1 Departments of Pediatrics, Indiana University School of Medicine,
Indianapolis, IN 46202, USA
2 Biochemistry and Molecular Biology, Indiana University School of Medicine,
Indianapolis, IN 46202, USA
3 Herman B. Wells Center for Pediatric Research, Indiana University School of
Medicine, Indianapolis, IN 46202, USA
* Author for correspondence (e-mail:myoder{at}iupui.edu)
Accepted 28 May 2003
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SUMMARY |
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Key words: Hematopoiesis, Embryonic yolk sac, CD41, Mouse
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INTRODUCTION |
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The platelet glycoprotein receptor IIb/IIIa
(IIbß3; CD41/CD61) is required for normal
platelet hemostatic function (Phillips et
al., 1988
). CD41 is a protein composed of two subunits that
interact with CD61 in the presence of calcium to form a functional adhesive
protein receptor. Damage to blood vessels results in the release of a variety
of intracellular mediators as well as initiation of the hemostatic cascade
that in combination activate CD41/CD61 to bind to a variety of proteins
including fibrinogen, fibronectin, von Willebrand factor and vitronectin
(Shattil et al., 1998
;
Coller, 1990
). Heritable
mutations in either CD41 or CD61 subunits results in a bleeding disorder in
human patients called Glanzmann's thrombasthenia
(Bellucci and Caen, 2002
).
CD41/CD61 was once considered to be a platelet specific-receptor complex
and has long been used as a phenotypic marker for the megakaryocyte-platelet
lineage (Phillips et al.,
1988). However, some published information suggests that CD41
expression may not be limited to the platelet lineage
(Berridge et al., 1985
;
Fraser et al., 1986
;
Tronik-Le Roux et al., 2000
;
Tropel et al., 1997
). CD41
expression was identified on a subpopulation of
CD34+CD41+CD42- human cord blood cells that
possessed colony forming cells (CFC) and lymphoid and myeloid repopulating
ability (Debili et al., 2001
).
In contrast, CD34+CD41+CD42- adult mobilized
peripheral blood cells were enriched in megakaryocyte and erythroid CFC
activity but lacked lymphoid repopulating ability. In murine studies, CD41
co-expression with Kit identified cells in late fetal liver and adult marrow
that possessed a variety of CFC activities and gave rise to T lymphocytes in
thymic organ cultures in vitro (Corbel and
Salaun, 2002
). Transplantation of adult marrow
Kit+CD41+ cells suggested that the sorted cells gave
rise to short-term but not long-term hematopoietic repopulation
(Corbel and Salaun, 2002
).
Other studies in fetal and adult mice suggested that CD41 was expressed on
most yolk sac progenitor cells at 9.5 days post coitus (dpc) and 10.5 dpc
aorta-gonad-mesonephros (AGM) hematopoietic progenitor cells but on few fetal
liver or adult marrow progenitors
(Mitjavila-Garcia et al.,
2002
). In addition, CD41 was expressed on most hematopoietic
progenitor cells derived in vitro from murine embryonic stem (ES) cells
(Mitjavila-Garcia et al.,
2002
). Finally, recent data suggest that CD41 expression may serve
as a marker for the onset of definitive hematopoiesis in the murine embryo and
in ES cell-derived hematopoietic cells in vitro
(Mikkola et al., 2002
).
In the present work, we have identified CD41 expression on yolk sac cells coincident with the first appearance of primitive erythroid progenitor cells (E7.0) and on all yolk sac definitive hematopoietic progenitor cells (E8.25). Fetal liver and adult marrow hematopoietic progenitor cells were found in both CD41dim and CD41lo/- populations and both populations demonstrated long-term repopulating ability in multiple lineages upon transplantation, however, repopulating ability was enriched in the CD41lo/- cells. These results demonstrate that CD41 is expressed on the first hematopoietic progenitor cells of the primitive and definitive lineages in the murine yolk sac. CD41 expression persists in some hematopoietic stem and progenitor cells in the fetal liver and adult marrow. These data provide new insight into the expression of a molecule previously thought to be lineage restricted and suggest novel roles for the CD41/61 receptor complex.
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MATERIALS AND METHODS |
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Hematopoietic cell isolation
Yolk sac
Whole embryo or yolk sac cells were dissected from the decidual tissue of
timed-pregnant dams (E6.5-9.5 gestation embryos) and extensively washed in
phosphate-buffered saline (PBS; Invitrogen, Grand Island, NY) as described
(Yoder and Hiatt, 1997).
Presomitic embryos were staged by the presence of tissue landmarks and later
stages by somite counting (Downs and
Davies, 1993
). Dissected yolk sacs were incubated for 60-90
minutes at 37°C in 0.1% collagenase/dispase (Sigma, St. Louis, MO) with
20% fetal bovine serum (FBS, HyClone, Logan, UT) in PBS.
Fetal liver
Fetal liver cells were collected from E12.5 embryos. The collected tissue
was mechanically dissociated into a single cell suspension using a 5 ml
pipette. The cells were washed in Iscove's Modified Dulbecco's Medium (IMDM)
with 10% fetal bovine serum (FBS), 2% penicillin/streptomycin (P/S), 2 mM
L-glutamine, and filtered through a 70 µm cell strainer.
Bone marrow
Bone marrow was collected from normal C57BL6/J mice and the low-density
mononuclear (LDM) cells isolated by density centrifugation as previously
described (Yoder et al.,
1993).
Lineage depletion of fetal liver and adult bone marrow cells
Removal of lineage antigen-expressing cells from fetal liver cells was
achieved as follows. 0.5-1 µg/ml of rat anti-mouse Gr-1 (granulocytes),
B220 (B lymphocytes), TER-119 (erythroid cells), and CD4/CD8 (T lymphocytes)
monoclonal antibodies (PharMingen, San Diego, CA) were added to the fetal
liver cell suspension for a 20 minute incubation on ice. The stained cell
populations were pelleted (300 g for 8 minutes), washed with
buffer, repelleted and resuspended in buffer. Goat anti-rat IgG magnetic
microbeads (Miltenyi Biotec, Auburn, CA) were added for a 20 minute incubation
on ice, and the lineage-depleted cell population was obtained using a magnetic
separation device as directed by the manufacturer (Miltenyi Biotec). This
procedure was repeated for the adult bone marrow cells, with the exception
that purified and fluorescein isothiocyanate (FITC)conjugated rat anti-mouse
Mac1 was also used. Lineage-depleted populations of cells were restained with
FITC conjugates of the same antibodies and analyzed for purity using a
FACSvantage instrument (Becton Dickinson).
Fluorescence activated cell sorting
Cells to be stained were incubated with the primary antibodies of interest
for 20 minutes on ice, pelleted at 300 g, and resuspended with
streptavidin-conjugated allophycocyanin (APC), if necessary. Labeled cells
were washed twice, pelleted at 300 g, and resuspended in IMDM
with 10% FBS, 1% P/S, and 2 mM L-glutamine. Cell sorting was accomplished
using a FACStar instrument (Becton Dickinson). Rat monoclonal antibodies used
in this study (all purchased from Pharmingen) included: FITC-conjugated
anti-mouse CD41, rphycoerythrin (PE)-conjugated anti-mouse Kit,
biotin-conjugated CD34, APC-conjugated streptavidin, purified and
FITC-conjugated CD4, purified and FITC-conjugated CD8, purified and
FITC-conjugated B220, purified and FITC-conjugated TER119, purified and
FITC-conjugated Mac1, purified, FITC- and PE-conjugated CD45.1, purified,
FITC- and PE-conjugated CD45.2, and all appropriate isotype control
antibodies.
HPP-CFC assay
Double-layer agar cultures were prepared as previously described
(Yoder et al., 1995). Briefly,
the recombinant hematopoietic growth factors human macrophage
colony-stimulating factor 1 (M-CSF; 100 U), interleukin 3 (IL-3; 200 U;) and
IL-l
(500 U), and rat stem cell factor (SCF; 100 ng) were all purchased
from Peprotech, Rocky Hill, NJ and added to 10x35 mm gridded tissue
culture dishes (Nalge Nunc, Naperville, IL) followed by the addition of 0.5%
agar (Bacto-agar; Difco, Detroit, MI). Sorted yolk sac, fetal liver or bone
marrow cells (500 cells/dish) were suspended in 0.3% agar and applied as an
overlay to the 0.5% agar/growth factor containing dishes. On day 14 of
culture, the plates were read and colonies greater than 0.5 mm were scored as
HPP-CFC. For all colony assays, cells were plated in triplicate and
experiments repeated 2-4 times. In all experiments, statistically significant
differences were determined using the Student's t-test with a level
of significance set at <0.05.
Primitive erythroid colony assay
Cells were plated in triplicate at 1x105 cells/ml in 0.9%
methycellulose-based medium (Stem Cell Technologies, Vancouver, CA) that
included IMDM, 2 mM glutamine, 1% P/S, 5% protein-free hybridoma medium-II
(PFHM-II: Gibco BRL), 50 µg/ml ascorbic acid (Sigma), 450 µM
monothioglycerol (Sigma), 200 µg/ml iron-saturated holo-transferrin
(Sigma), 15% plasma-derived serum (Animal Technology, Antech, TX) and 4 U/ml
human erythropoietin (Epo; Amgen, Thousand Oaks, CA). Cultures were incubated
at 37°C in 5% CO2, and colonies were counted on day 7.
Hematopoietic progenitor cell assay
Sorted cells were plated (500-1000 cells/dish) in triplicate in 0.9%
methylcellulose cultures as previously described
(Yoder et al., 1995). Briefly,
the cells were suspended in 1% methylcellulose, 30% fetal calf serum,
10-5 mol/l 2-mercaptoethanol (Sigma), 2 mmol/l glutamine
(Invitrogen), 4 U Epo, 200 U IL-3, and 100 ng SCF. The cells were incubated at
37°C in 5% CO2 in air and groups of more than 50 cells were
scored as colonies (CFU). The early appearing CFU-Meg (<E8.25 plated cells)
were identified as clusters of 3 or more large refractile cells on day 3 of
culture. These cells were plucked, applied to glass slides, and stained for
expression of acetylcholine esterase. These early CFU-Meg were no longer
detectable by day 7 of culture (when the definitive progenitors were
scored).
CFU-Meg and BFU-Meg assay
Sorted cells were plated at 500-1,000 cells/ml in 0.3% agar-based McCoy's
5A medium (Invitrogen, Grand Island, NY) which included 10% FBS (HyClone,
Logan, UT), 100 U/ml recombinant IL-3, and 50 ng/ml recombinant human Tpo
(Peprotech, Rocky Hill, NJ). Cultures were incubated at 37°C in 5%
CO2 in air (Long,
1984). After 7 days (for CFU-Meg) or 14 days (for BFU-Meg)
incubation, CFUMeg and BFU-Meg were scored for their colony morphology and
CD41 expression. The early appearing CFU-Meg (<E8.25 plated cells) did not
grow when plated in agar medium.
Hematopoietic transplantation assays
Sorted B.6Gpi-1a/BoyJ mice yolk sac cells (15,000 cells) were transplanted
via the facial vein into six sublethally irradiated (200 centigray) newborn
C57Bl6/J recipient mice as previously described
(Yoder et al., 1997a). Sorted
fetal liver or adult marrow populations from B.6Gpi-1a/BoyJ mice were isolated
as above and transplanted intravenously via the tail vein into 6-10 lethally
irradiated (11Gy in divided doses 4 hours apart) C57BL/6J recipient animals.
The sorted donor cells (500-2000) were mixed with 200,000 low-density bone
marrow competitor cells immediately prior to intravenous injection into the
recipient mice. At monthly intervals thereafter, peripheral blood was isolated
and analyzed for evidence of donor type (CD45.1) blood cells as well as
recipient type (CD45.2). After 6 months post-transplant, peripheral blood
cells were further analyzed for donor type B lymphocyte, T lymphocyte, and
granulocyte cells as evidence of multilineage engraftment.
Whole-mount immunolabeling for confocal microscopy
C57BL/6J mice were killed by cervical dislocation on days 6.5 to 8.5 of
development (day 0.5, morning of vaginal plug). The embryos were dissected and
washed three times in PBS, fixed 10 minutes in cold acetone then rinsed three
more times in PBS. Embryos were blocked in PBS containing 3% blotting grade
non-fat dry milk (Bio-Rad Laboratories, Hercules, CA) and varying amounts of
Triton X-100 (0.0125% for day 7 embryos and 0.025% for day 8 embryos) for 1
hour. Directly conjugated primary antibodies were then added to a final
concentration of 5 µg/ml for 12 to 18 hours at 4°C. TER119, Flk-1 and
CD41 purified antibodies were labeled with Rhodamine Red-X protein labeling
kit (TER119), Alexa Fluor 488 monoclonal labeling kit (Flk-1) or Alexa Fluor
647 monoclonal labeling kit (CD41) (Molecular Probes, Eugene, OR). Similarly
labeled isotype control antibodies produced no specific staining (not shown).
Embryos and yolk sacs were mounted in 70% glycerol/PBS either intact or
embryonic dorsal side up following open dissection according to a previously
published method (Drake and Fleming,
2000).
Confocal microscopy and image processing
Embryos were analyzed using a Bio-Rad MRC 1024 laser scanning confocal
microscope (Bio-Rad Microscopy Division, Cambridge, MA) equipped with a
krypton-argon laser (488, 568, 647 nm) and attached to a Nikon Diaphot
inverted microscope (Fryer Co, Huntley, IL). 3D series (Z series) were
obtained by imaging serial confocal planes (25-50 planes at 1-2 µm
intervals) at 512x512 pixel resolution with a Nikon 20x
oil-immersion objective (2 µm intervals) or a Nikon 60x 1.2-NA
water-immersion objective (1 µm intervals). Z-stacks were converted into 2D
projections with MetaMorph (Universal Imaging Corp., Downingtown, PA).
Composite two-dimensional (2D) image reconstruction from adjacent confocal
fields was performed using Adobe Photoshop 5.0 (Adobe Systems Inc., San Jose,
CA). The red, green and blue colors displayed on micrographs indicate that
labeling was revealed, respectively, with Rhodamine Red-X (TER119), Alexa
Fluor 488 (Flk-1), and Alexa Fluor 647 (CD41) fluorochromes.
Fibrinogen staining protocol
Single cell suspensions of YS cells were stained with 1 µl CD61 PE and
sorted into CD61+ and CD61- populations using the
FACStar instrument. Recovered cells were pretreated with 5 µl of 0.1 M DTT
(Invitrogen, Grand Island, NY) for 5 minutes followed by addition of 0.5 µl
of fibrinogen conjugated to Oregon Green (stock solution of 1.5 mg/ml in 0.1 M
sodium bicarbonate, pH 8.3; Molecular Probes, Eugene, OR). The cells were
incubated for 30 minutes at 37°C. To the fibrinogen and cell suspension, 1
µl CD41 conjugated to Alexa 647 was added for 30 minutes at 37°C.
Following the incubation, cells were pelleted, washed and resuspended in IMDM
with 10% FBS, 1% P/S, 2 mM L-glutamine for cell analysis on the FACSVantage
instrument. In some experiments, 1-8 µl of a anti-mouse CD41 blocking mAb
(1B5F(ab)'2, 1.6 mg/ml) was added to one aliquot and 1-8
µl of a rat isotype control (99-C7-B3, 1.8 mg/ml) was added (both
antibodies were generously provided by Dr Barry Coller, Rockefeller
University, New York) to a second aliquot of cells for 30 minutes at 37°C
prior to addition of fibrinogen.
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RESULTS |
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Emergence of the Ery P lineage and the definitive
progenitors
Expression of the erythroid lineage marker TER119 in a subset of the
CD41dim cells began soon after the onset of blood island
vasculogenesis (Fig. 4A-C).
Between E8.0 and E8.25 there was a significant increase in the number of
TER119+ cells and the level of TER119 expression
(Fig. 4A,D). Cells with the
highest level of TER119 expression had diminished levels of CD41 expression
(below dim), thus a reciprocal relationship appeared to exist between the
level of TER119 expression and the level of CD41 expression in the
CD41dim population (Fig.
4E-I). When CD41dim and TER119 co-expressing cells
(Fig. 4I) were sorted, the
frequency of EryP in E8.5 yolk sac
CD41dimTER119- cells was 83/2000 cells plated compared
to 41/2000 cells plated for the more mature
CD41lo/-TER119+ cells (n=2). These observations
suggest that maturing EryP and emerging primitive erythroblasts
downregulate CD41 expression during differentiation
(Fig. 4E-I). However, the level
of TER119 expression in the primitive erythroid lineage never attained that
displayed by definitive erythroid cells (e.g. contaminating maternal
erythrocytes in Fig. 3J).
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CFC potential of CD41+ cells in the late yolk sac, PSp,
fetal liver and adult marrow
Yolk sac, P-Sp, fetal liver and adult marrow
Kit+CD34+ cells are enriched for hematopoietic
progenitor cell activity (Ito et al.,
2000). We examined Kit+CD34+ cells in the
E9.5 yolk sac, E12.5 fetal liver and adult bone marrow for expression of CD41
(Fig. 5). Nearly all
Kit+CD34+ cells in the E9.5 yolk sac express CD41
(Fig. 5A). CD41 expression was
present on a high percentage of Kit+CD34+ cells in the
E12.5 fetal liver (Fig. 5E) but
expression was restricted to a small percentage of adult bone marrow cells
(Fig. 5F). Since adult bone
marrow hematopoietic stem cells are enriched in cells that fail to express
CD34, we also examined Kit+CD34- cells for CD41
expression and less than 1% of these cells expressed CD41 (data not shown).
CD41 expression was also examined in E9.5 Kit+CD34+ P-Sp
cells and the majority of cells (68±12%) were CD41+.
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Since hematopoietic progenitor cells were present in both CD41+ and CD41- subpopulations of fetal liver and adult marrow cells, we wished to determine which subpopulation was enriched in long-term repopulating activity. E12.5 fetal liver and adult marrow cells were first depleted of B and T lymphocyte and granulocyte lineage antigen-positive cells (see Materials and Methods). Cells were then fractionated into 4 subpopulations including Kit+CD34+ CD41+, Kit+CD34+ CD41-, Kit+CD34- CD41+, and Kit+CD34- CD41- cells. Sorted cells were transplanted into lethally irradiated congenic recipient mice along with fresh low-density marrow competitor cells. Fetal liver Kit+CD34- CD41+ and Kit+CD34- CD41- cells failed to demonstrate any (>0.5% donor-derived CD45.1 cells) long-term repopulating activity in the blood of recipient animals at 4 or 6 months post-transplant. Fetal liver Kit+CD34+ CD41+ displayed minimal but persistent and detectable long term repopulating activity (1.0±0.3%, n=5 mice) while Kit+CD34+ CD41- cells demonstrated significantly greater long-term donor-derived chimerism (27.3±24.4%, n=5) in the peripheral blood of the recipient mice at 6 months post-transplant (Fig. 6A). Thus, even though equal numbers of donor cells (500) were originally injected, higher repopulation was observed with Kit+CD34+ CD41- fetal liver hematopoietic cells. All of the adult marrow sorted cell populations possessed some degree of long-term repopulating ability (Fig. 6B), though the highest levels of donor blood cell chimerism were present in recipient mice transplanted with 2000 Kit+CD34- CD41- cells (42.6±28.3%, n=5). Evidence that both E12.5 fetal liver Kit+CD34+ CD41- and adult bone marrow Kit+CD34- CD41- cells contributed for more than 6 months to multiple blood cell lineages including B lymphocytes, T lymphocytes and granulocytes is depicted in Fig. 7 and Table 3. Thus, CD41 is expressed on some long-term reconstituting hematopoietic stem cells, however, repopulating ability is enriched in Kit+CD34+ fetal liver and Kit+CD34- adult marrow cells that are CD41-.
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DISCUSSION |
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The transient nature, unique morphology and gene expression, and restricted
cell types of the primitive erythroid lineage suggest that it is distinct from
the definitive erythroid lineage. Support for the separation of primitive and
definitive erythropoiesis is also provided by gene-targeting experiments where
deletion of certain genes (e.g. Runx1, Gata2) disrupts definitive
erythropoiesis with little change in primitive erythropoiesis
(Shivdasani and Orkin, 1996).
The early appearing macrophage progenitors identified in the present studies
(E8.0) are absent in Runx1 null embryos and are therefore probably
related to the definitive lineage (Lacaud
et al., 2002
). Whether the early appearing megakaryocyte
progenitors identified in the present studies are more related to the
definitive or primitive lineage remains to be determined, but these
progenitors are reported to display features that distinguish them from later
appearing definitive megakaryocyte progenitors
(Xu et al., 2001
). Studies
performed with murine embryonic stem (ES) cells suggest that primitive and
definitive hematopoietic progenitor cells emerge from a common
Flk-1+ precursor cell called the hemangioblast
(Kennedy et al., 1997
).
Expression of Flk-1 is first evident in the murine embryo in proximal lateral
mesoderm during gastrulation and these mesoderm cells give rise to both the
hematopoietic and endothelial lineages in the yolk sac
(Kataoka et al., 1997
;
Nishikawa, 1997
). While Flk-1
has been used to isolate primitive and definitive progenitor cells from ES
cell-derived hematopoietic cultures, Flk-1 has not been used to purify
EryP or early appearing CFU-Mac and megakaryocytic progenitors from
the early yolk sac. We have identified CD41 expression in the yolk sac in
Flk-1+ cells and have demonstrated that the
CD41dim-expressing yolk sac cells at E7.0 constitute the entire
population of in vitro clonable EryP-CFC. Thus, CD41 expression
serves as a marker for both the onset of hematopoiesis in general (early
macrophage and megakaryocytic progenitors), and specifically for primitive
erythroid progenitor cell emergence.
CD41 expression at a high level in E8.25 yolk sac cells identifies
essentially all of the definitive hematopoietic progenitor cells. Flk-1 and
vascular endothelial cadherin have previously been utilized to isolate
primitive and definitive hematopoietic cells in vitro from ES cell-derived
hematopoietic cultures but this combination has not been examined as a tool
for isolating primitive and definitive progenitors in the early yolk sac
(Kabrun et al., 1997;
Nishikawa et al., 1998
). The
core-binding factor Runx1 is expressed in endoderm, mesoderm and primitive
erythroblasts in the yolk sac (North et
al., 1999
). It remains to be determined whether expression of this
transcription factor will identify EryP or CFU-Mac in the early
yolk sac. Thus, CD41 is a unique marker for both primitive and definitive
hematopoietic progenitor cells in the earliest phases of murine development.
These observations both support and extend the recent reports of CD41
expression on hematopoieitc progenitor cells from human and murine fetal and
newborn subjects and the report that CD41 serves as a marker for the onset of
definitive hematopoiesis (Corbel and
Salaun, 2002
; Debili et al.,
2001
; Mikkola et al.,
2002
; Mitjavila-Garcia et al.,
2002
).
While essentially all the hematopoietic progenitor cells in the yolk sac
expressed CD41, CD41 expression diminished in progenitors present in the P-Sp,
fetal liver and adult bone marrow compartments. We have summarized the
immunophenotype of these progenitors in
Fig. 9. E9.0 yolk sac
repopulating stem cells are enriched in cells expressing CD34 and Kit and
nearly all of these cells expressed CD41 in the present studies
(Yoder et al., 1997b). CD34
and Kit are expressed on hematopoietic stem and progenitor cells in the AGM,
fetal liver and adult marrow (Morel et
al., 1996
; Sanchez et al.,
1996
). Of interest, fetal liver stem cell activity is enriched in
cells expressing CD34 and Kit while adult marrow stem cell repopulating
activity is highest in cells expressing Kit but not CD34
(Ema and Nakauchi, 2000
;
Ito et al., 2000
;
Zeigler et al., 1994
). We
observed that transplantation of lethally irradiated adult mice with sorted
fetal liver and adult bone marrow cells resulted in very low but persistent
long-term chimerism of the peripheral blood of recipient mice with donor cells
expressing CD41. However, the highest levels of donor cell chimerism achieved
in the competitive repopulation experiments resulted from transplantation of
Kit+CD34+ fetal liver and
Kit+CD34- adult marrow cells that did not express
detectable cell surface levels of CD41. Further studies to determine whether
CD41 is expressed on stem cells derived from the AGM, the site of development
of the first stem cells that engraft in adult mice are warranted.
|
The role CD41 is playing in the primitive and definitive progenitor cells
is unknown. One obvious role the CD41/61 receptor complex may play is in the
interaction of the progenitor cells with extracellular matrix molecules in the
yolk sac. The specific adhesion interactions between the earliest
hematopoietic and endothelial cells that are required for proper formation of
yolk sac blood islands remain unknown. Disruption of several integrin and
extracellular matrix molecule genes results in embryonic lethality due to
aberrant blood island and yolk sac vascular development (with or without
defects in hematopoiesis) (Francis et al.,
2002). Thus, CD41 may play a role in hematopoietic-endothelial
interactions important for early blood island organogenesis. However, mice
deficient in CD41 display no obvious defect in yolk sac, fetal liver or adult
marrow hematopoiesis other than a platelet adhesion abnormality similar to
that seen in human subjects with Glanzmann's thrombasthenia
(Tronik-Le Roux et al., 2000
).
Thus, further studies will be required to define the role of CD41 in
hematopoietic stem and progenitor function during murine embryogenesis and
throughout ontogeny.
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