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Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1283-1290
A Critical Role for PU.1 in Homing and Long-Term Engraftment by
Hematopoietic Stem Cells in the Bone Marrow
By
Robert C. Fisher,
Joshua D. Lovelock, and
Edward W. Scott
From the Institute for Human Gene Therapy, University of
Pennsylvania, Philadelphia, PA.
 |
ABSTRACT |
We have previously demonstrated that PU.1 is required for the
production of lymphoid and myeloid, but not of erythroid progenitors in
the fetal liver. In this study, competitive reconstitution assays show
that E14.5 PU.1 / hematopoietic progenitors
(HPC) fail to sustain definitive/adult erythropoiesis or to contribute
to the lymphoid and myeloid lineages. PU.1/
HPC are unable to respond synergistically to erythropoietin plus stem
cell factor and have reduced expression of c-kit,
which may explain the erythroid defect. Fluorescently labeled,
PU.1/, AA4.1+, fetal liver HPC
were transferred into irradiated recipients, where they demonstrated a
severely impaired ability to home to and colonize the bone marrow.
PU.1/ HPC were found to lack integrins
 4 (VLA-4/CD49d), 5 (VLA-5/CD49e), and
CD11b (M). Collectively, this study has shown that PU.1
plays an important role in controlling migration of hematopoietic
progenitors to the bone marrow and the establishment of long-term
multilineage hematopoiesis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ALL BLOOD CELLS originate from a common
progenitor termed the hematopoietic pluripotent stem cell (HSC). These
cells have the capacity to self-renew, provide radioprotection, and long-term repopulating activity.1 Fetal hematopoiesis is a dynamic developmental process dominated by migrating populations of
hematopoietic stem cells, progenitors, and precursors.2 The
earliest detectable site of hematopoiesis occurs in the yolk sac at day
7. This stage of fetal hematopoiesis, referred to as embryonic or
primitive hematopoiesis, is characterized by the generation of
nucleated erythrocytes and a limited number of primitive macrophages.
The adult/definitive hematopoietic system is thought to be generated
from an intraembryonic site. At approximately day 8, hematopoietic
activity can be detected in the para-aortic splanchopleura (PAS), which
later develops into the aortic-gonad-mesonephros region
(AGM).3,4 At day 9, the fetal liver (FL) becomes the active
site of fetal hematopoiesis and remains the dominant site until the
early neonatal period.5 The shift to the FL involves an
initial wave of colonization by primitive erythroid and erythro-myeloid progenitors emanating from the yolk sac, followed by further waves of
colonization consisting of circulating committed precursors and
uncommitted multipotent progenitors from the PAS/AGM
regions.6 Definitive HSC arise intraembyronically in the
AGM region at day 10 and subsequently migrate to the yolk sac and
FL.7-10 The frequency of HSC peaks in the FL at day 14.5 and then decreases as hematopoiesis gradually shifts to the bone
marrow, the principal site of hematopoiesis in the
adult.9,11
Regulation of HSC renewal, proliferation, and differentiation is still
a poorly understood process. HSC are thought to be subjected to both
stochastic and instructive elements within the hematopoietic
microenvironment. The microenvironment consists of cellular contacts
mediated by a myriad of different adhesion molecules and their cognate
ligands together with locally produced cytokines. These factors act
together to modulate stem cell differentiation, proliferation, and
survival. Changes in the microenvironment that occur as HSC migrate
from the FL to bone marrow may be responsible for a number of
pronounced phenotypic and functional differences between fetal and
adult HSC.1 The importance of the microenvironment has
recently been highlighted by the demonstration that the developmental potential of HSC could be reprogrammed by changing their
microenvironment.12 Little is known about the molecular
mechanisms that enable different hematopoietic microenvironments to
alter the functional properties of HSC during different stages of
gestation. However, one key regulatory step modulating HSC function is
transcription factor-dependent alterations in gene expression. Defining
which transcription factors modulate gene expression in HSC is,
therefore, fundamental to our understanding of hematopoiesis.
We and others have used gene targeting in mice to probe the function of
transcription factors during hematopoiesis.13 The transcription factor PU.1, a member of the ets family of DNA
binding proteins, is expressed only in the hematopoietic
system.14 Targeted mutagenesis of the PU.1 gene
causes a late gestational embryonic lethal phenotype and a profound
defect in yolk sac and FL hematopoiesis.15-17 Extensive
functional and phenotypic analyses have shown the total absence of lymphoid and myeloid lineages, but normal numbers of both
erythroid and megakaryocytic progenitors in the mutants. These results
suggest that PU.1 is required for the function of a multipotential
lymphoid-myeloid progenitor population in the FL. Furthermore, normal
levels of erythropoiesis and megakaryopoiesis in the FL and yolk sac
indicate that hematopoiesis was properly initiated at both sites.
Additional experimentation, consisting of FL adoptive transfer
experiments and the generation of embryonic stem (ES)
stem-cell-derived chimeric mice, established that the PU.1
mutation is cell intrinsic and cannot be rescued by a wild-type (WT)
microenvironment.17 The inability of
PU.1/ FL cells to offer short-term
multilineage reconstitution and radioprotection provided the first
indication of a possible dysfunction in the FL HSC compartment. The
short duration of the radioprotection assay prevented any further
conclusions concerning the long-term repopulating potential of
PU.1/ HSC. In addition, the ability
of PU.1/ ES cells to contribute to
the erythroid lineage in fetal chimeras but not in adult chimeras
suggests that the requirement for PU.1 may change during hematopoietic
development.17
In this report, we have initiated a series of experiments to study the
functional behavior of PU.1/ fetal
HSC and multipotent hematopoietic progenitor cells (HPC) in the adult
microenivironment. Competitive repopulation assays (CRA) showed that
PU.1/ HSC are incapable of
establishing long-term repopulation of the erythroid, lymphoid, or
myeloid lineages. The lack of contribution suggests that
PU.1/ fetal HSC are at a competitive
disadvantage compared with WT adult HSC in the bone marrow.
PU.1/ HPC failed to respond
synergistically to erythropoietin (Epo) and stem cell factor
(SCF), which play an important role during normal
definitive erythropoiesis. The expression of c-kit on
PU.1/ HPC was severely reduced. This
may explain the inability of PU.1/
HPC to sustain erythropoiesis in the bone marrow. Homing and engraftment studies demonstrated that
PU.1/ HPC are severely impaired in
their ability to migrate to and colonize the bone marrow.
PU.1/ HPC do not express a number of
adhesion molecules, including  -integrins VLA-4/CD49d and
VLA-5/CD49e, which have previously been shown to be important for HPC
function. The lack of adhesion molecule expression may explain the
dysfunctional properties of PU.1/ fetal HPC
in vivo. Collectively, this study establishes that PU.1 is required for
proper migration and engraftment of HPC in the adult bone marrow and
suggests a new role for PU.1 in maintaining definitive erythropoiesis.
| |
MATERIALS AND METHODS |
Mouse strains.
PU.1 WT (PU.1+/+ or PU.1+/)
and mutant (PU.1/) embryos were
generated and genotyped as previously described.15
C57BL/6-Ly 5.1 mice were obtained from Jackson Laboratories (Bar
Harbor, ME).
Antibodies/fluorescence-activated cell sorting (FACS) analysis.
Flow cytometric analysis was undertaken on single-cell suspensions
prepared from FL and peripheral blood samples as previously described.15 Cell samples were stained with either
fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated
monoclonal antibodies (MoAbs) as stipulated by the manufacturer
(Pharmigen, San Diego, CA). PE-conjugated MoAbs included RA3-6B2
(B220), RM4-5 (CD4), and M1/70 (CD11b/Mac-1). FITC-conjugated MoAb
included A20 (Ly5.1/CD45.1), 104 (Ly5.2/CD45.2), and 2B8
(c-kit/CD117).
Erythroid progenitor assay.
E16.5 WT or PU.1/ FL single-cell suspensions
(2 × 105) were plated in 1.3 mL of a serum-free
methylcellulose medium as described by Ratajczak et al.18
Briefly, a methylcellulose mixture was prepared containing a final
concentration of 0.9% methylcellulose, 1% delipidized bovine serum
albumin, 270 µg/mL saturated transferrin, 20 µg/mL insulin, 5.6 µg/mL cholesterol, 2 mmol/L L-glutamine, 0.01%
penicillin/streptomycin, and 100 µmol/L monothioglycerol. Methylcellulose cultures were supplemented with Epo (4 U/mL; Amgen, Thousand Oaks, CA) or Epo plus SCF (50 ng/mL; R&D Systems,
Minneapolis, MN) to stimulate the generation of erythroid progenitors.
Hemoglobin (Hbb) assays.
Cystamine-modified hemoglobin samples were analyzed by cellulose
acetate electrophoresis as previously described.17 After electrophoresis, cellulose acetate plates were stained in a 1% Ponceau
S solution (Sigma Laboratories, St Louis, MO) containing 5% trichloroacetic acid for 5 minutes and rinsed in 5% acetic acid.
Quantification was performed by scanning densitometry image analysis to
determine relative contributions of donor versus recipient hemoglobin isoforms.
In utero HPC transplants.
PU.1+/ (Ly5.2) and WT (Ly 5.1) pairs were
crossed to generate timed pregnancies. E14.5, AA4.1+ WT (Ly
5.1) HPCs were enriched by magnetic bead selection to a cell density of
105/5 µL as described below. Anesthetized pregnant
PU.1+/ females had their uterine horns
surgically exposed, and each embryo (Ly 5.2) was injected with
105 cells intraperitoneally via 60-µm glass needles
through the uterine wall. After replacement of the uterine horns and
suturing, the manipulated embryos were carried to term. Pups were
genotyped at 3 weeks of age and were subsequently analyzed at 6 weeks
to determine HPC contribution.
CRA.
Recipient animals (C57BL/6-Ly 5.1) were irradiated with a single dose
of 960 rads from a 137Cs source. Donor cells (Ly 5.2) were
prepared as pooled single-cell suspension (106/mL) in
phosphate-buffered saline (PBS) from E14.5, PU.1 WT (+/+ or +/ )
or mutant (/) FLs. Syngeneic competitive bone marrow cells (Ly 5.1) were prepared as a single-cell suspension in PBS. A
mixture of 70% (5 × 105), 80% (1 × 106), or 90% (2 × 106) Ly 5.2 FL cells
along with a radioprotective dose of syngeneic Ly 5.1 bone marrow cells
(2 × 105) were retro-orbitally injected into
anesthesized, lethally irradiated Ly 5.1 animals. After
transplantation, the animals were maintained on water containing 20 mL/L of Sulfamethoxazole and Trimethoprim. On a monthly basis,
approximately 0.2 mL of peripheral blood was obtained from recipient
mice by tail bleeds. The harvested blood was mixed with an equal volume
of 2% dextran sulfate, incubated at 37°C to precipitate
erythrocytes, and treated with ammonium chloride lysis buffer to lyse
any remaining erythrocytes. Mononuclear cells in the resulting
supernatant were washed twice with PBS and analyzed by flow cytometry
to detect Ly 5.2-derived lymphoid and myeloid cells. Contribution to
the erythroid lineage was assessed by analyzing the presence of
hemoglobin isoforms as described above.
Enrichment and fluorescein labeling of FL AA4.1+ HPC.
Single-cell suspensions were prepared in Dulbecco's modified Iscove's
medium (DMIM) from E14.5 FL and pooled as either WT or
PU.1/ samples as described above. The
AA4.1+ fraction was isolated from pooled FL cells by
magnetic bead positive selection using MidiMACS separation columns and
goat anti-rat IgG MicroBeads as recommended by Miltenyi Biotec (Auburn,
CA). Briefly, pooled FL cells were incubated at a cell density of 5 × 106 to 1 × 107 cells/mL with
saturating amounts of AA4.1 MoAb/MicroBeads for 30 minutes at 4°C.
The AA4.1 MoAb/MicroBeads were prepared by mixing an AA4.1 MoAb,
obtained from a hybridoma culture supernatant as previously
described,17 with goat antirat IgG MicroBeads for 15 minutes at 4°C. The samples were loaded onto the MidiMACS separation columns, washed, and eluted as specified by the
manufacturer. This procedure routinely resulted in a 100-fold
enrichment of AA4.1+ cells.
For homing experiments, pooled FL cells were fluorescently labeled with
carboxyfluorescein diacetate succinimidyl ester (CFDASE; Molecular
Probes, Inc, Eugene, OR), as described.19 Briefly, 1 × 107 cells that have been extensively washed in PBS
were incubated with a 10 µmol/L CFDASE solution for 10 minutes at
room temperature while slowly agitating. The labeling reaction was
quenched by adding an equal volume of newborn calf serum (NCS) and
washing extensively with PBS. Alternatively, FL cells were
fluorescently labeled using FITC (Isomer I; Molecular Probes) according
to Butcher and Weissman.20 Briefly, cells at a density of 3 to 4 × 107 were labeled with a 60 µg/mL solution of
FITC for 20 minutes at room temperature in a 1:1 mixture of complete
DMIM and 1× PBS (pH 7.4) with a final serum concentration of 5%.
After FITC labeling, the cells were washed with complete DMIM and
centrifuged through a serum gradient. Labeled cells were passed through
a Nitex membrane before selection with AA4.1 MoAb/MicroBeads.
Short-term homing and engraftment of HPC.
Fluorescein-labeled AA4.1+ cells (1 to 2 × 105) in 0.1 mL of PBS were transferred retro-orbitally into
lethally irradiated C57B/6 recipients as described above. Recipient
animals were killed 5 to 48 hours posttransfer, and single-cell
suspensions were prepared from the bone marrow, liver, spleen, and
thymus. For the bone marrow, cells were harvested from both the femur
and tibia and subsequently pooled. After transfer of the cell
suspensions to flat-bottom multiwell tissue plates, the number of
fluorescent cells in the entire sample was detected visually using a
Nikon (Melville, NY) Diaphot 300 inverted microscope with
an external fluorescent light source. Phase contrast visualization was
used to confirm the viable nature of the cells counted. Nontransfered fluorescein-labeled cells provided a staining control. After
quantification of total cell numbers per sample, results were
normalized and reported as fluorescent cells per 107 total cells.
Reverse transcriptase polymerase chain reaction
(RT-PCR) analysis of HPC.
Total RNA was isolated from E14.5 AA4.1+ pooled FL cells
using RNAzol (Tel-Test, Woodlands, TX) according to the manufacturer's specification. Random-primed cDNA was synthesized from 2.0 µg of
total RNA using the First Strand Synthesis Kit (Pharmacia Biotech, Uppsala, Sweden) as stipulated by the manufacturer. All
PCR reactions were performed using 30 cycles and the following
annealing temperature and primer pairs: 4/VLA-4
(62°C; forward, 5'-CTGCACAGCCACGG GTCGAAG-3'; reverse,
5'-GAGAT CTGAGAAG CCATCTGC-3'), 5/VLA-5
(57°C; forward, 5'-GGCTTCTCCGTCCAGT TTTAC-3'; reverse,
5'-GATGTAGACCCTG CCCAC CTCC-3'),  1
(62°C; forward, 5'-G TGAACAGTGAAGACAT GGACGC-3'; reverse, 5'-GGTGAGATTGAAGTGGGA GCACTC-3'),
7 (57°C; forward, 5'-CCATCTGGACAGCAATGGTGTC-3'; reverse,
5'-GGAGGCAGTGAGTAGCTTGAAGAG-3'), CD11b/M (57°C; forward, 5'-GACCCA
GGTTACCGTCTACTAC-3'; reverse, 5'-TTCAGCACTGGGGTCCTTTC AAGC-3'),
2/CD18 (62°C; forward, 5'-CTTCCTGGGATCT GCTGT
GTC-3'; reverse, 5'-CCAGC TTCTTGACGTTGTTGAGG-3'),
hypoxanthine phosphoribosyl transferase (HPRT; 62°C;
forward, 5'-CACAGGACTAGAACAC CTGC-3'; reverse,
5'-GCTGGTGAAAAGGACCTCT-3'). PCR products were fractionated
in 2% agarose and examined further by Southern blot analysis when
necessary using cDNA fragments as hybridization probes.
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RESULTS |
In utero transplantation of WT HSC rescues
PU.1/ animals from a lethal hematopoietic
defect.
PU.1/ embryos die by E17.5,
apparently due to a severe hematopoietic defect.15 The
mutant embryos are normal morphologically with unimpaired erythrocyte
and platelet production. Therefore, it is unclear why the embryos die.
To determine if the defects found in
PU.1/ embryos are purely
hematopoietic in origin, WT (Ly 5.1) hematopoietic progenitors were
transplanted in utero. Both donor WT × WT (Ly 5.1) and recipient
PU.1+/ × PU.1+/
(Ly 5.2) crosses were performed to generate timed embryos.
AA4.1+ HPC were enriched from E14.5 WT (Ly 5.1) pooled FL
cells and injected intraperitoneally into recipient embryos (Ly 5.2).
AA4.1 is a known surface marker expressed on HSC and multipotent
progenitors in the FL.21,22 The manipulated embryos were
brought to term, genotyped by Southern blot analysis, and analyzed for
hematopoietic contributions at 6 weeks of age.
PU.1/ animals were fully rescued by
the transplantation of WT HPC and were indistinguishable from
PU.1+/+ or
PU.1+/ littermates. Flow cytometric
analysis of B cells (B220+), monocytes
(CD11b+), and T cells (CD4+) demonstrated that
only donor-derived progenitors (Ly 5.1+) contributed to
hematopoiesis in these animals (Fig 1). In
contrast, donor cells rarely contributed to hematopoiesis in
PU.1+/+ or PU.1+/
recipients. A total of 3 rescued
PU.1/ and 6 transplanted WT animals
were examined with equivalent results. The rescue experiments
demonstrate that the lethal nature of the PU.1/ defect is of hematopoietic
origin. Furthermore, hematopoiesis can be supported by a
PU.1/ microenvironment.
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| Fig 1.
In utero transplant of WT hematopoietic progenitors.
E14.5 embryos (Ly 5.2) from PU.1+/ matings were
injected with 105 WT AA4.1+ HPC (Ly 5.1). The
resulting animals were then genotyped by Southern blot at 3 weeks of
age. At 6 weeks of age, bone marrow underwent 2-color flow cytometric
analysis for B-cell (B220+/Ly 5.1+) and
monocyte (CD11b+/Ly 5.1+) origin, and the
thymus was analyzed for T-cell (CD4+/Ly
5.1+) origin.
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PU.1/ HSCs lack long-term
repopulating activity in radioablated adult mice.
Previous adoptive transfer experiments determined that
PU.1/ fetal HPC are unable to provide
radioprotection to lethally irradiated adult recipients.17
However, the recipient animals did have detectable levels of
donor-derived erythrocytes, but no lymphoid or myeloid engraftment,
before dying 2 to 3 weeks postirradiation. The early death of the
recipient animals receiving PU.1/ FL cells
prevented any further conclusions being drawn concerning HSC and/or HPC
function. Therefore, a competitive repopulation assay was used to
examine the functional status of
PU.1/ HSC in the adult bone marrow
microenvironment. This assay system is designed to test the ability of
HSC to offer multilineage reconstitution in vivo in the presence of a
competing radioprotective dose of syngeneic bone marrow
cells.23
E14.5 FL contain the highest level of multilineage repopulating
activity.9 Therefore, CRAs were performed with pooled WT (PU.1+/+ or PU.1+/
littermates) or PU.1/ E14.5 FL
samples (Ly 5.2) in competition with a radioprotective dose of
syngeneic bone marrow (Ly 5.1). Two groups of 8 recipients received
70% (5 × 105) WT or
PU.1/ FL cells plus 30% (2 × 105) syngeneic bone marrow, 2 groups received 80% (1 × 106) FL cells with 20% (2 × 105)
syngeneic marrow, and a final 2 groups received 90% (2 × 106) donor to 10% (2 × 105) syngeneic.
After transplantation, the recipients were bled on a monthly basis and
analyzed for donor-derived contribution to the erythroid, lymphoid, and
myeloid lineages. Donor-derived erythrocytes were detected by
differences in Hbb isoforms. Cellulose acetate electrophoresis was used
to distinguish differences between the Hbbd (Ly 5.2, donor)
and the Hbbs (Ly 5.1, syngeneic) isoforms. Relative
contributions of the hemoglobin isoforms were quantified by scanning
densitometry. The presence of donor-derived lymphoid and myeloid cells
was ascertained by 2-color flow cytometry using lineage- and Ly
5.2-specific MoAbs. The CRA were repeated a total of 3 independent times.
Reconstitution of the erythroid lineage by WT and
PU.1/ FL cells is shown in
Fig 2A. Reconstitution by donor-derived
erythrocytes is expressed as the percentage of donor-derived hemoglobin
(Hbbd) in the peripheral blood. At 1 month posttransplant,
WT FL cells were contributing to erythrocyte production at nearly the
expected ratios based on input percentage. By 2 and 3 months
posttransplant, the WT FL cells were contributing at equal to or better
than the expected ratios. In contrast, FL cells isolated from
PU.1/ littermates only produced
erythrocytes for approximately 1 month and at greatly reduced levels.
Results for reconstitution of the lymphoid and myeloid compartments are
shown in Fig 2B. Unlike the contribution to the erythroid lineage by
PU.1/ HPC, there were no
detectable PU.1/ (Ly
5.2+)-derived B cells (B220+), T cells
(CD4+), or monocytes (CD11b+)
posttransplantation (Fig 2B). Reconstitution was monitored for a total
of 6 months with no detectable contribution by
PU.1/ HSC. For WT FL cells, a
substantial portion of mononuclear cells in the peripheral blood
expressed Ly 5.2 and lineage markers (B220, CD4, and CD11b) for the
full course of the experiment. These results demonstrate that
PU.1/ FL HPC are incapable of
contributing to long-term erythropoiesis, lymphopoiesis, or
myelopoiesis in the bone marrow.
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| Fig 2.
CRA of the hematopoietic lineages by WT and
PU.1/ E14.5 FL cells. Irradiated Ly 5.1 adult mice (Hbbs) were transplanted with either 70% (5 × 105), 80% (1 × 106), or 90% (2 × 106) E14.5 FL progenitors from donor WT
(PU.1+/+,/) or
PU.1/ embryonic littermates (Ly 5.2, Hbbd). A radioprotective dose of 2 × 105
normal adult bone marrow cells was included as a source of competitive,
syngeneic HPC. Peripheral blood was taken from recipient animals on a
monthly basis for 6 months postengraftment. (A) Contribution of donor
HPC to peripheral blood erythrocytes. Cellulose acetate
electrophoreisis was used to separate donor from recipient hemoglobin
isoforms. Relative contribution was determined by scanning densitometry
and expressed as the percentage of donor contribution to total
hemoglobin production. Data from 3 independent experiments are depicted
on the graph. Solid lines represent animals that received WT donor HPC.
Dashed lines represent animals that received
PU.1/ donor HPC. (B) Contribution of donor
HPC to the lymphoid and myeloid lineages. Contribution to the lymphoid
and myeloid lineages was determined by flow cytrometric analysis using
an Ly 5.2-specific MoAb to identify donor-derived cells and
lineage-specific MoAbs to characterize the B-cell
(B220+), T-cell (CD4+), and monocyte
(CD11b+) populations. Representative FACS profiles are
shown for peripheral blood samples obtained at 2 months
posttransplantation for lethally irradiated Ly 5.1+ adult
mice receiving either pooled WT or PU.1/
E14.5 FL HPC (Ly 5.2+).
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PU.1/ FL erythroid progenitors fail
to respond to SCF stimulation.
The inability of E14.5 PU.1/ FL
HPC to support long-term erythropoiesis contrasts with the normal
number of FL or yolk sac erythroid progenitors in
PU.1/ embryos and with the capacity
of PU.1/ ES cells to establish
erythropoiesis in fetal chimeras.16,17 The differing
requirement for PU.1 alludes to a fundamental difference between fetal
and adult erythropoiesis. One critical regulatory signal governing
erythropoiesis that has been shown to change during development
emanates from the stem cell factor receptor (c-kit).24,25 In vivo administration of
anti-c-kit MoAbs showed that primitive erythropoiesis in the
yolk sac and FL is independent of c-kit function, whereas
definitive erythropoiesis depends on c-kit activity. To examine
the ability of SCF to promote erythropoiesis, a serum-free progenitor
assay system, was chosen to test the synergistic activity between Epo
and SCF for the generation of erythroid progenitors (colony-forming
unit-erythroid [CFU-E]).18 The assay was
performed in duplicate on 6 different embryos of each genotype. E16.5
WT FL cells produced 9 ± 6 CFU-E per 106 cells in
response to Epo alone. PU.1/ cells
produced an equivalent 7 ± 2 CFU-E with Epo alone. WT cells had a
synergistic response to a combination of Epo and SCF to produce 551 ± 230 CFU-E per 106 cells. In contrast,
PU.1/ cells still only produce 13 ± 1 CFU-E in response to Epo plus SCF, indicating a lack of
synergy. Day E16.5 was chosen for this analysis because our previous
studies quantifying FL erythroid progenitors showed normal numbers of
PU.1/ CFU-E when assayed in the
presence of serum with a cocktail of growth factors.17 Flow
cytrometry analysis was performed to ascertain the effect of the
PU.1 mutation on c-kit expression. Very few E16.5
PU.1/ FL cells express detectable
levels of c-kit on their surface (Fig 3). This analysis has shown that the
lack of c-kit expression by E16.5
PU.1/ FL cells may explain the inability of
PU.i/ erythroid progenitors to
synergistically respond to Epo plus SCF.
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| Fig 3.
c-kit expression on WT and
PU.1/ E16.5 FL cells. c-kit
expression by WT and PU.1/ E16.5 FL cells was
examined by flow cytometry using a c-kit-specific MoAb.
Representative data for staining by isotype-match control or an
anti-c-kit MoAb for individual WT or
PU.1/ E16.5 FL cells are shown.
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The effect of PU.1 on homing and engraftment of FL progenitors in the
adult bone marrow.
One possible explanation for the lack of long-term hematopoiesis in
recipient adult animals is that PU.1/
HPC are more prone to programmed cell death. TUNEL assay of E14.5 whole
FL cell suspensions or AA4.1+ enriched progenitor cell
populations, containing a mixture of HSC and multipotent progenitor
cells, showed no detectable apoptotic cells in either population (data
not shown). These results suggest that an increased rate of apoptosis
is an unlikely explanation for the aberrant behavior of the HPC
compartment in PU.1/ FL. An
alternative possibility to explain the inability of
PU.1/ HSC to establish long-term
reconstitution in adult animals would be a defect in the ability of
progenitor cells to home to and engraft in the adult bone marrow.
To monitor migration, pooled FLs from WT
(PU.1+/+,+/) and
PU.1/ E14.5 embryos were
fluorescently labeled with either CFDASE or FITC. After fluorescent
labeling, tagged cells were enriched for AA4.1+ progenitors
by magnetic bead selection and injected retro-orbitally into lethally
irradiated adult mice. Recipient animals were killed at 5, 16, or 48 hours posttransplant. Bone marrow, spleen, thymus, and liver were
examined for the presence of transplanted fluorescent cells
(Table 1). The results for the spleen and
thymus were essentially constant for all time points, with
approximately 2-fold fewer PU.1/
AA4.1+ HPC migrating to these tissues. A striking
difference was observed between the WT and
PU.1/ AA4.1+ FL
progenitors in the bone marrow. At 5 hours posttransplant, there was a
2.5-fold reduction in PU.1/ HPC
migrating to the bone marrow. By 16 hours posttransplant, there were
8.5-fold fewer PU.1/ HPC in the
marrow. The gap widened to an 11-fold decrease in the marrow by 48 hours. In contrast, after 48 hours, there was a 9-fold increase in
PU.1/ AA4.1+ progenitors
detected in the adult liver when compared with WT. The data presented
in Table 1 represent a single homing assay that has been repeated 4 independent times with similar results. Therefore, HPC from
PU.1/ FLs are severely impaired in
their ability to home to and engraft the adult bone marrow. The altered
migration of the mutant progenitor cells results in their abnormal
accumulation in the liver after transplantation.
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Table 1.
Quantification of Fluorescently Tagged E14.5 WT and
PU.1/ AA4.1+ FL Cells Migrating
to the Bone Marrow, Spleen, Thymus, and Liver
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Adhesion molecule profile of hematopoietic progenitors isolated
from PU.1/ E14.5 FL.
The observed defect in the ability of
PU.1/ progenitors to home to the bone
marrow prompted an evaluation of a number of adhesion molecules thought
to mediate hematopoietic progenitor cell migration. Initial flow
cytometry experiments demonstrated that the intergrin CD11b was missing
and suggested that integrins 4 and 5 were affected on HPC, but
not in the FL as a whole (data not shown). Integrins 4/VLA-4 and
5/VLA-5 have been strongly implicated in the attachment of
hematopoeitic progenitors to bone marrow stroma and associated
extracellular matrix.26-28 To more closely examine integrin
4 and 5 expression, RT-PCR analysis was undertaken on total
cellular RNA isolated from either whole FLs or AA4.1+
enriched cell populations from WT and
PU.1/ embryos. RT-PCR followed by
Southern blotting was chosen as the most sensitive method to examine
expression given the difficulty of obtaining large numbers of
PU.1/ HPC.
Figure 4 shows that the integrin
CD11b/M is not expressed by
PU.1/ AA4.1+ cells, whereas
expression of its heterodimeric binding partner, intregin 2/CD18, is
reduced. Both the 4 and 5 integrins were expressed in whole
PU.1/ FLs, but not in the
AA4.1+ PU.1/ FL HPC, even
after Southern blot analysis of the PCR reactions products (Fig 4).
Expression of the integrin 1, which has been demonstrated to affect
HSC migration to the FL,29 was unaffected by the
PU.1 mutation. Another integrin, 7, possessing a role in
lymphocyte homing,30 showed a minor reduction in
expression. These results are consistent with the flow cytometric
characterization of FL cells from
PU.1/ embyros. Thus, a number of
functionally important integrins are not properly expressed in the
absence of PU.1, which may explain the aberrant homing and engraftment
properties of PU.1/ HPC.
View larger version (37K):
[in this window]
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| Fig 4.
Adhesion molecule profile of E14.5 AA4.1+
FL cells. Total RNA was isolated from either whole FLs or
AA4.1+ enriched cells from E14.5 WT and
PU.1/ embryos. RNA expression was examined
using primers specific for integrins CD11b, 4,
5, 1, 2, and
7. Expression of the house keeping gene HPRT was used to
normalize variation between RNA samples. Results are displayed as
either ethidium bromide-stained PCR products or, to enhance
sensensitivity of the expression analysis, the RT-PCR products were
further analyzed by Southern blot hybridization using cDNA fragments as
hybridization probes.
|
|
| |
DISCUSSION |
Phenotypic and functional analysis of
PU.1/ embryos demonstrated severe
abnormalities in fetal hematopoiesis occurring in both the FL and yolk
sac.15-17 This analysis showed a cell-intrinsic defect in
lymphoid and myeloid development. In contrast, both primitive and
definitive erythropoiesis, along with megakaryopoiesis, appeared normal
in the yolk sac and FL. A second targeted mutation of the PU.1
allele demonstrates a similar phenotype with respect to fetal
hematopoiesis.31 However, mice containing the second PU.1 mutation can be kept alive for 2 weeks by high-dose
antibiotics. During those 2 weeks, T cells and abnormal neutrophils can
develop. Recent work with our mutation demonstrates that similar
neutrophils32 and T cells32a can
be cultured. Therefore, with respect to hematopoietic development, the
two PU.1 mutations appear almost identical. Strain differences
may contribute to the variable onset of lethality. Rescue of
PU.1/ animals in utero by WT HSC
(Fig 1) demonstrates that mutant embryos die from hematopoietic
deficiencies. Furthermore, PU.1/
micoenvironments can foster proper development of WT hematopoietic progenitors. These data suggest a role for PU.1 in a multipotential lymphoid-myeloid progenitor population in the FL. Our prediction is
that one or more PU.1-regulated genes are required for efficient commitment and/or differentiation of multipotent progenitors to either
the B-lymphoid or myeloid lineage. This hypothesis has recently been
supported by work that demonstrates that PU.1 can irreversible commit
retroviral transformed avian multipotent progenitor cells to the
myeloid lineage.33 The normal levels of erythroid and
megakaryocytic progenitors found in
PU.1/ embyros suggests that
PU.1/ HSC form in the AGM region and are able
to colonize the yolk sac and FL.15,16 However,
PU.1/ HSC do not provide radioprotection for
lethally irradiated recipients. In addition,
PU.1/ ES cells contribute to fetal
but not adult erythropoiesis in chimeric animals.17 These
data suggest that PU.1/ HSC may be
functionally impaired and point to a possible role for PU.1 during
adult erythropoiesis.
The competitive reconstitution experiments were designed to allow
donor-derived hematopoiesis to be evaluated over the course of several
months. In the presence of a radioprotective dose of competitor adult
bone marrow cells, there were significant levels of
PU.1/ derived erythrocytes detectable
for 1 month (Fig 2A). These results show that
PU.1/ FL HPC can migrate to the adult
bone marrow and contribute to erythropoiesis, but they are quickly
out-competed by syngeneic WT cells. This suggests that, on a per cell
basis, PU.1/ fetal HPC are much less
efficient than WT bone marrow cells. The absence of donor-derived
PU.1/ erythrocytes beyond 2 months in
radiation chimeras is entirely consistent with the lack of contribution
to the erythroid lineage by PU.1/ ES
cells in adult chimeras.17 When reconstitution of the
lymphoid and myeloid lineages was examined, no contribution was seen
from PU.1/ HSC (Fig 2B). FL HSC
should be highly favored in these competitive reconstitutions due to
their 4- to 5-fold greater long-term repopulating capacity.9,22,34 Furthermore, the lack of any detectable Ly
5.2+ hematopoietic cells beyond 2 months in the recipients
indicates the importance of PU.1 for the function of HSC in the adult
bone marrow. It is still unclear exactly how far along the lymphoid and/or myeloid differentiation pathways
PU.1/ HSC can proceed in a
competitive microenvironment.
In situ immunochemical expression analysis of the murine bone marrow
has documented PU.1 protein expression in immature erthyroblasts. PU.1
expression is downregulated upon terminal differentiation into
erythrocytes.35 Continued expression of PU.1 in
erythroblasts leads to growth immortalization and subsequent conversion
to an erythroleukemic state.14 One key regulatory cascade
operative during definitive erythropoiesis requires synergy between the cytokines Epo and SCF.36 Both Epo and SCF have been shown
to be necessary for erythroid differentiation, proliferation, and survival.37-40 Serum-free clonogenic assays can directly
measure synergistic and/or additive effects of growth
factors.18 It is important to examine growth factor
responsiveness using a defined assay system due to wide degree of
functional redundancies between cytokines and cytokine receptors. For
example, thrombopoietin can rescue CFU-E colonies in EpoR knockout
mice.39 Serum-free clonogenic assays of E16.5
PU.1/ FL erythroid progenitors showed
their lack of synergy to Epo plus SCF. Flow cytometric analysis of
PU.1/ E16.5 FL cells showed little or
no expression of c-kit (Fig 3). The possibility of
PU.1-dependent expression of c-kit in erythroid progenitors is
supported by recent analysis that has shown an ets family
member, in conjunction with c-myb, is involved in the regulation of the
c-kit promoter.41 One postulate is that, during repopulation, PU.1/ erythroid
progenitors are at a competitive disadvantage in the bone marrow due to
their inability to respond to differentiative, proliferative, and
survival signals emanating from EpoR and c-kit.
We next examined the homing potential of
PU.1/ HPC with a quantitative homing
assay. PU.1/ HPC were 11-fold less
efficient at homing/colonizing to the adult bone marrow after 48 hours
(Table 1). There was an accompanying 9-fold increase in the frequency
of PU.1/ HPC found in the adult
liver. Cell migration through the bone marrow microenvironment has been
hypothesized to be a 2-step process of initial cell binding followed by
transenthothelial migration into the hematopoietic
compartment.42 For both steps, the importance of cell-cell
interactions is clear. This prompted an evaluation of adhesion molecule
expression by HPC isolated from PU.1/ E14.5
FLs (Fig 4). PU.1/ HPC lack expression of a
number of integrins, including CD11b, 4/VLA-4, and 5/VLA-5, but
retain expression of 1, 2, and 7.
Of particular interest was the absence of 4/VLA-4. Considerable data
have implicated 4/VLA-4 as a key regulatory surface molecule
mediating HPC/stromal cell contact in vitro and controlling HPC homing
in vivo.43-45 The lack of 4/VLA-4 expression in
PU.1/ HPC is supported by a previous study
that identified a functional ets regulatory element in the 4/VLA-4
promoter.46 However, the investigators in
this study failed to determine which ets family member bound
this element. Flow cytrometric and RT-PCR analyses have shown that
4/VLA-4 is still expressed outside the AA4.1+ HPC
population in PU.1/ FL (Fig 4). Thus,
these data suggest a direct role for PU.1 in regulating 4/VLA-4
expression in HPC. Another ets family member may be involved in
4/VLA-4 expression outside the HPC compartment. However, the lack of
4 is not sufficient to explain the PU.1/
homing defect, because HSC migration to the bone marrow during fetal
development appears to be normal in the absence of 4
expression.47 The ability of a recombinant fibronectin
fragment containing binding sites for 4, 5, and CD44 to inhibit
multilineage engraftment has indicated a potential role for these
adhesion molecules in interactions with the bone marrow
stroma.48 Therefore, the 11-fold decrease in bone marrow
homing/colonization of PU.1/ HPC is
most likely the aggregate effect of several missing adhesion molecules.
The decrease may also reflect a reduction in the absolute number of HPC
in AA4.1+ population of
PU.1/ animals that are capable of
homing to any tissue. Accumulation of
PU.1/ HPC in the adult liver raises
the interesting possibility of an active tropic mechanism to explain
this observation. Both PU.1/ embryos
and fetal chimeras made with PU.1/ ES
cells indicate that mutant HSC home to and function in the FL
microenvironment. 1 integrin has been shown to be required for
proper homing of HSC to the FL.29 Without 1, cells fail to enter the FL and remain in the circulation, suggesting that 1 may
be directly involved in liver homing.
PU.1/ progenitors retain 1
expression, but lack expression of other integrins. Perhaps this
absence of other integrins causes
PU.1/ progenitors to retain their
liver tropism resulting in their abnormal accumulation in the adult
liver after transplantation. The data presented in this study
demonstrate that PU.1 is required for fetal HSC to establish and
maintain long-term hematopoiesis in the bone marrow. In addition, we
have shown that PU.1 is necessary for long-term definitive
erythropoiesis. Therefore, the PU.1/
mouse provides an ideal model system to study the mechanism of hematopoietic progenitor migration to and multilineage engraftment of
the adult bone marrow. We are currently in the process of reintroducing missing integrin molecules into PU.1/
HPC via retroviral transduction to ascertain their effect on progenitor
cell function in the bone marrow. These future studies should provide
insight into adhesion molecule-dependent events that control
hematpoietic stem and progenitor cell migration and subsequent
multilineage repopulation in the bone marrow microenvironment.
| |
ACKNOWLEDGMENT |
The authors thank Irving Weissman (Stanford University, Stanford,
CA) for the 4 and 7
integrin cDNA fragments and H. Scott Baldwin (University of
Pennsylvania, Philadelphia, PA) for the 5 integrin cDNA
fragment. We also thank Hamid Bassiri (University of Pennsylvania) for
advice on detecting apoptosis, Andrew D. Wells (University of
Pennsylvania) for the CFDASE labeling protocol, Mariusz Ratajczak
(University of Pennsylvania) for assistance in setting up the
serum-free progenitor assay system, and Debbie Knobleman for assistance
with the in utero transplants.
| |
FOOTNOTES |
Submitted March 5, 1999; accepted April 21, 1999.
Supported by National Institutes of Health Grants No. CA72769 and
HL58716 to E.W.S. E.W.S. is a Leukemia Society of America Scholar.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Edward W. Scott, PhD, Institute for Human
Gene Therapy, Stellar Chance Laboratories, Room 401a, 422 Curie Blvd,
University of Pennsylvania, Philadelphia, PA 19104-6140; e-mail:
scotte{at}mail.med.upenn.edu.
| |
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A. Janowska-Wieczorek, M. Majka, J. Kijowski, M. Baj-Krzyworzeka, R. Reca, A. R. Turner, J. Ratajczak, S. G. Emerson, M. A. Kowalska, and M. Z. Ratajczak
Platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment
Blood,
November 15, 2001;
98(10):
3143 - 3149.
[Abstract]
[Full Text]
[PDF]
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P. Keller, J. L. Payne, G. Tremml, P. A. Greer, M. Gaboli, P. P. Pandolfi, and M. Bessler
Fes-Cre Targets Phosphatidylinositol Glycan Class a (Piga) Inactivation to Hematopoietic Stem Cells in the Bone Marrow
J. Exp. Med.,
September 3, 2001;
194(5):
581 - 590.
[Abstract]
[Full Text]
[PDF]
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F. Colucci, S. I. Samson, R. P. DeKoter, O. Lantz, H. Singh, and J. P. Di Santo
Differential requirement for the transcription factor PU.1 in the generation of natural killer cells versus B and T cells
Blood,
May 1, 2001;
97(9):
2625 - 2632.
[Abstract]
[Full Text]
[PDF]
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L. A. Garrett-Sinha, R. Dahl, S. Rao, K. P. Barton, and M. C. Simon
PU.1 exhibits partial functional redundancy with Spi-B, but not with Ets-1 or Elf-1
Blood,
May 1, 2001;
97(9):
2908 - 2912.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION