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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2605-2612
Enhanced In Vivo Regenerative Potential of HOXB4-Transduced
Hematopoietic Stem Cells With Regulation of Their Pool Size
By
Unnur Thorsteinsdottir,
Guy Sauvageau, and
R. Keith Humphries
From the Terry Fox Laboratory, British Columbia Cancer Agency,
Vancouver, British Columbia, Canada; and the Department of Medicine,
University of British Columbia, Vancouver, British Columbia, Canada.
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ABSTRACT |
After bone marrow transplantation (BMT), there is a rapid
regeneration to normal pretransplantation levels in the number of hematopoietic progenitors and mature end cells, whereas hematopoietic stem cell (HSC) numbers recover to only 5% to 10% of normal levels. This suggests that HSC are significantly restricted in their
self-renewal behavior and hence in their ability to repopulate the host
stem cell compartment. Previously, we have reported that HSC engineered to overexpress the homeobox transcription factor HOXB4 have a large repopulation advantage over untransduced cells as assessed at 4 months in a murine transplantation model (Sauvageau et al, Genes
Dev 9:1753, 1995). This phenomenon has now been
examined in detail for periods extending to 12 months in cohorts of
mice transplanted with various numbers of HOXB4-transduced HSC.
In all mice analyzed, HOXB4-transduced HSC were capable of
fully reconstituting the HSC compartment, resulting, on average, in some 14-fold greater numbers of HSC than observed when transplanting control, non-HOXB4-transduced bone marrow cells. These data
indicate that HOXB4 is a limiting factor in the regeneration of
HSC to normal levels after BMT. Furthermore, we show that
HOXB4-transduced HSC did not expand above levels normally
observed in unmanipulated mice, indicating that its overexpression does
not override the regulatory mechanisms that maintain the HSC pool size
within normal limits.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS IS the process by which
mature blood cells are continuously generated throughout adult life
from a small number of totipotent hematopoietic stem cells (HSC). The
HSCs have the key properties of being able to self-renew and to
differentiate into mature cells of both lymphoid and myeloid lineages.
Although the genetic mechanisms responsible for the control of
self-renewal and differentiation outcomes of HSC divisions remain
largely unknown, a number of studies have implicated a variety of
transcription factors as key regulatory components of these
processes.1
Among such factors are the mammalian Hox homeobox gene family
of transcription factors, consisting of 39 members arranged in 4 clusters (A, B, C, and D), initially described as important regulators
of pattern formation in a variety of embryonic tissues.2 These genes are structurally related by the presence of a 183-bp sequence, the homeobox, that encodes a helix-turn-helix DNA binding motif.3 Apparent stage- and lineage-specific expression of numerous HOXA, B, and C genes has now been
demonstrated for both hematopoietic cell lines4 and primary
hematopoietic cells.5-7 For example, we have shown that
members of the HOXA and HOXB cluster genes are
preferentially expressed in the CD34+ fraction of human
bone marrow cells that contains most if not all of the hematopoietic
progenitor cells.7 Further detailed analysis of Hox
gene expression in functionally distinct subpopulations of
CD34+ cells has shown that genes, primarily located at the
3' end of the clusters (eg, HOXB3 and HOXB4), are
preferentially expressed in the subpopulation containing the most
primitive hematopoietic cells.7
Using a murine bone marrow transplantation (BMT) model, we previously
obtained evidence indicating that retroviral overexpression of
HOXB4 in hematopoietic cells can greatly enhance the
regeneration of the HSC compartment after BMT,8 thus
implicating HOXB4 as a regulator of self-renewal divisions of
HSC. In these initial studies, the effects of HOXB4
overexpression were assessed at 20 weeks posttransplantation, at which
time the HSC compartment had regenerated to slightly above normal
pretransplantation levels or some 47-fold higher than achieved in
control transplant recipients. This enhanced regenerative ability was
further suggested by a significant expansion of
HOXB4-transduced HSC after transplantation into secondary
recipients. From these limited initial studies it was unclear whether
HOXB4 overexpression in steady-state hematopoiesis would lead
to continuing expansion of HSC over time, suggesting that it could
override the normal processes that control the population size and
self-renewal potential of HSC. Also unanswered was whether HOXB4 could act on a wide spectrum of transduced HSC or, as
opposed, on a limited subset, as would be reflected in polyclonal
versus monoclonal or oligoclonal expansion, respectively.
To address these questions, the current studies have examined the size
and the degree of polyclonality of the regenerated pool of HSC in mice
transplanted with HOXB4-transduced bone marrow cells as a
function of time for a period extending up to 1 year after transplantation.
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MATERIALS AND METHODS |
Animals.
Recipients were 7- to 12-week-old male or female (C57Bl/6J × C3H/HeJ)F1 [(B6C3)F1] mice and donors were (C57Bl/6Ly-Pep3b × C3H/HeJ)F1 [(PepC3)F1] mice. (B6C3)F1 and (PepC3)F1 mice are
phenotypically distinguishable by their cell surface expression of
different allelic forms of the Ly5 locus; (B6C3)F1 are homozygous for
the Ly5.2 allotype and (PepC3)F1 are heterozygous for the Ly5.1/Ly5.2 allotypes. These mice were bred from parental strain breeders originally obtained from Jackson Laboratories (Bar Harbor, ME), maintained in microisolator cages, and provided with sterilized food
and acidified water in the animal facility of the British Columbia
Cancer Research Center.
Retroviral generation and infection of primary bone marrow cells.
Retroviral vectors carrying the HOXB4 cDNA under the control of
the viral long terminal repeat and/or a neomycin gene cassette under
the control of an internal PGK promoter were constructed, and
high-titer viral producers were generated in the GPE-86 packaging line
as previously described.8 Bone marrow cells were obtained from (PepC3)F1 (Ly5.1) mice who had received 4 days previously an
intravenous injection of 150 mg/kg body weight of 5-fluorouracil (5-FU), prestimulated in Dulbecco's modified Eagle medium (DMEM) containing 15% fetal calf serum (FCS), 6 ng/mL of murine interleukin-3 (mIL-3), 100 ng/mL murine Steel factor (mSF), and 10 ng/mL human IL-6
(hIL-6) for 48 hours and then cocultivated on irradiated viral producer
cells using identical medium with the addition of 6 µg/mL polybrene
for an additional 48 hours. mSF, hIL-6, and mIL-3 were used as diluted
supernatant from transfected COS cells as prepared in the Terry Fox
Laboratory. Loosely adherent and nonadherent bone marrow cells were
recovered from the cocultures by repeated washing of dishes and then
counted using a hemocytometer. Unless otherwise specified, all media,
serum, and growth factors were obtained from StemCell Technologies Inc
(Vancouver, British Columbia, Canada).
Transplantation of retrovirally transduced bone marrow.
For bone marrow transplantation procedures, lethally irradiated (950 cGy, 110 cGy/min, 137Cs  -rays) (B6C3)F1 (Ly5.2) recipients were
injected intravenously with 2 × 105 bone marrow cells
derived from (PepC3)F1 (Ly5.1/Ly5.2) immediately after their
cocultivation with HOXB4- or neo-viral producer cells. Donor-derived repopulation in recipients was assessed using flow cytometry to measure the proportion of leukocytes in bone marrow, thymus, spleen, and peripheral blood that expressed the Ly5.1 surface
antigen recognized by the fluorescein isothiocyanate (FITC)-conjugated anti-Ly5.1 monoclonal antibody (kindly provided by Dr G. Spangrude, Salt Lake City, UT).
In vitro clonogenic progenitor assays.
In vitro myeloid and pre-B clonogenic progenitor assays were performed
as previously reported.9
Competitive repopulation unit (CRU) assay.
The CRU assay10 was used to evaluate the regeneration of
HSC in neo and HOXB4 mice. Briefly, bone marrow cells
from neo or HOXB4 mice that were transplanted earlier
with transduced cells derived from (PepC3)F1 (Ly5.1/Ly5.2) mice were
injected at different dilutions into lethally irradiated (B6C3)F1
(Ly5.2) mice together with a life-sparing dose of 1 × 105 competitor bone marrow cells from (B6C3)F1 (Ly5.2)
mice. The level of lymphoid and myeloid repopulation with
Ly5.1+ donor-derived cells in these secondary recipients
was evaluated more than 13 weeks later by flow cytometry analysis of
peripheral blood, as described.11 Recipients with greater
than 1% donor-derived peripheral blood lymphoid and myeloid leukocytes
as determined by the side scatter distribution of Ly5.1+
cells (ie, lymphoid low side scatter; myeloid high side scatter) were
considered to be repopulated by at least 1 lympho-myeloid repopulating
(CRU) cell. CRU frequency in the test cell population was then
calculated by applying Poisson statistics to the proportion of negative
recipients at different dilutions, as described
previously.10 Secondary recipients were killed 16 weeks
after transplantation.
Southern and Northern blot analyses.
DNA was isolated from bone marrow, spleen, and thymus of neo
and HOXB4 mice using DNAzol (GIBCO BRL, Burlington, ON,
Canada). High molecular weight DNA was digested with
Kpn I, which cuts in the long terminal repeat region to release
the integrated provirus, or with EcoRI or BamHI, which
each cut the provirus once to release DNA fragment(s) specific to the
proviral integration site(s). DNA fragments were separated on a 0.9%
agarose gel, transferred to nylon membrane (Zeta-Probe; Bio-Rad,
Hercules, CA), prehybridized, hybridized, and washed as
described.8 Total cellular RNA was isolated using TRIzol
(GIBCO BRL), separated on a 1% formaldehyde/agarose gel, transferred
to nylon membrane (Zeta-Probe), prehybridized, hybridized, and washed
as described.8 Probes used were a Xho I/Sal
I fragment of pMC1neo,12 the full-length HOXB4
cDNA, a 2.0-kb Pst I fragment containing the chicken  -actin
gene, and the 1.8-kb Kpn I/HindIII genomic fragment of
the murine SH2-containing inositol phosphatase (SHIP)
gene.13
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RESULTS |
Retroviral transduction and transplantation of murine bone marrow
cells.
The MSCV recombinant retroviral vector containing the HOXB4
cDNA (Fig 1A) was used to transfer and
overexpress this gene in mouse bone marrow cells. To assess the
long-term effects of HOXB4 overexpression on hematopoietic
regeneration, lethally irradiated mice were transplanted with
HOXB4- or neo-transduced bone marrow cells. Cohorts of
mice from 3 independent transplantation experiments (hereafter called
HOXB4 and neo mice) were assessed for regeneration of
various hematopoietic compartments at different times after transplantation, beginning as soon as 16 weeks and as late as 52 weeks
after transplantation (Fig 1B). Each recipient was transplanted with an
inoculum of 2 × 105 bone marrow cells, as recovered
from retroviral infection cultures. This cell dose is estimated to
contain approximately 35 HSC, as previously measured for recovered
cells under identical infection conditions, using the CRU
assay.8 The gene transfer efficiencies to the transplanted
bone marrow as assessed by the proportion of G418-resistant clonogenic
cells varied between experiments and were 30% to 58% and 70% to 74%
for HOXB4- and neo-transduced cells, respectively (Fig
1B). Assuming a retroviral infection efficiency of CRU (HSC) no greater
than that of clonogenic progenitor cells, each recipient would have
received an estimated maximum of 16 to 24 transduced (neo or
HOXB4; see Table 3) CRU, plus an approximately equal number of
nontransduced CRU. Findings from the 20-week timepoint in experiment
no. 1 have been previously reported8; selected summary data
from that timepoint are provided in Figs 2
and 3 to facilitate comparison with the new
data from mice of that same transplant cohort now assessed at 52 weeks
posttransplantation.
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| Fig 1.
Structure of the HOXB4 and control neo
retroviruses and the experimental outline. (A) Diagrammatic
representation of the integrated HOXB4 and neo
proviruses. Expected size of the full-length viral transcripts and also
those initiated from the PGK promoter are shown, as are the sites for
the various restriction enzymes used in this study. (B) Experimental
outline showing the number of HOXB4 and neo mice from 3 transplantation experiments that were used in this study and the time
posttransplantation when they were analyzed. Also shown is the initial
gene transfer to the transplanted bone marrow inoculum received by
neo and HOXB4 mice in these transplantations. (C)
Southern blot analysis of DNA isolated from bone marrow and thymus of
some of the neo (killed 32 weeks posttransplantation) and
HOXB4 mice (killed 32, 41, and 52 weeks posttransplantation)
used in this study to demonstrate the presence of the integrated
provirus. DNA was cut with Kpn I, which releases the
neo (2.7 kb) and the HOXB4 (3.9 kb) proviruses, and the
blot was successively hybridized to probes specific for the neo
and HOXB4 genes (full-length HOXB4 cDNA was used as a
probe). The endogenous murine HOXB4 is detected at 1.3 kb by
the HOXB4 probe and provides a single gene copy control of
loading. In some of the HOXB4 mice, in addition to the
full-length HOXB4 provirus, a weaker 2.7-kb proviral signal is
detected with the neo probe but not with the HOXB4
probe. Because this fragment failed to hybridize to the HOXB4
probe, it likely represents a rearrangement resulting in the loss of
the HOXB4 gene from some of the integrated proviruses. Kp,
Kpn I; E, EcoRI; B, BamHI; SD, splice donor;
SA, splice acceptor; CFC, colony-forming cells; B, bone marrow; T,
thymus.
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| Fig 2.
Effects of HOXB4 overexpression on the number of
myeloid and pre-B colony-forming cells after BMT. Results shown are the
means ± SD of the numbers of in vitro myeloid colony-forming cells in
bone marrow (top) and spleen (middle) and of IL-7-responsive
B-lymphoid progenitor cells (pre-B CFC) in the bone marrow (bottom) of
individual neo ( ) and HOXB4 ( ) mice at various
time points after transplantation. The number of neo and
HOXB4 mice analyzed at each time point are shown in Fig 1B.
Consistent with their preferential derivation from
HOXB4-transduced cells, a major proportion of myeloid and pre-B
lymphoid progenitor cells in HOXB4 mice were G418-resistant
(HOXB4 mice, 59% ± 9% v neo mice, 37% ± 10% for all 3 experiments).
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| Fig 3.
Variation in CRU numbers in recipients of neo-and
HOXB4-transduced bone marrow cells over 1-year period. The
number of CRU in femurs of cohorts of HOXB4 recipients from 3 different experiments ([] experiment no. 1, [ ] experiment no.
2, and [ ] experiment no. 3) and neo recipients from 2 different experiments ( and  ) were evaluated using the CRU assay.
The results shown are expressed as the mean ± 95% confident interval
of the CRU numbers in 1 femur of neo and HOXB4 mice at
the various time points after transplantation. The number of
neo and HOXB4 mice analyzed at each time point are
shown in Fig 1B. The shaded area represents the normal number of CRU
measured in the femoral cavity of an unmanipulated (C57Bl/6J × C3H/HeJ)F1 mouse.9
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Effect of HOXB4 overexpression on progenitor and mature populations
in vivo.
Hematopoietic regeneration in both neo and HOXB4 mice,
from all 3 transplantation experiments, was essentially completely donor-derived, because greater than 85% of bone marrow, spleen, thymic, and peripheral blood leukocytes were of transplant origin (Ly5.1+) at all time points analyzed. A contribution by
transduced cells to this reconstitution was evident by Southern blot
analysis that readily detected the neo-or the
HOXB4-proviruses in the bone marrow and thymuses of these mice
(Fig 1C).
In contrast to differences in HSC levels (see below), the bone marrow,
spleen, and thymus nucleated cell counts, as well as the peripheral
blood white and red blood cell counts, were similar in HOXB4
and neo mice (Table 1).
Furthermore, fluorescence-activated cell sorting (FACS) analysis showed
that the absolute numbers of bone marrow and splenic myeloid
(Mac-1+), erythroid (Ter119+), B (ie, proB
[B220+CD43+], immature B
[B220+IgM+], and mature B
[IgM+IgD+]), and CD4 and CD8 thymic T cells
subpopulations were all also within normal range in HOXB4 mice
(Table 2).
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Table 2.
Absolute Numbers of Various Phenotypically Defined
Hematopoietic Populations in neo and HOXB4 Mice 32 Weeks After Transplantation
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We had previously reported that the bone marrow myeloid progenitor
numbers, as assayed in semisolid cultures supplemented with growth
factors, were increased by 5-fold in recipients of HOXB4-transduced cells when compared with neo control
mice. This increase, initially detected in recipients analyzed at 20 weeks posttransplantation,8 was not observed in
HOXB4 mice from transplantation experiments no. 2 or 3 (Fig 2).
However, the number of myeloid progenitors in the spleen were always
increased in these mice (Fig 2), with an overall increase of total body
myeloid progenitor numbers of less than 2-fold in all mice analyzed.
Evaluation of bone marrow IL-7-responsive B-lymphoid progenitor cells
at 16, 32, and 41 weeks after transplantation showed that their numbers were slightly increased in the HOXB4 mice compared with
neo control mice. However, at none of these time points was
this increase statistically significant (Fig 2). Together, these data
indicate that long-term overexpression of HOXB4 has mild to
moderate effects on the number of clonogenic progenitors and
essentially no effect on the number of mature end cells in recipient mice.
Effect of HOXB4 overexpression on long-term repopulating cells.
To determine the frequency of long-term lympho-myeloid repopulating
cells (or HSC) in neo and HOXB4 primary mice, the CRU assay was used,10 which combines principles of limiting
dilution together with competitive repopulation. Bone marrow cells were harvested from HOXB4 and neo mice at 5 different time
points (Fig 1B), spanning as early as 16 weeks to as late as 52 weeks
posttransplant. These cells were then transplanted at several different
dilutions into secondary recipients that were themselves analyzed 12 to 15 weeks later for donor-derived (ie, Ly5.1+)
lympho-myeloid repopulation.
At all time points examined and in all experiments, CRU numbers were
markedly higher, on average some 14-fold, in HOXB4 mice when
compared with neo control mice, whose CRU numbers achieved values that were approximately 10% of normal untransplanted mice (Fig
3). The low recovery of CRU in recipients of neo-transduced bone marrow is consistent with previous studies using nontransduced unmanipulated adult bone marrow.14-17 The results with
HOXB4-transduced cells thus stand in sharp contrast
with recovery to the normal pretransplantation level at
all times posttransplant analyzed.
Interestingly, once normal levels of CRU were reached in the
HOXB4 mice, which is at least as early as 16 weeks, the CRU did not
expand further or become exhausted, as indicated by the similar number
of CRU cells present at 20 versus 52 weeks posttransplantation in
experiment no. 1 (solid box in Fig 3) and at 16 versus 32 weeks in
experiment no. 2 (solid diamond in Fig 3). Northern blot analysis of
primary recipients (data not shown) and secondary mice used for CRU
determination (Fig 4C) confirmed continued expression of the transduced
HOXB4 gene. Thus, plateauing of CRU numbers was not associated
with extinction of HOXB4 gene expression. Taken together, these
results clearly indicate that HOXB4 appears to be a limiting
factor in the regeneration of CRU numbers to normal levels after BMT
and that its overexpression does not override the regulatory mechanisms
that maintain the CRU pool size within normal limits and neither does
it lead to rapid exhaustion of CRU.
Polyclonal expansion and hematopoietic regeneration by
HOXB4-transduced CRU.
To prove that the enhanced regeneration of CRU cells in the
HOXB4 mice was indeed caused by preferential expansion of
HOXB4-transduced CRU cells and to analyze the degree of
polyclonality of the regenerated pool of CRU cells, we performed
Southern blot analyses of proviral integration sites in DNA isolated
from various hematopoietic tissues of primary and secondary
HOXB4 and neo mice. Results from representative primary
recipients and their corresponding secondary recipients used for CRU
assay are shown in Fig 4.
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| Fig 4.
Southern blot analysis of proviral integration
patterns in primary and secondary recipients of neo- and
HOXB4-transduced bone marrow cells. DNA samples isolated from
various hematopoietic organs of primary recipients killed 52 (A) or 32 (B) weeks posttransplantation and their secondary recipients (killed 16 weeks posttransplantation) were first digested with EcoRI and
then BamHI, both of which cut the integrated provirus once,
generating a DNA fragment specific for each proviral integration site.
The number of transduced clones detected with either enzyme were the
same; thus, the results for only 1 of the enzymes is
shown. The membranes were first hybridized to a
neo-specific probe for detection of proviral fragments and
subsequently with a probe specific for the SHIP gene to provide
a single copy control of loading. Exposure times were 48 hours for the
neo and SHIP probes. To demonstrate that the proviral
fragments contained the HOXB4 cDNA, the blots were also
hybridized with full-length HOXB4 cDNA probe, which generated
the same proviral banding pattern as the neo probe (data not
shown). Each mouse is identified with a specific number derived from
the time that the primary recipient was killed, and indicated above
that number are the number of bone marrow cells received by each
secondary recipient as well as the estimated number of CRU cells that
they received. Expression of the 3.9-kb HOXB4-containing
message in the bone marrow of secondary recipients is shown in (C). The
percentages of the donor-derived repopulation (ie,
Ly5.1+) in the secondary neo mice were as
follows: 32-1 (44%) and 32-2 (18%). In secondary HOXB4 mice,
the percentages were as follows: 32-1 (60%), 32-2 (76%), 32-3 (83%),
32-4 (52%), 32-5 (4%), 32-6 (23%), 32-7 (18%), 32-8 (8%), 32-9 (51%), 32-10 (5%), 32-11 (0%), 52-1 (20%), 52-2 (69%), 52-3 (73%), 52-4 (47%), 52-5 (5%), 52-6 (36%), 52-7 (47%), 52-8 (30%),
and 52-9 (20%). B, bone marrow; S, spleen; T, thymus.
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In the bone marrow, spleen, and thymus of primary HOXB4
recipients, which were killed 52 and 32 weeks posttransplantation, multiple proviral integrations could be detected (Fig 4A and B). Variations in the proviral signal intensities detected in most of these
tissues are a signature for multiple proviral integrations and thus
demonstrate polyclonal regeneration by HOXB4-transduced long-term repopulating cells, rather than multiple proviral
integrations into the same cell. In contrast, moderate
proviral integration complexity was observed in the primary neo
recipients, consistent with oligoclonal repopulation by
neo-transduced cells (Fig 4B). This difference between
neo and HOXB4 mice cannot be contributed to higher
numbers of transduced stem cells being initially transplanted to the
HOXB4 mice, because the neo control and the
HOXB4 mice received similar numbers
(Table 3), thus underscoring the
repopulating advantages of HOXB4-transduced CRU cells over
untransduced cells.
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Table 3.
Summary of the Clonal Analyses of Primary and Secondary
Recipients of neo- or HOXB4-Transduced Cells Presented
in Fig 4, Demonstrating Enhanced Polyclonal Long-Term Repopulation by
HOXB4-Transduced Stem Cells
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Southern blot analysis of proviral integration patterns in bone marrow
(myeloid) and thymus (lymphoid) of secondary recipients was used to
further analyze the clonality of the regenerated pool of transduced
lympho-myeloid repopulating (CRU) cells present in primary
HOXB4 and neo mice at the time of their death (Fig 4A
and B). In all of the 9 secondary recipients of the primary donor mouse
killed at 52 weeks posttransplantation that were scored positive for
donor-derived repopulation by FACS (Ly5.1+), HOXB4
proviral integration(s) were detected in their bone marrow and/or
thymus (Fig 4A). Furthermore, the intensities of the proviral signals
correlated, for most of the mice, with their level of donor derived
(Ly5.1+) repopulation (Fig 4A). As can be seen in Fig 4A
and summarized in Table 3, a total of at least 15 different
HOXB4-transduced clones could be detected in these secondary
mice. In addition, at least 4 of these mice had a common proviral
integration pattern in their bone marrow and thymus (mice 52-2, 52-4, 52-6, and 52-9 [bone marrow signal very faint due to low amount of
DNA]), indicating the lympho-myeloid repopulating potential of the
regenerated CRU cells. The lack of detection of common proviral
intergration patterns both in bone marrow and thymus in the other
secondary mice can be explained in some cases by the low amount of DNA
analyzed (mouse 52-7 in thymus), the lack of bone marrow sample (mouse
52-3), or very low donor-derived repopulation levels (mouse 52-5, Ly5.1+ PBL only 5%). Thus, even as late as 52 weeks
posttransplantation, the hematopoietic regeneration in the primary
HOXB4 mice was polyclonal and without apparent dominance of any
HOXB4-transduced clone.
Similarly, analysis of the proviral intergration sites in the secondary
recipients receiving bone marrow cells from 1 of the HOXB4
recipients killed 32 weeks posttransplantation showed a complete
concordance between detection of donor-derived hematopoietic regeneration by FACS analysis and contribution to regeneration by
HOXB4-transduced cells, again indicating selective or
competitive regeneration of the HOXB4-transduced cells over
nontransduced cells (Fig 4B). Of the 10 secondary recipients (32-1 through 32-10) that were positive for lympho-myeloid repopulation by
FACS, 8 had detectable HOXB4 proviral integration in their bone
marrow and thymus (Fig 4B). In the case of the 2 negative mice (32-5 and 32-10), which both had low donor repopulation (Ly5.1+
PBL, 4% and 5%, respectively), the expression of HOXB4 could, however, be detected by Northern blot analysis of their bone marrow (Fig 4C). Several of these secondary recipients, including those that
were estimated to receive between 1 and 2 CRU cells (mice 32-6, 32-8, and 32-9), had a common proviral integration in their bone marrow and
thymus, thus again confirming the lympho-myeloid repopulating potential
of the regenerated HOXB4-transduced CRU cells (Fig 4B).
Self-renewal of HOXB4-transduced CRU cells was also
demonstrated by detecting a common lympho-myeloid repopulating clone in
all of the secondary recipients (32-1 to 32-4) receiving high cell dose
(43 CRU/mouse) and in 1 mouse (32-6) receiving fewer CRU cells (2 CRU/mouse). However, interestingly, this clone could not be detected in
bone marrow, spleen, or thymus of the primary HOXB4 mouse (Fig
4B), indicating that this cell, despite extensive self-renewal
division, did not contribute significantly to bone marrow, spleen, or
thymic repopulation in this primary HOXB4 recipient at the time
of death. The detection of 4 different HOXB4-transduced
lympho-myeloid repopulating clones and at least 9 others with either
lymphoid or myeloid potential in these secondary HOXB4 mice
(Fig 4B and Table 3) strongly suggest that the enhanced CRU
regeneration in the primary HOXB4 mouse killed 32 weeks
posttransplantation was also a polyclonal event.
In contrast to the HOXB4 mice, those secondary neo
recipients that were scored positive for donor-derived lympho-myeloid
repopulation (estimated to receive ~5 CRU cells) were only positive
for proviral integrations in their bone marrow (Fig 4B). These data for
the neo mice thus stand in sharp contrast to that of the
HOXB4 mice, in which transduced cells with lymphoid-myeloid
repopulating potential can be detected in secondary mice that received
approximately 70 times lower number of bone marrow cells than these
secondary neo mice (1.4 × 104 cells for the
HOXB4 v 1 × 106 for the neo;
Fig 4A and B).
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DISCUSSION |
Using retroviral gene transfer and the murine BMT model, we have
previously obtained evidence that HOXB4 overexpression can markedly enhance CRU regeneration as assessed 5 months
posttransplantation of transduced bone marrow cells.8 In
this current study, we extend those initial observations to provide
significant new insights into the kinetics, control, and magnitude of
this phenomenon. Our current findings show that the enhanced
regeneration of the CRU compartment by the HOXB4-transduced CRU
is evident as early as 16 weeks posttransplantation and persists for at
least 1 year after transplantation. This increase in regenerative
behavior is such that normal, pretransplantation levels of CRU are
achieved and maintained levels some 14-fold higher than observed with
non-HOXB4-transduced stem cells. Moreover, the apparent
stabilization in the CRU pool at normal pretransplantation levels
suggests that the expansion of HOXB4-transduced CRU in these
mice is ultimately subjected to existing in vivo control mechanisms.
HOXB4 was also demonstrated to act on multiple CRUs, because
the regenerated pool of HOXB4-transduced CRUs in the
transplanted mice was highly polyclonal even as late as 1 year after
transplantation. Furthermore, detailed analysis of various mature cell
populations in these mice strongly suggests that overexpression of
HOXB4 neither alters myeloid or lymphoid differentiation nor
leads to dominant outgrowth of any type of hematopoietic cells.
Serial transplantation studies have suggested that the transplantable
HSCs may fail to fully regenerate the HSC compartment, because the
self-renewal capacity of HSCs may be intrinsically limited or at least
subjected to exhaustion.14,15 In contrast to depletion of
the HSC pool, recent studies have suggested that the failure to fully
regenerate the HSC compartment after transplantation could be the
result of negative feedback mechanisms activated when progenitors and
mature cells have been regenerated to their normal levels, which
prematurely inhibit further HSC expansion.16,17 HOXB4 overexpression might thus render HSC less sensitive to
this negative feedback mechanism.
Using fibroblasts engineered to overexpress HOXB4, it was
recently shown that these cells acquire the capacity to grow in low
concentrations of serum.18 An alternative explanation for the HOXB4 effect described in the present studies is that
HOXB4 might alter the sensitivity of HSC to extrinsic factors
acting during hematopoietic regeneration, thus allowing for a greater expansion of the HOXB4-transduced HSC compartment.
Several studies have indicated that, in steady-state hematopoiesis, the
proliferation of HSC is tightly controlled. In mice, HSC numbers remain
relatively constant throughout most of their adult life, although in
very old mice (>2 years) their numbers appears to increase, possibly
due to accumulation of genetic lesions.19 The HSC
population in mice has also been demonstrated to be quiescent (or
slowly cycling), because the vast majority of these cells are resistant
to cytotoxic agents such as 5-FU or hydroxyurea.20 The
stabilization of the CRU pool in HOXB4 mice at normal levels suggests that, although CRU cells overexpressing HOXB4 have
enhanced regenerative potential, their ability to respond to this
regulatory mechanism is not altered. However, because the cycling
status of CRU cells in HOXB4 recipients is currently unknown,
other regulatory mechanisms acting to maintain stable levels of
HOXB4-transduced CRU cells cannot be ruled out.
Despite a profound and consistent effect on the expansion of primitive
hematopoietic cells, overexpression of HOXB4 did not promote
preferential expansion along any hematopoietic lineage or lead to
leukemia, despite evidence of persistent HOXB4 expression for
at least 52 weeks. These results stand in sharp contrast to our
published data for the retroviral overexpression of either HOXB3 or HOXA10 in a similar transplantation
model.9,21 The different outcomes on hemopoiesis generated
by overexpression of these 3 different HOX genes strongly
indicate that these proteins activate and/or repress different sets of
target genes in hematopoietic cells. The effects of HOXB4
overexpression during hematopoietic regeneration can thus be viewed as
more restricted than those generated by overexpression of either
HOXB3 or HOXA10, suggesting that targets open to
HOXB4 are restricted to primitive hematopoietic cells.
Together, the results presented in this report document that as few as
10 to 20 HOXB4-transduced HSC are sufficient to regenerate the
HSC pool size to pretransplantation levels and, more importantly, once
that level is reached, the number of HOXB4-transduced HSC is
controlled over time. These findings now suggest that it is possible to
engineer HSC that possess repopulating potential 10- to 20-fold higher
than that of unmanipulated HSC. In addition, these findings provide
important insights into the control and molecular mechanism of stem
cell self-renewal and point to potential application in stem
cell-mediated therapies such as those where HSC numbers may be limiting.
| |
ACKNOWLEDGMENT |
The authors are grateful to Patricia Rosten and Wieslawa Dragowska at
the Terry Fox Laboratory for expert technical assistance. We also
acknowledge Drs Margaret Hough, Jana Krosl, Jeffrey Lawrence, Corey
Largman, Connie Eaves, and Peter Lansdorp for insightful discussions
during the course of these studies.
| |
FOOTNOTES |
Submitted December 8, 1998; accepted May 31, 1999.
Supported by the National Cancer Institute of Canada with funds from
the Canadian Cancer Society and the Terry Fox Foundation; the Medical
Research Council of Canada; and the National Institutes of Health
(Grant No. DK48642). U.T. was the recipient of a University of British
Columbia Graduate Fellowship. G.S. is the recipient of a Clinician
Scientist Fellowship of the Medical Research Council of Canada.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to R. Keith Humphries, MD, PhD, Terry Fox
Laboratory, 601 W 10th Ave, Vancouver, BC, V5Z IL3, Canada; e-mail:
keith{at}terryfox.ubc.ca.
| |
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