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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3210-3217
Long-Term Contribution to the Myeloid Compartment by
Lineage-Committed Stem Cells
By
Chiann-Chyi Chen,
Amariliz Rivera,
Naomi Ron,
Natalie Sutkowski,
Joseph P. Dougherty, and
Yacov Ron
From the Department of Molecular Genetics and Microbiology,
University of Medicine and Dentistry of New Jersey, Robert Wood Johnson
Medical School; and the Graduate Program in Microbiology and Molecular
Genetics, Rutgers University, Piscataway, NJ.
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ABSTRACT |
The current paradigm concerning the kinetics of hematopoiesis is
that only the most primitive pluripotential bone marrow stem cells can
support prolonged hematopoiesis whereas more differentiated, lineage-committed stem cells can only contribute to a particular lineage for a limited period of time. In this study, we present evidence that in mice, the spleen contains a long-lived
myeloid-committed stem cell population(s) that continuously replenishes
the mature myeloid lineage for at least 9 months. After
myeloid-specific retroviral-mediated gene transfer, the exogenous gene
could be detected in thioglycollate-induced macrophages and
granulocytes by Southern blot analysis and by in situ polymerase chain
reaction on an individual cell basis. The targeted stem cell population does not repopulate the bone marrow in secondary recipients and did not
give rise to cells other than cells of the myeloid lineage. It
therefore represents the first nonpluripotential stem cell population
capable of replenishing a hemopoietic lineage for a long period of
time. The ability to target a myeloid-specific stem cell could
facilitate gene therapy of congenital disorders of the myeloid system
such as lysosomal storage diseases. It also offers a unique opportunity
to assess the immunologic consequences of expressing an exogenous gene
of choice exclusively in the myeloid lineage.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ALL HEMATOPOIETIC lineages including the
myeloid lineage develop from pluripotential stem cells (PSC, also
called primitive stem cells) which in adult mice are found in bone
marrow (BM) and spleen.1 The accepted definition of a PSC
is that it is capable of self-renewal and that it can differentiate
into all lineages of the hematopoietic system.2-4 Thus far,
a cell surface marker unique for PSC has not been identified and
therefore they have not yet been purified to homogeneity. PSC are rare
in BM (1 in 2,000 to 5,000 cells) and are contained within a population of BM cells with the phenotype
Thy-1.1lo,Lin-,Sca-1+.2,3,5-7
When this population is purified by negative selection with antibodies
and complement followed by positive selection by fluorescence-activated
cell sorter (FACS), it contains all BM PSC.2,3,5-7 However,
the PSC constitute only 10% of that population.6,8 The
rest of the cells in this population are a mix of various
uncharacterized, more differentiated stem cells.
The first characterization of BM stem cells was described in the early
1960s by Till and McCulloch9 as cells that form spleen
nodules containing highly replicating hematopoietic stem cells, which
they termed colony-forming-units-spleen (CFU-S), 8 to 12 days
postreconstitution of irradiated recipients with adult BM cells. It was
later realized that the spleen nodules only contained myeloid and
erythroid precursors and only have a transient capacity to replenish
the myeloid and erythroid lineages.10 More recent studies
have shown that only the most primitive pluripotential BM stem cells
can support hematopoiesis for long periods of time.2,11-13 This notion is mostly based on experiments in which the reconstitution potential of the PSC-containing subpopulation
(Thy-1.1loLin-Sca-1+ cells) was
compared with that of the remaining BM cells. In these experiments,
only the PSC-enriched population could protect a lethally irradiated
host indefinitely.2,6,7
Further support for this notion came from studies on the kinetics of
hematopoiesis. Studies using retroviral-mediated tagging of BM-derived
PSC with a reporter gene have shown that for a short time immediately
after the reconstitution of a lethally irradiated mouse, many stem cell
clones contributed to hematopoiesis. However, a few weeks later, only
very few clones were contributing to the continuous replenishment of
all hematopoietic lineages.14,15 These results suggested
that for a short time after reconstitution, both lineage-committed as
well as PSC contributed to hematopoiesis, but the more mature,
lineage-committed cells are short-lived and therefore disappear within
a few weeks. Similar conclusions were reached by Harrison et
al.16 Using a competitive repopulation assay, these
investigators showed that the appearance of all three hematopoietic
lineages is highly correlated with respect to time, which they
interpreted as an indication that most donor cells arise from the same
PSC, and therefore, lineage-committed precursors present in the
inoculum could not have contributed significantly to any of the
lineages. In contrast to the finding regarding BM-derived stem cells,
in this paper we describe the identification of a splenic population of
very long-lived myeloid-committed stem cells that can continuously
replenish the myeloid lineage. Lipopolysaccharide (LPS)-stimulated,
T-cell-depleted spleen cells obtained from normal or B-cell-deficient
mice were infected with a retroviral vector containing the neomycin
phosphotransferase (neo) marker gene and were used to rescue
lethally irradiated syngeneic recipients. In thioglycollate-induced
granulocytes and macrophages, 0.5 to 1 copy/cell of the neo
gene could be detected by Southern blotting for at least 9 months after
reconstitution. In situ polymerase chain reaction (PCR) showed the
presence of the gene in greater than 80% of induced granulocytes and
macrophages in the peritoneal cavity 12 months after reconstitution.
The targeted stem cell in the spleen was not a self-renewing PSC
because secondary, lethally irradiated recipients reconstituted with BM
obtained from primary recipients never contained the exogenous gene in
myeloid or any other cell type. Further indication that PSC were not
targeted was the fact that the lymphoid lineage (T cells) were
invariably negative. Because the average life of macrophages is only a
few days,17 and that of mature granulocytes is only a few
hours,18 these results indicate that the targeted
lineage-committed stem cell population was the main replenishing source
for the myeloid lineage for at least 9 months. Based on these results
and the previous studies cited above, the splenic cells targeted in
these experiments would have to be classified as myeloid precursors at
an earlier differentiation stage than CFU-S cells.
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MATERIALS AND METHODS |
Mice.
C57Bl/6, (BALB/C × C57Bl/6)F1 and B-cell-deficient (µ knockout) C57Bl/6 male19 and female mice were purchased
from the Jackson Laboratory (Bar Harbor, ME).
Retroviral vectors and virus-producing cell line.
pN220 and pAsADA
(Fig 1) are Moloney murine leukemia virus
(MLV)-based retroviral vectors that contain the neo gene, which
is expressed from the MLV long terminal repeat (LTR) promoter.
pAsADA contained, in addition to the neo
gene, the human adenosine deaminase (ADA) gene driven
by its own minimal promoter.21,22 N2 and AsADA
virus-producing cells were previously described.22 Briefly, the NIH 3T3-based ecotropic murine packaging cell line
GP+E-8623 were transfected with 5 µg of pN2 or
pAsADA retroviral vectors using the polybrene/dimethyl
sulfoxide shock method, followed by selection with G418 (0.35 mg/mL;
GIBCO-BRL, Grand Island, NY). The retroviral titer for both
virus-producing cell lines ranged from 1.5 to 2.0 × 107 CFU/mL.
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| Fig 1.
Schematic diagram of the retroviral vectors used in gene
transfer. Retroviral vectors N2 and AsADA are Moloney murine leukemia
virus-based vectors. N2 contains the neo gene, expressed from
the viral LTR promoter. AsADA contains neo, expressed from the
viral LTR promoter, and the human ADA gene, expressed from its
endogenous promoter, As. Relevant restriction enzyme sites and
the distance between them are indicated.  + represents
the packaging signal. The 930-bp EagI-AvaI fragment in
the neo coding region was used as a probe in experiments
described in Figs 2 and 3.
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Gene transfer.
The gene transfer protocol was performed as follows: enriched B-cell
populations were prepared from the spleens of C57Bl/6, (BALB/C × C57Bl/6)F1 or B-cell-deficient (µ knockout) C57Bl/6 mice by
depleting T cells with monoclonal antibody J1j, (rat IgM anti-mouse
Thy-1.2),24 plus complement treatment. The remaining cells
were stimulated for 24 hours with 50 µg/mL LPS and then cocultivated
with a monolayer of irradiated (1,600 R) N2 or AsADA virus-producing cells in the presence of 6 µg/mL polybrene. The nonadherent cells were collected and washed 24 hours later, and 4 to
15 × 106 of the cells were injected
intravenously into each lethally irradiated (1,000 R) (BALB/C × C57Bl/6)F1 recipient mouse.
Cell preparation, Southern blotting analysis, and ADA assay.
Thioglycollate was injected intraperitoneally 24 or 96 hours before the
isolation of myeloid cells to induce the recruitment of granulocytes
and macrophages to the peritoneum of the reconstituted recipients. Some
of the peritoneal exudate cells (PEC) were fixed onto glass slides for
in situ PCR and hybridization as described below. Spleen cells were
isolated from the same mice from which the PEC were isolated. Part of
the spleen cells were injected into the lethally irradiated secondary
recipient mice and some of the spleen cells were treated with the
monoclonal antibody J11d (rat IgM anti-mouse heat stable
antigen)24 or J1j and complement before the extraction of
genomic DNA, followed by Southern blotting or ADA assay. Southern
blotting was performed according to standard procedures. For detection
of the neo marker gene, 10 µg/lane of SacI-digested
genomic DNA was screened using a 32P-labeled probe, an
EagI-AvaI fragment from the neo coding region. To determine the clonality of the transduced population, 10 µg/lane of BamHI- or HindIII-digested genomic DNA was screened
using the same probe as above.
The ADA assay was described previously.22 Briefly, 1 × 106 target cells were lysed by multiple
freezing/thawing cycles, and samples were separated by electrophoresis
on cellulose acetate plates. Enzyme activity was developed by an agar
overlay containing adenosine, nucleoside phosphorylase, xanthine
oxidase, phenazine methosulfate, and dimethylthiazol diphenylterazolium
bromide.
In situ PCR and hybridization.
PEC were fixed onto glass slides with 4% paraformaldehyde, washed, and
dehydrated. The cells were then permealized with proteinase K and
sealed with the PCR mixture containing the neo specific primers
5 -CAGGATGATCTGGACGA and 3-TGGATGCCGACGGATTTGCA. Cycling conditions were 94°C, 1 minute; 55°C, 1 minute; and 72°C, 1 minute 30 seconds for 30 cycles. After the PCR reaction was completed, the slides were washed and incubated with the hybridization mixture containing a 404-bp neo-specific biotinylated probe
complementary to the PCR-amplified sequence excluding the primer
region. Streptavidin was then added (to amplify the signal) followed by
biotin-conjugated alkaline phosphatase. Color was developed using
bromochloroindoyl phosphate (BCIP)/nitro blue tetrazolium
(NBT).25 The cells were analyzed on a
Bright-field phase, differential interference contrast microscope
(Zeiss, Germany). Images were captured using a Dage CCD72 camera and
Dage DSP2000 digital signal processor (Michigan City, IN) (capable of
on-chip integration for low-light situations), and a Macintosh Quadra
700 (Cupertino, CA) with a Scion LG-3 frame grabber board.
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RESULTS |
Retrovirus-mediated gene tagging of a long-lived myeloid precursor
population in the spleen.
Previously, we developed a very efficient gene transfer protocol for
the introduction of exogenous genes into highly purified lymph node
(LN) B and T cells for other purposes.22,26,27 We noticed
that when T-cell-depleted spleen cells (rather than LN cells) were
used as the target cells in this system, cells harboring the exogenous
gene could also be detected in nonlymphoid tissues such as liver and
lungs as well as in spleen and LN, and in some experiments, in low
levels also in the thymus (data not shown). Because signals were
obtained from organs that normally do not contain significant numbers
of B cells, this raised the possibility that some spleen-residing stem
cells were also targeted and gave rise to tissue-residing myeloid
cells. To check this possibility, thioglycollate-induced granulocytes
and macrophages obtained from animals reconstituted with retroviral
vector-targeted BM cells were directly assayed for the presence and
expression of an exogenous gene introduced by the retroviral vector.
LPS-stimulated, T-cell-depleted spleen cells obtained from (BALB/c × C57Bl/6)F1 or C57Bl/6 mice were infected by cocultivation with
packaging cell line producing the N2 vector virus (Fig 1) and
adoptively transferred into lethally irradiated F1 recipients. Gene
transduction was dependent on LPS stimulation but independent of T-cell
depletion, although T-cell depletion results in a higher transduction
efficiency (data not shown). A total of 30 recipient mice were injected
with thioglycollate 6 to 9 months after adoptive transfer to induce
granulocyte and macrophage migration into the peritoneal cavity. As
assessed by  -naphthyl acetate esterase staining
(macrophage-specific) versus naphthol AS-D chloroacetate esterase
staining (granulocyte-specific), 24 hours after injection of
thioglycollate, greater than 95% of the cells obtained from the
peritoneum were granulocytes, whereas 96 hours after thioglycollate injection, greater than 95% of the cells were macrophages (data not
shown). Six and nine months posttransfer, approximately 1.0 to 2.0 proviral copies/cell were detected in both peritoneal granulocytes and
macrophages by Southern blotting (Fig 2A).
DNA extracted from granulocytes (24 hours PEC) was analyzed in lanes 1 and 3 at 6 and 9 months postreconstitution, respectively (Fig 2A).
Lanes 2 and 4 show the same analysis but for macrophages (96 hours
PEC). Lanes 5 through 8 represent DNA from the spleens of the same
animals. Lanes 9 and 10 represent DNA from the same spleen cells used
in lanes 5 and 8, respectively, but after treatment with the monoclonal antibody J11d and complement. J11d is a cytotoxic antibody specific for
the murine heat stable antigen. It removes 80% to 90% of splenic B
cells but spares macrophages. The results indicate that in this experiment, approximately half of the signal (as measured by gel densitometry) obtained from the spleens was due to resident
macrophages.
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| Fig 2.
Gene transfer into the myeloid lineage but not
pluripotential hematopoietic stem cells. (A) Southern blot analysis of
PEC and spleen cells from four representative lethally irradiated mice
reconstituted with 15 × 106 N2-infected T-cell-depleted
spleen target cells 6 and 9 months earlier. Genomic DNA was prepared
from spleen and PEC 24 and 96 hours after injection of thioglycollate
and digested with the restriction enzyme SacI and probed using
a neo-specific probe. Lanes 1 and 3 represent DNA from
granulocytes (24 hours) and lanes 2 and 4 represent DNA from
macrophages (96 hours) as indicated by the lettering below the lanes.
The last four lanes represent SacI-digested pN2 plasmid DNA
equivalent to 0.5, 1.0, 2.0, and 5.0 copies/cell, respectively. The
middle lanes represent DNA prepared from whole spleen cells (lanes 5 through 8) or from J11d-treated spleen cells (lanes 9 and 10) obtained
from the same mice used for harvesting PEC. (B) Genomic DNA extracted
from PEC or spleen cells obtained from three representative secondary
mice previously reconstituted with 8 × 106 N2-infected,
B- and T-cell-depleted BM or spleen cells obtained from primary
recipients were analyzed by Southern blotting. Lanes 1 and 2 represent
DNA from macrophages (96 hours PEC) taken from mice reconstituted with
BM cells from positive primary mice. Lane 3 represents DNA from
macrophages (96 hours PEC) taken from a mouse reconstituted with spleen
cells from a positive primary mouse. Lanes 4 through 6 represent the
same mice except that the DNA was extracted from spleen rather than
PEC. The last four lanes represent SacI-digested pN2 plasmid
DNA equivalent to 0.5, 1.0, 2.0, and 5.0 copies/cell, respectively. (C)
Genomic DNA extracted from PEC, spleen, or thymus obtained from mice
reconstituted 4 months earlier with 4 × 106 N2-infected,
T-cell-depleted BM taken from syngeneic, C57Bl/6 µ knockout mice.
Lanes 1, 2, and 3 represent DNA from macrophages (96 hours PEC),
spleen, and thymus, respectively. Lanes 4 through 7 represent
SacI-digested pN2 plasmid DNA equivalent to 0.125, 0.25, 0.5, and 1.0 copies/cell, respectively.
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The donor origin of the hematopoietic system in the target animals was
verified by flow cytometric analysis of both macrophages and B cells in
recipient F1 mice reconstituted with parental C57Bl/6 target cells
using major histocompatibility (MHC) class I- and II-specific
antibodies. The degree of chimerism was checked directly on PEC with an
anti-H-2Dd antibody and on spleen cells with MHC class
II-specific anti-I-Ek,d antibodies. C57Bl/6 do not express
I-E and therefore spleen cells from a C57Bl/6  (C57Bl/6 × BALB/c)F1 should not stain with this antibody.
Figure 3 clearly indicates that the myeloid
and lymphoid (B cells) lineages in the reconstituted animals were
derived entirely from donor stem cells (H-2b+,
I-E ). Taken together, these results indicate a very
efficient infection of a precursor cell population that contributes to
the myeloid lineage for at least 9 months.
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| Fig 3.
Myeloid and B cell in C57Bl/6 (BALB/c × C56Bl/6)F1 chimeras are of donor origin. PEC and spleen cells obtained
from C57Bl/6 (BALB/c × C57Bl/6)F1 or (BALB/c × C57Bl/6)F1 were stained with fluorescein isothiocyanate-labeled
34-4-21S (anti-Dd) and 14-4-4 (anti-I-Ek,d)
antibodies, respectively, and analyzed by FACS.
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Pluripotential hematopoietic stem cells were not infected during gene
transfer.
The next set of experiments was aimed at determining whether
pluripotential hematopoietic stem cells as opposed to rather later-stage, lineage-committed progenitor cells were targeted. Two
assumptions could be made if pluripotential cells were targeted in the
initial inoculum. The first is that all hematopoietic lineages in the
reconstituted animals should harbor the marker gene. This was not the
case in any of the mice tested. Southern blots were always negative for
the marker genes in B-cell-depleted LN (Fig 2A, lane 12), and in most
experiments was also undetected in thymus (Fig 2A, lane 11). In some
experiments, a weak signal can be detected in the thymus (Fig 2C, lane
3), which is not surprising because this organ does contain a small
fraction of BM-derived macrophages. The second is that BM cells from
the reconstituted animals should be able to transfer the marker gene
into a secondary lethally irradiated host. To test this, BM or spleen
cells from positive primary recipients were used to reconstitute
secondary lethally irradiated recipients. To ensure that the cells
transferred into secondary recipients were depleted of mature B cells
that might contain the exogenous gene, donor BM cells were first
treated with J11d and complement (to remove B cells) and also with J1j (anti-Thy-1 monoclonal antibodies, to remove mature T cells) and complement. Over 30 secondary recipients reconstituted with BM obtained
from primary positive mice were examined and proviral sequences could
never be detected by Southern blotting in 96 hours PEC (Fig 2B, lanes 1 and 2) and spleen (Fig 2B, lanes 4 and 5).
In contrast to BM, the spleen should contain the targeted myeloid
precursor cells and therefore, in parallel, secondary irradiated animals were reconstituted with 8 × 106 B- and
T-cell-depleted spleen cells obtained from positive primary reconstituted mice. In these experiments, all secondary recipients were
positive for proviral sequences both in 96 hours PEC (Fig 2B, lane 3)
and in spleen (Fig 2B, lane 6). The signals are weaker here (0.1 to 0.4 copies/cell for PEC) probably because only 8 × 106
spleen cells were transferred; however, all six mice tested were positive. T cells isolated from spleen, LN, and thymus of these mice
(secondary recipients) were invariably negative (data not shown). These
results strongly suggest that pluripotential stem cells were not
targeted during the first infection.
Because the infection protocol using LPS-activated, T-cell-depleted
spleen cells as target cells results in the transduction of both
myeloid precursors and mature B cells, the next set of experiments was
designed to rule out the possibility that some of the signal is due to
residual B cells. In these experiments, spleen cells obtained from
B-cell-deficient mice (µ knockout mice) were used as target cells.
In this case no infected B cells are transferred and the donor stem
cells cannot generate any B cells; therefore, the recipient animals
were totally devoid of donor origin B cells. As seen in Fig 2C, PEC
obtained from such mice 4 months after cell transfer contained an
average of 1 copy/cell of the exogenous gene, similar to the results
obtained with normal spleen (Fig 2A). Spleen cells contained an average
of 0.125 copies/cell which is, as expected, much lower than when normal
spleen cells were used as target cells because in this case there are
no infected B cells in the inoculum. Very faint bands could be seen
from thymocytes on overexposure.
Absence of replication-competent virus in reconstituted recipients.
To rule out the possibility that the results reflect a lateral spread
of vector virus due to activation of an endogenous retrovirus, assays
were performed to determine whether replication-competent virus could
be detected in PEC or LPS-stimulated spleen cells isolated from
reconstituted mice 24 or 96 hours after thioglycollate injection. For 7 days, 1 × 106 PEC or spleen cells were cocultivated
with either 2 × 105 NIH 3T3 cells or 1 × 106 primary syngeneic spleen cells. Supernatant was
obtained from these cultures every 24 hours and tested for reverse
transcriptase (RT) activity according to standard
procedures.28 If replication-competent virus was present,
it should have been propagated in fresh cells, and supernatant from
these cultures should be positive for RT activity. In parallel, each
supernatant sample was added to fresh cultures of NIH 3T3 cells for a
second round of expansion for an additional 7 days, and RT activity was
also checked every 24 hours. Figure 4
represents the results from supernatants obtained from day 3 of the
primary and secondary cultures, and as can be seen, all of the samples
were negative except for the positive control. RT activity was negative
for all time points tested. Moreover, the NIH 3T3 cultures from both
rounds of amplification were also scored for transfer of the
neo-containing N2 vector by selection with G418 (350 µg/mL).
If replication-competent virus was present, it should provide viral
proteins for passage of N2 vector virus. No G418-resistant colonies
were detected (data not shown), providing additional support that
replication-competent virus was not present in vector positive animals,
and ruling out horizontal spread of the vector virus due to activation
of a latent endogenous retrovirus.
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| Fig 4.
RT assay for the detection of replication competent
virus. PEC and spleen cells were obtained 24 and 48 hours after
thioglycollate injection and cocultured with either 3T3 cells or
LPS-stimulated spleen cells. Supernatants were obtained every 24 hours
for 7 days and added to fresh 3T3 cells. Two samples of supernatants
harvested at day 3 of the first and second cultures were checked for
the presence of RT (columns 3 through 10). Columns 1 and 2 represent
control supernatants taken from cultures of N2 producer line, spleen
cells, PEC, and media control, as marked in the figure.
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Lack of expression of a transduced human ADA gene in granulocytes and
macrophages.
We have previously shown that the human ADA gene can be
efficiently introduced into, and expressed in mature B and T cells using the AsADA retroviral vector (Fig 1).22,26
However, in the experiments mentioned earlier in which we first used
T-cell-depleted spleen as target cells, although the gene could be
detected in tissues other than lymphoid organs, expression of the ADA
protein could not be detected in these tissues. One explanation for
this could be that the relative number of transduced cells in
nonlymphoid organs is too low to allow protein detection. Because the
ADA assay is quite sensitive, we preferred an alternative explanation, which is that the ADA gene when introduced into early
progenitor cells is shut off during the differentiation to mature
granulocytes and macrophages. To test this, the same viral vector used
in previous studies (Fig 1)22,26,27 was used to infect
T-cell-depleted spleen cells. As shown in
Fig 5A, gene transfer into the myeloid lineage, as assessed by Southern blotting, was as efficient for the
human ADA gene as it was for the neo gene. The
transferred ADA gene could be detected in spleen and PEC 6 months after adoptive transfer of infected target cells (1 copy/cell
for PEC and 0.2 copies/cell for spleen cells, respectively). The human
ADA gene allows for a simple enzymatic measurement of protein
expression and it can be distinguished from the endogenous murine ADA
because of its different electrophoretic mobility.22,26 As
shown in Fig 5B, we could not detect any human ADA expression in PEC
using an ADA-specific enzymatic assay. On the other hand, as expected, human protein expression was detected in spleen cells because the
spleen contains large numbers of B cells which are also transduced in
this protocol. The lack of expression in myeloid cells is not surprising. It is not uncommon that xenogeneic tissue-specific promoters are turned off during differentiation.29 Also,
the human ADA gene is driven by a minimal promoter which was
shown to be active in mature lymphocytes and in various lymphoid cell lines, but to the best of our knowledge, it was never tested in myeloid
cells.30 However, the fact that there is no expression of
the ADA gene in PEC provides further support to the notion that
the proviral signal observed by Southern blotting in PEC is only due to
positive myeloid cells and not to contaminating B cells, which is
consistent with the esterase staining results. The levels of expression
of the endogenous murine ADA are obviously much higher than the human
gene because most of the cells in the spleen express the endogenous
murine ADA.
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| Fig 5.
Lack of expression of the exogenous gene in granulocytes
and macropahges. (A) Southern blotting of genomic DNA prepared from PEC
24 and 96 hours after thioglycollate treatment and spleen from two
representative mice reconstituted 6 months earlier with 20 × 106 cells of T-cell-depleted spleen cells infected with
pAsADA. DNA was digested with SacI and probed with a
neo-specific probe. SacI-digested pAsADA plasmid DNA
equivalent to 0.25 and 1 copies/cell are as indicated. (B) Human and
mouse ADA protein assay. Cell lysates prepared from 1 × 106 cells from the same spleen and PEC samples used for
Southern blotting were electrophoresed on a cellulose acetate plate to
separate human from murine ADA enzymes. ADA activity was detected by
the standard colorimetric enzymatic assay. PEC were analyzed 24 hours
(granulocytes) and 96 hours (macrophages) after thioglycollate
induction, respectively.
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In situ PCR of peritoneal exudate cells.
The results obtained by Southern blotting can only indicate the average
number of exogenous gene copies per cell. They cannot indicate whether
most cells harbor 1 copy or whether a few cells harbor many copies of
the gene. To determine the exact percentage of myeloid cells that
harbor the transduced gene, in situ PCR was used to amplify the
transduced neo gene followed by specific hybridization of
biotinylated neo-specific probes. This allows the visualization
of positive signals and the morphology of the target cell on a cell per
cell basis. At least 80% of both granulocytes (Fig 6C) and macrophages (Fig 6D) from
reconstituted animals harbored at least 1 copy of the transduced
neo gene. Cells from control animals were totally negative (Fig
6A and B).
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| Fig 6.
Detection of provirus sequences by in situ PCR
amplification. Peritoneal granulocytes and macrophages were obtained 24 and 96 hours, respectively, after thioglycollate injection from control
(A and C) or from mice reconstituted with targeted spleen cells (B and
D) 12 months earlier and subjected to in situ PCR hybridization using
biotinylated probes. Amplified neo-specific sequences were
visualized on a cell per cell basis using an alkaline phosphatase-based
colorimetric assay.
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Clonal analysis of the targeted myeloid population.
Previous retroviral gene tagging of pluripotential stem cells suggested
that at any particular time point, very few clones contribute to the
generation of the various hematopoietic lineages.31,32 To
test the degree of clonality in the mature myeloid lineage after
adoptive transfer of retrovirus-tagged spleen cells, the same DNA used
in the Southern blotting analysis for the detection of the proviral DNA
in PEC-derived granulocytes and macrophages was digested with either
BamHI or HindIII. These enzymes only cut once within
the retroviral vector. Therefore, if the mature PEC arise from one or
few precursor clones, one would expect to see only a few bands in a
Southern blot when neo-specific probes are used. The results
shown in Fig 7 clearly show that the
populations of peritoneal granulocytes and macrophages are not
oligoclonal. Discrete bands were not detected in the Southern blot
analysis after exposure for 1 to 2 days, even though the blot was
sensitive to below 0.125 copies of the exogenous gene/cell. After
longer exposure, some faint bands (corresponding to 0.001 copies/cell) could be detected, which might suggest that some infected clones contribute a little more than others to the mature myeloid population.
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| Fig 7.
Clonal analysis for the populations of PEC-derived
granulocytes and macrophages. The PEC isolated from the same mice
described in Fig 2A were used for clonal analysis. Genomic DNA was
prepared from PEC 24 and 96 hours after injection of thioglycollate and
digested with the restriction enzyme BamHI or HindIII
and probed using a neo-specific probe. Lanes 1, 3, 7, and 9 represent DNA from granulocytes (24 hours) and lanes 2, 4, 8, and 10 represent DNA from macrophages (96 hours) as indicated by the lettering
below the lanes. Genomic DNA from the N2 producer cell
(lanes 5 and 11) and 1 copy of plasmid DNA, pN2 (lanes 6 and 12), were used for oligoclonal and monoclonal controls,
respectively. The last four lanes represent SacI-digested
pN2 plasmid DNA equivalent to 0.125, 0.25, 0.5, and 1.0 copies/cell, respectively.
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DISCUSSION |
In this study we present evidence for the existence of a
long-lived, spleen-residing, myeloid-committed stem cell population. These stem cells can be targeted with a retroviral vector after stimulation of T-cell-depleted spleen cells with LPS. Because current
retroviral vectors, including the ones used in this study, require at
least one round of cell replication to integrate into the cell's
genome, the targeted stem cells are induced to proliferate either
directly or indirectly by LPS. There was no gene transduction in the
absence of LPS; however, transduction was independent on the presence
of B cells because spleen cells from B-cell-deficient (µ knockout)
mice were as efficiently transduced as normal B cells. This
suggests that the targeted cells themselves are responsive to LPS. The
evidence that this cell population is not pluripotential is three-fold.
First, the targeted cells do not home back to the BM and therefore,
secondary BM transfers from positive mice into normal
irradiated recipients do not transfer the exogenous gene. Second,
whereas at least 80% of mature thioglycollate-induced granulocytes and
macrophages were positive for the exogenous gene, as determined by in
situ PCR, newly generated T and B lymphocytes were not. Third, clonal
analysis of the targeted macrophages and granulocytes populations (Fig
7) indicated that the cells were not of oligoclonal origin, which
should have been the case if pluripotential cells were
targeted.14,15 However, it is not clear how homogenous the
targeted stem cell population is and it can represent either a pan
myeloid stem cell population or a pool of more than one precursor cells
such as macrophage- and granulocyte-committed stem cells.
Surprisingly, these myeloid-committed stem cells contributed to the
replenishment of the mature myeloid population for at least 9 months,
as assessed by Southern blot analysis and for 12 months as assessed by
in situ PCR hybridization. Their ability to replenish the
mature myeloid cell populations for prolonged periods is seemingly
antithetical to the current dogma that cells other than PSC can only
contribute to the hematopoietic lineage for a limited period. However,
this dogma is based on studies using BM cell transfers and our findings
are based on spleen cell transfers. It is therefore possible that the
targeted population we describe only resides in the spleen. It is
equally plausible that this population is present in BM but copurifies
with the enriched BM population that contains pluripotential cells.
Only 10% of this population are PSC and the rest of the cells are at present not well characterized.2,6 The myeloid precursor(s) we describe might be part of the uncharacterized fraction of this population. If these myeloid precursor cells also reside in BM, why
they were not detected by others is unclear. One possibility could be
that they do not respond to any of the stimuli used by others and would
have been detected if LPS was used. This possibility is currently under
investigation.
Aside from the implication these findings have on our understanding of
hematopoiesis, the ability to specifically introduce exogenous genes
into the myeloid lineage offers an opportunity to study the
consequences of expressing an exogenous gene exclusively in the myeloid
lineage using somatic cell gene transfer. So far, retroviral-mediated
gene transfer into the myeloid lineage has mainly been achieved by the
transduction of BM stem cells and the efficiency was rarely higher than
5%.33 Other disadvantages of using BM cells is that PSC
comprise a rare population in BM, they are very hard to purify, they
have not been ideal targets for gene transfer, and they are not lineage
specific. Even if the efficiency of gene transfer into pluripotential
cells improves in the future, transduction of exogenous genes into
these cells would probably result in the expression of the gene in all
hematopoietic cell lineages, which may not always be desirable. In
contrast, transduction of myeloid-committed stem cells allows specific
expression in the myeloid compartment. This cell would therefore make a
more suitable target for gene therapy-based treatment of genetic
disorders inherent to the myeloid lineage such as Gaucher's disease.
Restricted expression in the myeloid lineage might also facilitate
selective induction and potentiation of immune responses against tumor
or viral antigens.
| |
FOOTNOTES |
Submitted February 26, 1998;
accepted June 22, 1998.
Supported in part by the National Multiple Sclerosis Society Grant No.
2626A2/1 (Y.R.), Milstein family foundation (Y.R.), and by an
Individual National Research Service Award, Grant No. GM19331 (A.R.).
Address reprint requests to Yacov Ron, PhD, Department of Molecular
Genetics and Microbiology, University of Medicine and Dentistry of New
Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway,
NJ 08854; e-mail: yron{at}umdnj.edu.
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.
| |
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Abstract
Introduction
Abstract