progeny for many years after return to the
donor.
Although CD34+CD38 cells are ideal
candidates for gene therapy to treat abnormalities of the hematopoietic
system, they are not predicted to be easily transduced by retroviral
vectors. Moloney murine leukemia-based retroviral vectors presently
provide the most efficient method for permanent insertion of genes into
mammalian cells, but require target cell division for
integration.11 CD34+CD38 cells
from bone marrow are largely quiescent and are not rapidly recruited
into cell cycle by the cytokines that have been cloned to
date,7,12-14 so they might be refractory to transduction by Moloney-based vectors.
Long-lived human hematopoietic progenitors from CD34+
populations can be transduced with retroviral vectors, and generate
marked progeny in immune-deficient (beige/nude/xid [bnx])
mice for up to 1 year. However, the levels of transduction and
engraftment of more highly purified stem cells had not been previously
evaluated in the bnx/hu xenograft model of human
hematopoiesis.15-17 In the current studies, we used the
bnx/hu system to compare engraftment and marking of
primitive, reconstituting cells within CD34+ and
CD34+CD38 populations isolated from the same
bone marrow samples. Cells were transduced ex vivo for 1 hour (used as
a baseline control for engraftment) or for 72 hours. After the
transduction periods, cells were cotransplanted with IL-3 producing
stroma into sibling bnx mice in groups of three to four as
previously published.15-17 Nine to 11 months
post-transplantation, marrow was harvested from the mice and analyzed
for the extents of human hematopoietic cell engraftment, clonogenic
progenitor content, and vector marking of tissues and individual T-cell
and myeloid clones. In contrast to the CD34+ populations,
the CD34+CD38 cells were not transduced to
significant levels by the retroviral vector LN in 72-hour incubations.
The period of ex vivo culture and transduction was extended to 7 days,
with and without flt3 ligand (FL), a cytokine previously shown to be a
maintenance and stimulatory factor for primitive hematopoietic
progenitors.18-20 We hypothesized that a longer
preincubation with stromal support and FL might permit cell cycle
progression in CD34+CD38 cells, enhancing
retroviral-mediated transduction. The presence of FL was required in
the 7-day cultures to sustain the capacity of the cells to give rise to
long-term hematopiesis in the mice. Multiple long-lived,
vector-transduced cells were found in two mice that had received human
CD34+CD38 cells transduced on days 5, 6, and
7 in the presence of FL. CD34+CD38 cells
transduced for the same period of time without FL, or for only 3 days,
were not transduced.
Our data suggest that there is a population of long-term engrafting
cells in the CD34+ population that is easily transduced and
can sustain hematopoiesis for up to 11 months in immune-deficient mice.
The more primitive CD34+CD38 cells mediated
levels of engraftment comparable to the CD34+ population,
but were infrequently marked by retroviral vectors. This system
provides a stringent assay that will allow identification of improved
methods for ex vivo culture and transduction of multipotent human
hematopoietic stem cells.
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MATERIALS AND METHODS |
Supernatant production from vector-producing fibroblasts.
Marking of human hematopoietic cells was performed by using supernatant
from the LN retroviral vector, packaged in the producer cell line
PG13.21 Vector-producing fibroblasts (VPF) were grown in
Dulbecco's Modified Eagles Medium with high glucose (GIBCO-BRL, Gaithersburg, MD), supplemented with 10% fetal calf serum (FCS). VPF
were grown to subconfluency at 37°C in a humidified incubator with
5% CO2, then transferred to 32°C and grown for 48 hours.22 After the vector production period, supernatant
was collected, filtered through a 0.45µm cellulose acetate syringe
filter (Schleicher & Schuell, Keene, NH), and stored at
70°C. The PG13/LN supernatant had a titer of 5 × 106 infectious virions/mL, assayed on the human
cell line HT29 (American Type Culture Collections [ATCC], Rockville,
MD) and was determined to be free of recombinant helper virus by marker
rescue assay on 3T3 (ATCC) and HT29 cells as described.23
Transduction of human hematopoietic cells.
Human bone marrow cells were collected from screens used to filter
marrow during the harvest of normal donors for allogeneic transplantation. Use of the samples was approved by the Childrens Hospital Committee on Clinical Investigations (Los Angeles,
CA). CD34+ progenitors were isolated by
incubation with the monoclonal antibody (MoAb) HPCA-1 (Becton
Dickinson, San Jose, CA), followed by goat antimouse conjugated
immunomagnetic beads (Dynal, Oslo, Norway) as described.16
CD34+CD38 cells were isolated from human
marrow by pre-enrichment of CD34+ cells by using MiniMACS
columns (Miltenyi, Auburn, CA), followed by
fluorescence-activated cell sorting (FACS) acquisition with a stringent
gate (less than half of the fluorescence of the PE isotype control) as
previously described.7 Transduction with stromal support
was conducted in the presence of the cytokines IL-6 (10 ng/mL, R&D
systems, Minneapolis, MN), SCF (c-kit ligand, 50 ng/mL, R&D
Systems), and IL-3 (10 ng/mL, Biosource International, Camarillo, CA),
with and without inclusion of murine FL (FL, 50 ng/mL, generously
donated by DNAX, Palo Alto, CA). Cells were cultured at a
maximal concentration of 1 × 105 cells/mL medium, in
T-25 vent-cap flasks (Costar, Cambridge, MA). Supernatant from PA317/LN
and PG13/LN vector-producing fibroblasts, prepared as described above,
was added to the CD34+ and
CD34+CD38 cells on stromal support, at 24 hour intervals, on days 1, 2, and 3 of culture versus on days 5, 6, and
7 as described.16 Engraftment controls for designated
experiments were CD34+ and
CD34+CD38 cells incubated in the
transduction medium in suspension culture with vector supernatant for
one hour before transplantation into bnx mice. After
transduction, a small portion of the CD34+ cells were
plated in 14-day methylcellulose colony-forming assay with and without
the selective agent G418 (Geneticin, 0.9 mg/mL active: screened lots
from GIBCO/BRL), as described.15 The
CD34+CD38 cells were not plated in
colony-forming assay, because we had previously determined that they
are not clonogenic within that time period.7 The remainder
of each sample was transplanted into a cohort of immune-deficient
(bnx) mice for long-term engraftment and analysis of the marked
progeny of primitive human hematopoietic cells.
Transplantation of immune-deficient mice.
Six-week-old bnx mice bred at Childrens Hospital (Los Angeles,
CA) were used as transplant recipients in all experiments. Transplantation of transduced human hematopoietic progenitor cells was
done as described.15-17 In each experiment, 2,000 CD34+CD38 cells versus 5 × 105 CD34+ cells were transplanted into groups
of 3 to 4 mice per arm. Mice were killed by 75% CO2 25%
O2 narcosis, 9 to 11 months after transplantation with
human cells. Samples of each tissue were taken for DNA preparation for
analysis of vector integration. Bone marrow was flushed from the tibiae
and femurs of each mouse into 1x phosphate-buffered saline (PBS) and
dispersed with a fine needle. The bone marrow cells were then plated at
37°C for 2 to 4 hours in Iscoves Modified Dulbecco's Medium (IMDM)
with 20% heat-inactivated FCS to remove murine stromal elements by
adherence.
Human-specific colony-forming assay and isolation of clones.
After the adherence step, bnx/hu bone marrow cells were
collected, counted, and plated in human-specific methylcellulose-based colony-forming assay with or without G418 as described.15
Human IL-3 was added to a concentration of 10 ng/mL in basal
methylcellulose medium. This medium had previously been shown to
support the exclusive growth of human hematopoietic progenitors, if no
murine stromal cells had contaminated the colony-forming unit (CFU)
dish.15 Methylcellulose, FCS, and BSA were screened to
provide maximal CFU-GEMM development from human
CD34+ cells. A total of 5 × 104 and 1 × 105 plastic nonadherent cells from engrafted and
control mice were plated in duplicate in 1 mL of medium in gridded
culture dishes (Nunc, Naperville, IL), with and without G418 (0.9 mg/mL
active compound, screened lots from GIBCO-BRL). Recombinant human
erythropoietin (Epoietin alpha [Epo], Amgen Corp, Thousand Oaks, CA)
was added to all plates on day 4 of culture to a concentration of 1 U/mL. Colonies were enumerated on day 21, then clones that had attained a size of at least 200 cells in the presence of G418 were plucked from
the methylcellulose in a volume of 40 µL, and flushed
into 1 mL PBS (Irvine Scientific, Santa Ana, CA) in individual
microcentrifuge tubes. The cells were then pelleted and DNA was
isolated as described.17
Clonal analysis by inverse polymerase chain reaction (PCR).
Genomic DNA isolated from each tissue sample and individual human CFU
recovered from the mice was analyzed for the presence of provirus by
PCR for the neo gene as described.15 After
confirmation of vector integration, the clonal integration pattern was
assessed by subjecting DNA from each colony to amplification in the
inverse PCR reaction as described.17 The resulting PCR
products were electrophoresed on a 2% gel (1% Seakem LE
agarose and 1% Nu-Sieve; FMC Bioproducts, Rockland, ME)
and transferred to nylon membrane (Hybond-N+; Amersham,
Arlington Heights, IL). Hybridization was done in SSPE Hybridization
buffer (10X Denhardt's,24 5X SSPE,24 and 0.5%
SDS24) for at least 4 hours at 55°C, with an
oligonucleotide probe (5 GGCAAGCTAGCTTAAGT) specific for LTR sequences,
end-labeled with  -32 P-ATP by T4 kinase (GIBCO-BRL).
After hybridization, blots were washed twice for 5 minutes each at room
temperature in SSPE wash buffer #1 (2X SSPE; 0.1% SDS). Next, blots
were washed for 10 minutes at 55°C in SSPE wash buffer #2 (5X SSPE,
0.1% SDS). Exposures to high-performance autoradiography film
(Hyperfilm-MP, Amersham) were performed for 5 minutes to 2 days.
Antibody labeling and FACS analysis.
Single-cell suspensions from the marrow and spleens of long-term
engrafted bnx mice were preincubated for 15 minutes on ice with unconjugated mouse immunoglobulin (MsIgG; Coulter, Hialeah, FL)
before addition of antibody. Directly conjugated antibodies used to
identify human-specific cell surface antigens were: HLE-1 (anti-CD45,
Becton Dickinson); My9-RD1 (anti-CD33, Coulter); Leu-12 (anti-CD19,
Becton Dickinson); Leu-3a (anti-CD4, Becton Dickinson); and Leu-2a
(anti-CD8, Becton Dickinson). The antimouse CD45 antibody (Pharmingen,
San Diego, CA) was used to identify murine leukocytes. After a
15-minute antibody binding period on ice, cells were depleted of red
blood cells by resuspension in Ortho Lysis Buffer (Becton Dickinson),
washed, and fixed in 1% paraformaldehyde. Samples were acquired on a
Becton Dickinson FACScan and analyzed with the Cellquest software
package (Becton Dickinson). Ten thousand events were acquired for each
sample. In all experiments, parallel staining and FACS analyses were
performed on normal human and nontransplanted bnx mouse bone
marrow controls, to confirm that none of the human-specific antibodies
cross-reacted with murine cells.
Statistical analyses.
All analyses were performed with the Excel 5.0 software
(Microsoft Corp, Seattle, WA). Average values are listed
with standard deviations. Standard error of the mean is used in the
text in cases where all data points are listed in table format. The
significance of each set of values was assessed using the two-tailed
t-test assuming equal variance.
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RESULTS |
Transduction of human colony-forming progenitors.
Human CD34+ and CD34+CD38 cells
were isolated from normal human bone marrow as
described.7,18 The CD34+CD38
cells were acquired by FACS selection as shown in
Fig 1. In the first series of experiments
(n=4), the engraftment and transduction of CD34+ and
CD34+CD38 cells from the same donors
were compared after long-term hematopoiesis in immune-deficient mice. A
portion of each CD34+ and
CD34+CD38 sample was transplanted into the
mice after only 1-hour incubation in suspension culture with cytokines
and retroviral supernatant. The 1-hour transductions were performed to
provide baseline values and engraftment controls to assess the
potential extent of loss of long-term hematopoietic capacity in the
remainder of each population, after a 72-hour ex vivo culture and
transduction period. To accomplish the 72-hour transduction,
CD34+ and CD34+CD38 cells were
plated onto allogeneic, irradiated human stromal cells in transduction
medium including IL-6, IL-3, and SCF. An equal volume of supernatant
from the LN vector was added at the initiation of culture, and again 24 and 48 hours later, as described.16 After transduction, a
small portion of the CD34+ cells was plated in 14-day
methylcellulose colony-forming assay (CFA) with and without the
selective agent G418, as described,15 to ensure that the
transduction of colony-forming cells had worked adequately. The
CD34+CD38 cells were not plated in CFA,
because we had previously determined that they were not clonogenic
within a 2-week time period.7 No G418-resistant CFUs were
obtained from nontransduced marrow plated as controls in these
experiments, or from cells cultured in suspension with supernatant
addition for only 1 hour. The levels of gene transfer into
colony-forming progenitors from the CD34+ populations
transduced for 72 hours, assessed by G418-resistant CFU, ranged from
21.3% to 33.4%, with an average of 27.3 ± 5.1 (n=4).
These data indicate that the ex vivo culture conditions were favorable
for gene transfer into committed, clonogenic progenitors. The levels of
transduction of more primitive progenitors, contained within the
CD34+ and CD34+CD38 populations,
were assessed in the long-term bnx/hu xenograft assay.15-17
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| Fig 1.
Acquisition gate used to define
CD34+CD38 cell population. Region R1 was
defined as the lymphoid gate containing small, agranular cells, as
previously published.7 Quadrants are defined by fluorescein isothiocyanate (FITC) and PE-labeled isotype controls. Region R2 was
used to define CD34+CD38 cells for FACS
acquisition. This region contains CD34+ cells with
PE-CD38 fluorescence less than half of the isotype control.
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Human cell engraftment in immune-deficient mice transplanted with
CD34+ versus CD34+CD38 cells.
The extents of transduction and survival of human cells more primitive
than colony-forming progenitors were assessed after long-term
engraftment in immune-deficient mice. After 72-hours transduction, the
nonadherent cells and the entire adherent population from each
transduction flask were combined and transplanted into cohorts of
sublethally conditioned bnx mice. The cells were allowed to
engraft and to contribute to hematopoiesis in the mice for 9 to 11 months. Then the mice were killed and the marrow was harvested from the
hindlegs. The percentage of human CD45+ cells in the marrow
of each mouse transplanted with human CD34+ or
CD34+CD38 cells was assessed by
fluorescent-activated cell sorting (FACS) analysis
(Table 1). Significantly higher percentages
of human CD45+ cells were obtained in the marrow of mice
that had received CD34+ cells cultured ex vivo for one hour
(5.9% ± 1.5%, N=4) as opposed to 72 hours (2.7% ± 0.5%,
N=10, P = .03). Mice that had received CD34+CD38 cells transduced for 1 hour had an
average human CD45+ cell content of 6.2% ± 3.6 % in
their marrow (n = 5), and there was an average of 3.2% ± 2.8% in
mice that had received human CD34+CD38 cells
transduced for 72 hours (n = 14, P = .07). The extent of engraftment obtained from the sets transplanted with CD34+
and CD34+CD38 cells was not significantly
different. Similar levels of homing and subsequent survival of human
cells in the murine bone marrow were obtained from 2.5 logs less
CD34+CD38 cells than CD34+
cells.
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Table 1.
Human Hematopoietic Lineages Recovered From the Bone
Marrow of bnx Mice 9 to 11 Months
Post-Transplantation
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Comparison of human hematopoietic lineages recovered from mice
transplanted with CD34+ and
CD34+CD38 cells.
The human hematopoietic lineages that had developed during the 9 to 11 month engraftment period were determined by incubating samples of
marrow from each bnx/hu mouse with a panel of MoAbs. The
specificity of each antibody used was determined by testing on human
peripheral blood controls and on marrow from nontransplanted bnx mice. Only antibodies that bound specifically to the
human cells and did not cross-react with the murine cells were used in
the panel. As shown in Table 1, the human hematopoietic lineages that
developed (CD4, CD8, and CD33) in mice transplanted with human
CD34+ or CD34+CD38 cells did not
vary significantly. As we have previously reported,15-17 no
human B lymphocytes developed from any human hematopoietic cell
population engrafted in the bnx mice.
Analysis of tissue and individual, long-lived, clonogenic human
progenitor cell marking.
Human-specific CFAs were plated as described,15 using 3 × 105 marrow cells recovered from each
bnx/hu mouse. The number of human-specific colony-forming
progenitors that developed from each sample is shown in
Table 2. The highest average number of human clonogenic progenitors was obtained from mice that had received CD34+CD38 cells cultured ex vivo for only 1 hour (84.6 ± 19.5). The number was not significantly higher than
the average number of progenitors obtained from the CD34+
cells cultured ex vivo for 1 hour (45.8 ± 16.0, P = .18).
The numbers of human clonogenic progenitors recovered from mice
transplanted with human CD34+ and
CD34+CD38 cells cultured ex vivo for 72 hours did not differ significantly from one another (36.4 ± 8.1
v 55.6 ± 20.7, P = .46).
The levels of transduction of long-term engrafting, clonogenic
progenitors by the neo gene of the LN vector were assessed by
determining the percentages of human-specific CFU that were resistant
to the selective agent G418 (Table 2). No
transduction was observed in cells exposed to the retroviral vector
supernatant for only 1 hour. The highest number of G418-resistant
clonogenic human cells was obtained from mice that had received human
CD34+ cells transduced over a period of 72 hours (Table 2).
Eight of 10 mice from this group contained G418-resistant clonogenic human cells. In contrast, only 2 of 14 mice that had received human
CD34+CD38 cells transduced under the same
conditions harbored G418-resistant human progenitors, with an average
percentage of 0.62 ± 0.52, as opposed to 10.34 ± 2.48 in
the 72-hour CD34+ group (P = .0003).
These data show that, although CD34+ and
CD34+CD38 cells engraft and sustain
long-term hematopoiesis in immune-deficient mice, only the
CD34+ population can be reliably transduced by using
stromal support, 25% FCS, and the cytokines IL-3, IL-6, and SCF.
Long-lived progenitors from the CD34+CD38
population were only sporadically transduced.
Genomic DNA was extracted from the marrow and tissues recovered from
each of the long-term engrafted bnx mice, and subjected to
PCR for the neo gene, to determine whether or not gene marking of long-lived human hematopoietic cells had occurred in human hematopoietic progenitors or their differentiated progeny residing within each organ (representative blots are shown in
Fig 2). In this set of experiments, marking
was observed primarily in the marrow, with no cells positive for the
neo gene in the other organs, except for several spleen
samples. The results of the PCR of whole tissues were concordant with
detection of G418-resistant progenitors.
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| Fig 2.
PCR for the neo gene in bnx/hu bone
marrow and tissues. DNA was extracted from the bone marrow and tissues
of each long-term engrafted mouse, and subjected to PCR to detect the
presence of the neo gene, to determine whether or not gene
marking of long-lived human hematopoietic cells had occurred in human
hematopoietic progenitors or their differentiated progeny residing
within each organ. Hybridization with a neo-specific
oligonucleotide probe was done to ensure that the product band had been
correctly amplified. Tissues that were positive and negative for the
presence of the neo gene were amplified as controls in each
reaction. The results from one set of mice are shown.
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Transduction of human CD34+ versus
CD34+CD38 cells on days 1, 2, and 3 versus
5, 6, and 7 with and without inclusion of FL.
Because the transduction protocols that had been successful for
transducing CD34+ progenitors were inadequate to mediate
gene transfer into CD34+CD38 cells, we next
tried a strategy that would allow more time for the primitive cell
population to be induced to cycle. We compared transduction on days 1, 2, and 3 to transduction on days 5, 6, and 7, in the presence and
absence of FL. After the designated transduction period, 2,000 cells
were transplanted per bnx mouse. Tissues and bone marrow
cells were harvested and analyzed after 9 to 11 months engraftment.
The levels of engraftment and the numbers of transduced and total human
colony-forming progenitors recovered from the marrow of each mouse are
shown in Table 3. The mean level of human
cell engraftment was 1.85% ± 0.54% in mice that had received human CD34+CD38 cells transduced on days 1, 2, and
3 in 3/6/S, and 3.1% ± 0.93% in mice that had received cells
transduced on days 1, 2, and 3 in 3/6/S/FL (P = .27, not a
significant difference). CD34+CD38 cells
maintained in culture for 1 week, and transduced on days 5, 6, and 7, required the presence of FL during culture to sustain the ability to
reconstitute the bnx mice. Cells that were cultured for 7 days in 3/6/S gave an average engraftment of only 0.08% ± 0.05%
human CD45+ cells, whereas cells cultured 7 days in
3/6/S/FL produced a significantly higher average extent of engraftment
(3.15% ± 0.72%, P = .005, Table 3).
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Table 3.
Engraftment, Transduction, and Long-Term Clonogenic
Capacity of Human CD34+CD38 Cells
Transduced on Days 1, 2, and 3 Versus 5, 6, and
7
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The number of engrafted human CD45+ cells per 1 million
bnx bone marrow cells was calculated from the FACS data
obtained (Table 1), and an estimate of the total number of cells that
would be engrafted in the marrow of the entire mouse was extrapolated
(Table 4). The numbers given assume that
all transplanted cells engrafted in the marrow, and that the number of
marrow cells per mouse is 8 × 107.9,25,26
The fold expansion that the 2,000 transplanted
CD34+CD38 cells had undergone to reach the
final engraftment levels, as detected by FACS analysis, is given. It is
likely that only 10% to 20% of the marrow cells had actually homed to
the marrow,27 in which case the fold expansions given in
Table 4 would be greatly underestimated. In accordance with
observations made in LTCIC and NOD/SCID systems,9,12,28 the
capacity for cell proliferation from
CD34+CD38 cells is immense, greater than
1,000-fold.
The clonogenic capacity of human cells recovered from mice transplanted
with human CD34+CD38 cells transduced for 3 versus 7 days with and without FL was assessed next. In accordance with
the engraftment results, the cells cultured on stromal support for only
three days ex vivo were not affected by the presence or absence of FL.
The average number of human CFU recovered per 3 × 105
bnx bone marrow cells was 34.8 ± 12.2 from cells
maintained ex vivo in 3/6/S and 121 ± 51.7 from those kept
in 3/6/S/FL (P = 0.14). In contrast, there was a significant
difference in the average number of clonogenic progenitors recovered
from mice transplanted with CD34+CD38 cells
cultured for seven days with and without FL
(Fig 3). The average number of human CFU
recovered per 3 × 105 bnx bone
marrow cells was 2.25 ± 1.4 from cells cultured in 3/6/S and 212.8 ± 29.5 from those cultured in 3/6/S/FL (P = .0004; Table 3).
The numbers of clonogenic human progenitors that grew from 1 × 106 bone marrow cells recovered from mice in each
transduction group are compared in Table 4. Maintaining the
CD34+CD38 cells for 7 days in medium
containing IL-3, IL-6, and SCF without inclusion of FL resulted in
significantly lower levels of progenitor maintenance than obtained from
any other ex vivo culture condition.
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| Fig 3.
Survival of clonogenic human progenitors in
bnx mice transplanted with
CD34+CD38 cells cultured for 3 versus 7 days with and without inclusion of FL. Human-specific colony-forming
assays were plated from marrow recovered from the long-term engrafted
mice from experiments #5 and #6. Colonies were counted after 14 to 21 days of growth.
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The levels of transduction of human hematopoietic cells engrafted in
the different mice were determined by analyzing total marrow and
tissues by neo PCR. Marked bone marrow was only detected in two
mice that had received the CD34+CD38 cells
cultured for 7 days in the presence of FL, with transduction on days 5, 6, and 7 (Table 3). No marking was seen in any tissue from mice that
had received cells cultured for 3 days, with or without FL, or from
mice that had received human cells cultured for 7 days in the absence
of FL. The neo PCR results from selected tissues of one
of the sets of mice are shown in Fig 4.
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| Fig 4.
PCR for detection of the LN vector in bnx/hu
bone marrow and tissues. DNA was extracted from the bone marrow and
tissues of mice from experiments 5 and 6, after 9 to 11 months
engraftment. Each sample was subjected to PCR to detect the presence of
the neo gene. Hybridization with a neo-specific
oligonucleotide probe was done to ensure that the product band had been
correctly amplified. Tissues that were positive and negative for
the presence of the neo gene were amplified as controls in each
reaction. The results from one experiment are shown.
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Determination of the number of individual, marked human hematopoietic
stem and progenitor cells by inverse PCR.
We next determined the number of individual, marked human stem or
progenitor cells that had contributed to hematopoiesis over the 9 to 11 month engraftment period, in mice selected from each transduction
group. Single-colony inverse PCR, a sensitive technique that can be
performed on individual clones containing at least 200 cells, was
used.17 Cryopreserved marrow from mice that harbored marked
human cells, previously detected by PCR for the neo gene, was
thawed and labeled with antibodies to human CD45 plus CD33 to isolate
myeloid cells, and antibodies to human CD45 plus CD3 to isolate T
lymphocytes. Human T cells and myeloid progenitors (acquired with a
lymphoid gate) were then deposited individually into 96-well plates by
using the automated cell deposition unit (ACDU) of a FACSVantage
(Becton Dickinson), and grown into small colonies as
described.17 The clonal integration patterns obtained from
human colonies recovered from five mice transplanted with human
CD34+ cells transduced for 72 hours in 3/6/S showed
oligoclonal marking of long-lived lineage-restricted progenitors with
limited expansion within the marrow, as we have previously
described.17,29 The mouse that was transplanted with human
CD34+CD38 cells cultured for 72 hours in
3/6/S, and that had 17/240 G418-resistant human CFU (shown in Table 2),
had clonal integration patterns in individual colonies that revealed
that at least four different myeloid progenitors had been transduced
(Fig 5A). Two G418-resistant myeloid
colonies were obtained from the second mouse transplanted by the same
marrow, and both colonies were derived from a fifth progenitor, not
detected in the first mouse. No marked T lymphocytes were found in
either mouse, and 12 other mice in experiments one through four that
had received human CD34+CD38 cells
transduced under the same conditions contained no marked human cells 9 to 11 months post-transplantation. These data show that, while
occasionally marking of a small portion of a
CD34+CD38 cell population can occur on
stromal support in 72-hour transductions with IL-3, IL-6, and SCF, the
method is not a reliable way to achieve high extents of transduction of
this primitive hematopoietic cell population.
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| Fig 5.
Clonal analysis by inverse PCR of marked human T-cell and
myeloid colonies recovered from mice transplanted with
CD34+CD38 cells. (A)
G418-resistant human CFU-GM was recovered from the marrow of
2/14 mice transplanted with CD34+CD38
cells subjected to a 72-hour transduction. The clonal diversity of the
individual, marked human myeloid colonies was assessed by single-colony
inverse PCR. Colonies shown are from the mouse that had 17/240
G418-resistant progenitors (Table 2). (B) Human T-cell
and myeloid colonies were grown from mice transplanted with
CD34+CD38 cells transduced for 7 days with
FL (Table 3). Colonies from two of the four mice were shown to contain
the neo gene by PCR, then were futher subjected to
single-colony inverse PCR. Panels B and C show amplified inverse PCR
products from marked T-cell and myeloid colonies obtained from each
mouse. T = T lymphoid clones. M = myeloid clones.
|
|
Next, mice transplanted with human CD34+CD38
cells transduced for 7 days in the presence of FL were analyzed. The
two mice that had neo-positive cells in their marrow (Fig 4)
had both T lymphocytes and myeloid progenitors that bore LN provirus,
as determined by single-cell cloning and neo PCR. Inverse PCR
from the neo-positive clones recovered from those mice revealed
that in the first mouse, at least five different
CD34+CD38 cells had been transduced, and had
survived in the mice to generate clonogenic progeny after 10 months
(Fig 5B). The second mouse harbored marked human myeloid and T-cell
colonies derived from at least three different, transduced
CD34+CD38 cells (Fig 5C).The blot shown in
Fig 5C has one band that is of similar molecular weight in all samples.
However, the amplified product of the second LTR and flanking
genomic DNA is of a different size in three samples, indicating that
those progenitors were derived from unique precursors with different
proviral integration sites. No T-cell and myeloid clones bearing the
same proviral marker were detected in the current studies.
In conclusion, addition of FL to medium containing IL-3, IL-6, and SCF
during 168-hour culture periods was necessary to allow maintenance of
the capacity of the human progenitors to sustain subsequent long-term
hematopoiesis. The extended transduction with FL allowed multiple
progenitors to be marked, an event that had occurred in only one out of
26 mice transplanted with CD34+CD38 cells
cultured for only 72 hours (experiments 1-6).
| |
DISCUSSION |
From this series of studies, it was shown that CD34+ cells
from human marrow contain a population of cells that can be transduced by retroviral vectors, and generate gene-marked progeny in
immune-deficient mice for up to 11 months. In contrast,
CD34+CD38 cells engraft to comparable
extents in the mice, but are not easily transduced by Moloney Murine
Leukemia Virus-based vectors in a 72-hour period, in the presence of
the cytokines IL-3, IL-6, and SCF. Adding FL to the transduction
medium, and increasing the duration of ex vivo culture from 3 to 7 days, with vector addition on days 5, 6, and 7 slightly increased the
chance of obtaining transduction of human
CD34+CD38 cells capable of sustaining
long-term hematopoiesis in the mice. We and others had previously shown
that FL is a survival factor for primitive hematopoietic
cells.18-20 During a 7-day culture period, FL may be acting
to sustain survival of the primitive cells until a small-percentage
exit quiescence and become susceptible to transduction by Moloney
Murine Leukemia-based retroviral vectors, which require cycling of the
target cells to allow integration.11
In support of the theory that FL may be acting at the level of survival
of primitive cells, we observed that few human CFU were recovered from
long-term engrafted bnx mice transplanted with human
CD34+CD38 cells cultured for 7 days with
stromal support, in the presence of IL-3, IL-6, and SCF. However,
addition of recombinant FL to the same culture conditions supported a
significant increase in maintenance of the clonogenic capacity of the
CD34+CD38 cells during the 7-day
transduction period. The fact that cells cultured ex vivo for 7 days in
IL-3, IL-6, and SCF without FL had minimal maintenance of clonogenic
capacity, despite the presence of stromal support during the culture
period implies that the levels of FL produced by irradiated stroma are
insufficient to sustain the long-term clonogenic capacity of cells that
are able to give rise to long-term hematopoiesis in mice, during
extended culture periods.
The vector titer per cell (multiplicity of infection) was higher when
transductions were done by using CD34+CD38
cells, but a higher level of transduction was not achieved. These data
indicate that it is not the amount of vector that is rate limiting for
effective gene transfer into the primitive population, but rather
factors intrinsic to the cells. The
CD34+CD38 population has been reported to
have lower expression of the receptors that mediate vector entry into
the cell.30 The degree of quiescence of the target cell
population is also a confounding factor.31 We have
previously determined that CD34+CD38cells
with the phenotype used in this study are not easily recruited into
cycle by cytokines, but the entry occurs individually over time in
culture.7,18,28 This apparently stochastic property of the
primitive cell population may explain why there was 1 out of 14 mice
that had several long-lived human hematopoietic stem or progenitor
cells transduced during a 72-hour culture. Random entry of one
primitive cell into cycle could generate several dividing progeny that
would each be susceptible to retroviral-mediated transduction.
There are two possibilities to explain the presence in the mice of
marked human hematopoietic cells from CD34+ populations and
the paucity of marked cells in mice transplanted with transduced
CD34+CD38 populations. One possibility is
that the primitive CD34+CD38 cells were
transduced more efficiently when surrounded by the CD34+/CD38+ cells, which might have provided
some accessory factors that enhanced cell cycle progression, allowing
retroviral integration. The second possibility is that there is a
population of CD34+CD38dim cells that are
easily transduced (in contrast to the stringently gated
CD38 population used in the current studies), and are
able to maintain long-term hematopoiesis in immune-deficient mice.
There have been several reports that the
CD34+/CD38+ population can mediate only
short-term hematopoiesis.8,9,25 However, in those reports,
a population expressing CD38 with more intensity than the cells used in
our studies was included in the CD38 fraction, and may
represent long-lived committed progenitors. The
CD34+CD38+ fractions described in the previous
publications thus excluded the CD34+/CD38dim
fraction, whereas our CD34+ populations included it, and
our CD34+CD38 populations stringently
excluded it. Future studies will determine whether cells isolated from
the CD34+CD38dim region can sustain engraftment
for 1 year in bnx mice, and are responsible for the higher
level of marking that is obtained from the total CD34+
populations.
In the current studies, 2,000 CD34+CD38
cells engrafted bnx mice to levels comparable to 5 × 105 CD34+ cells. Our previous studies had shown
the CD34+CD38 population, acquired as shown
in Fig 1, to represent 0.02% of the total mononuclear
fraction.7 CD34+ cells are present at an
average of 1% of the mononuclear cells. The comparable extents of
engraftment we obtained from the cell numbers transplanted indicate
that the most primitive cells within each population, capable of
engrafting bnx mice, might not be present at the frequencies
predicted by simple analysis of cell surface expression. The group
headed by John Dick has done elegant studies to quantitate the SCID
mouse reconstituting cell (SRC) by limiting dilution, and found that
they are present at a frequency of one SRC in 617 CD34+CD38 cells.9,25,26 We have
observed that 1,000 CD34+CD38 cells give
rise to detectable human cell engraftment in 38% of the mice tested (n = 8), whereas 2,000 cells engraft 94% of the mice (n = 34), after a
72-hour transduction period with stroma and cytokines (Dao and Nolta,
unpublished data). Calculation of reconstituting cell
numbers from our experiments is complicated by the fact that some
period of ex vivo culture and transduction is always done, and may
alter the capacity of the cells to home to the correct sites or to
sustain long-term engraftment as has been shown with murine
marrow.27 However, in the current studies, we found that
CD34+CD38 cells cultured for 72 hours did
not give rise to significantly different levels of CD45+ or
clonogenic progenitor cell engraftment than the same population of
cells held in suspension culture for only 1 hour before
transplantation.
The current data show that CD34+CD38 cells
are resistant to transduction with Moloney-based retroviral vectors.
Our studies provide a system for evaluating the success of new methods
for hematopoietic stem cell transduction. To achieve transduction, the
cells must be prompted into mitosis, while maintaining
pluripotentiality, or alternatively must be transduced by vectors that
integrate into nondividing cells. Alternate cytokines, transduction
systems such as fibronectin support to replace the irradiated monolayer of stroma, and vectors such as the lentiviral system described by
Naldini et al32-36 may prove to be more useful for
transduction of CD34+CD38 cells.
| |
FOOTNOTES |
Submitted August 1, 1997;
accepted October 8, 1997.
Supported by a grant from the NIH NHLBI (SCOR #1-P50-HL54850-03).
J.A.N. was also supported by the John Connell Gene Therapy Foundation.
G.M.C. was supported in part by a Translational Research Grant from the
Leukemia Society of America (#6360-97) and Grants No. NCI # 2CA14089-21
and 5P01 CA59318-05.
Address reprint requests to Jan A. Nolta, PhD, Division of Research
Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles,
4650 Sunset Blvd, Mailstop #62, Los Angeles, CA 90027.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
C. H. Hannum at DNAX generously provided the recombinant Flt3 ligand
used in these studies. Don Kohn, Ken Weinberg, Craig Jordan, and
Robertson Parkman provided useful discussion. This work was made
possible by Sally Worttman, head of our animal facility, and Renee
Traub-Workman, due to their dedication in maintaining an immaculate
bnx mouse colony.
| |
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Abstract
Introduction
Abstract