1 Department of Cell and Matrix Biology, MCRI, 3052 Parkville Victoria,
Australia
2 Department of Experimental Medicine I, University Erlangen-Nürnberg,
91054 Erlangen, Germany
3 Department of Ophthalmology, University Erlangen-Nürnberg, 91054
Erlangen, Germany
4 Max-Planck-Institute of Psychiatry, 80804 München, Germany
5 Department of Experimental Pathology, Lund University, 22363 Lund,
Sweden
6 Department of Genetics, University Erlangen-Nürnberg, 91054 Erlangen,
Germany
* Author for correspondence (e-mail: bent.brachvogel{at}mcri.edu.au)
Accepted 22 March 2005
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SUMMARY |
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Key words: Annexin A5, Perivascular cells, Pericytes, Adult stem cells
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Introduction |
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PVCs are essential for the development of functional vessel walls and
contribute to the structural integrity and contractility of vessels. However,
developmental origins of PVCs remain unclear. Different speculations were
proposed, such as derivation from mesenchymal and epicardial cells or neural
crest (Gerhardt and Betsholtz,
2003), but also transdifferentiation of endothelial cells is
discussed (DeRuiter et al.,
1997
). Recent data support the idea of a common progenitor of
perivascular and endothelial cells the FLK1-positive angioblast
(Ema et al., 2003
;
Yamashita et al., 2000
).
Pericytes were originally defined by their morphology and close contact to
endothelial cells located within a shared basement membrane
(Sims, 1986;
Tilton, 1991
). They represent
a heterogeneous population of cells involved in normal and pathological
angiogenesis, such as diabetic microangiopathy
(Cogan et al., 1961
;
Hirschi and D'Amore, 1996
),
atherosclerosis (Bostrom et al.,
1993
) or cancer (Wesseling et
al., 1995
). Recruitment and coverage of vessels with pericytes is
essential for the development of vasculature, as has been shown by genetic
ablation of PDGFB signaling. In mice with disrupted signaling, the expansion
and spreading of pericytes is impaired and leads to perinatal lethality due to
leakage and hemorrhage (Leveen et al.,
1994
; Lindahl et al.,
1997
; Soriano,
1994
). Presently, no `pan-specific' marker is available that
defines the pericyte phenotype unambiguously
(Gerhardt and Betsholtz,
2003
). Common markers such as smooth muscle actin
(Nehls and Drenckhahn, 1991
),
NG2-proteoglycan/HMW-MMA (Ozerdem et al.,
2001
), PDGFRß receptor
(Lindahl et al., 1997
) and the
regulator of G-protein-signaling RGS5
(Bondjers et al., 2003
) define
subsets of pericytes in a time- and tissue-dependent manner. Recently, the
promoter-trap transgene XlacZ4 revealed a specific coexpression in vSMCs and
pericytes (Tidhar et al.,
2001
).
Previous studies have shown that PVCs can differentiate into various cell
types such as adipocytes, chondrocytes, fibroblasts and macrophages
(Canfield et al., 2000;
Diaz-Flores et al., 1992
).
These cells may therefore reflect in some aspects the phenotype of mesenchymal
stem cells (MSC) originally isolated from bone marrow stroma
(Caplan, 1991
). Owing to
general difficulties with isolation and characterization of pericytes, it is
not yet possible to purify these cells from mouse. Therefore, detailed
analysis of pericytes and PVCs in the mouse system is still pending.
Annexin A5 represents a typical member of the annexin family, characterized
by the ability to bind to phospholipid membranes in the presence of
Ca2+ (Moss et al.,
1991). Although numerous functions of annexin A5 have been
described in vitro, its in vivo role still remains unclear
(Brachvogel et al., 2003
).
Expression analysis of the annexin A5 gene (Anxa5) by the use of an
Anxa5-lacZ fusion gene in vivo showed that this gene is
expressed in vasculature-associated cells and at later stages (E13.5-P1) in
mesenchymal condensations, chondrocytes as well as all skeletal elements
(Brachvogel et al., 2001
).
In this report, we used the Anxa5-lacZ promoter trap first to describe the onset of Anxa5-lacZ expression early in development and second to characterize the Anxa5-lacZ+ cells of different developmental stages in vitro and in vivo. Surprisingly, Anxa5-lacZ expression is initially detected in cells associated with the primary vascular plexus. Later, expression is restricted to cells associated with endothelial cells. By using fluorescence-activated cell sorting (FACS) we were able to purify and characterize Anxa5-lacZ+ PVCs for the first time from mouse. The purified cell populations revealed unique expression profiles of markers characteristic for pericytes and mesenchymal stem cells. Additionally, these isolated PVCs have a capacity for differentiation into mesenchymal cell lineages. In the future, this will enable us to define the developmental ontogeny and plasticity of the heterogeneous, poorly characterized perivascular cell population.
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Materials and methods |
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Immunohistochemistry
Embryos and tissues were processed for frozen sections and immunostaining
as described previously (Ringelmann et
al., 1999). Primary antibodies detecting smooth muscle actin,
desmin (Sigma), NG2-proteoglycan (Chemicon), PECAM/CD31, Sca-1, CD34
(Pharmingen), PPAR
2 (Affinity BioReagents), collagen I (Sigma),
collagen II (Lab Vision), collagen IX (generously provided by Vic Duance,
Cardiff) and collagen VI (kindly provided by R. Timpl, Martinsried) were used
with corresponding secondary antibodies conjugated with Cy2, Cy3 or Cy5
(Jackson). Nuclei were stained by DAPI according to suppliers protocol
(Sigma). Immunolabeled sections were stained for ß-galactosidase activity
at room temperature in X-gal solution (1 mg/ml) for 16-24 hours (Sigma)
according to Hogan et al. (Hogan et al.,
1994
). Sections were stained with oil Red O and Hematoxylin as
described earlier (Nuttall et al.,
1998
).
Detection of ß-galactosidase activity
Whole-mount staining of embryos was performed after fixation in 0.2%
glutaraldehyde for 10 minutes and stained in X-gal solution at room
temperature for 24 hours. Treated embryos were dehydrated by increasing
concentrations of ethanol and embedded in paraffin wax according to standard
procedures. Embryos were cut into 9 µm sections (Leica RM 205), dried for 1
hour at 60°C and de-waxed in xylene. Specimens were counterstained in
eosin for morphological assessment.
Purification of lacZ-expressing cells
Cells were isolated from heterozygous embryos of different stages
(E8.5-E16.5) or adult brain meninges (dura, arachnoid and pia mater). In
embryos, the heart was removed. Residual embryos and meninges were digested
with 0.4% collagenase II (Worthington) for 60 minutes at 37°C, followed by
2% trypsin (Sigma)/100 U DNAse I (Roche) for 30 minutes at 37°C.
Subsequently, cell suspensions were filtered through a 100 µm nylon mesh
(Becton Dickinson). Suspensions were stained for ß-galactosidase activity
as described (Miles et al.,
1997). Briefly, 1 x106 cells were suspended in 20
µl PBS and added to 20 µl of 2 mM
fluorescein-di-(ß-D-galactopyranoside) FDG (Sigma). Cells were incubated
at 37°C for 75 seconds and subsequently 500 µl of ice-cold PBS were
added. Cells were stored on ice for 3 hours. The suspension was stained with
propidium iodide (PI, 1 µg/ml) to label dead cells during flow cytometry.
lacZ+ cells were detected by the FITC channel, besides
FSC/SSC and PI. Cells were sorted by fluorescence-activated cell sorting
(MoFlow, Cytomation) and viable PI/lacZ+
cells were collected. Cells derived from wild-type embryos or adult brain
meninges were used as negative control.
In vitro culture of sorted cells
Purified lacZ+ cells from adult brains or embryos were
plated on gelatin-coated 24-well plates (5 x104 cells/well)
in proliferation medium [MCDB-131, 5% FCS, EGF (10 ng/ml), bFGF (2 ng/ml),
insulin (5 µg/ml), PDGF-BB (5 ng/ml) (Gibco)]. Medium was changed every
second day. Aggregate cultures of lacZ+ cells were
performed as described (Johnstone et al.,
1998). Briefly, 2 x105 of confluent cells were
centrifuged in a 15 ml polystyrene tube and incubated in chondrogenic [DMEM,
10% FCS, 100 nM dexamethason, insulin-transferrin-sodium selenite media
supplement, 5.35 µg/ml linoleic acid, 1.25 µg/ml bovine serum albumin
(Sigma), 100 ng/ml hBMP2 (Sigma)] or osteogenic medium [DMEM, 10% FCS, 100 nM
dexamethason, 10 mM glycerophosphate, 10 mM CaCl2, 250 µM
ascorbate-2-phosphate (Sigma)] at 37°C and 5% CO2. Cell
aggregates were cultured in tubes for 14 or 21 days and medium was changed at
3-day intervals.
Ultrastructural analysis
Tissue specimens were fixed in 2.5% glutaraldehyde in 0.1 M phosphate
buffer (pH 7.4) for 24 hours at 4°C, postfixed in 1% buffered osmium
tetroxide and processed for embedding in epoxy resin (EPON 812). Sections (2
µm) were analyzed by transmission electron microscopy (LEO 906E,
Oberkochen; Germany).
Regeneration of muscle after crush injury
Regeneration studies of tibialis anterior muscle (TA) were carried out as
described (Mitchell et al.,
1992). Briefly, mice (15-20 weeks old) were anesthetized and a
transversal crush injury was induced. Immediately after damaging the muscle,
25 µl of FDG-sorted PVCs from meninges of 2-month-old mice (5
x104 cells/25 µl) were injected into damaged region with a
25 µl syringe. The wound was closed with 6-0 suture-silk; the mice were
allowed to recover in a warmed environment and placed in standard cages. Mice
were sacrificed 13 days after injury and the treated muscle was processed for
frozen sections.
Regeneration of bone marrow by Anxa5-lacZ+ PVCs
The Anxa5-lacZ+ mutant was generated on a
C57BL/6 x129SvJ background, displaying the Ly5.2 marker on the surface
of bone marrow cells (BMCs). lacZ+ PVCs of adult meninges
were isolated from heterozygous animals (2-4 months of age). Supporting BMCs
were isolated from C57BL/6-Ly-5.1 mice. Supporting BMCs (9
x105 cells) were mixed with
Anxa5-lacZ-positive PVCs (1 x105 cells) and
injected (100 µl) into the tail vein of C57BL/6-Ly-5.1 recipient, that had
been given an irradiation dose of 900 Rad
(Kawada and Ogawa, 2001). As
control, BMCs from Anxa5-lacZ+-Ly-5.2 were mixed
with supporting BMCs from C57BL/6-Ly-5.1 and injected into irradiated
C57BL/6-Ly-5.1 recipients. Peripheral blood was isolated 4 weeks after
transplantation from the retro-orbital plexus of anaesthetized recipients and
C57BL/6-Ly-5.1 mice. Cells (106) were stained with PE-conjugated
Ly5.1 or FITC-conjugated Ly5.2 antibodies (Pharmingen) and incubated on ice
for 30 minutes. Cells were washed three times in PBS/5% FCS and stained for
dead cells with PI. Analysis was performed by flow cytometry detecting PE- and
FITC-specific fluorescence.
Expression analysis
Total RNA of embryos or sorted cells was isolated by Trizol purification
(Chomczynski and Sacchi, 1987)
and concentrated by ethanol precipitation. RNA was reverse transcribed (Roche)
and mRNAs of specific genes were detected by PCR using optimized primers: Flk1
(270 bp; X70842, positions 315-584), Kit (404 bp; AY536431, positions
1292-1695), Sca1 (409 bp; NM_010738, positions 35-443), CD34 (303 bp;
BC066820, positions 122-424), VE-cadherin (230 bp; NM_009868, positions
52-281), Pax7 (492 bp; NM_011039, positions 1043-1534), Myod1 (130 bp;
NM_010866, positions 762-891), PDGFRß (451 bp; BC055311, positions
3290-3740), NG2 (236 bp, AF352400, positions 4290-4525), Anxa5 (300 bp;
MUSANXV, positions 209-508) and Gapdh (443 bp; XM_284895, positions
532-974).
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Results |
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Anxa5 expression represents a characteristic marker for perivascular cells
Owing to the variable expression of markers in PVCs, their specificity
varies depending on the tissue- and development-related context
(Gerhardt and Betsholtz,
2003). Therefore, staining for SMA does not represent an
unequivocal marker for PVCs and their precursors. By contrast, electron
microscopy allows the identification of cells by their localization within
vessel structure. Specific staining for ß-gal activity in
Anxa5-lacZ-expressing cells results in the formation of an
unusual cytoplasmatic deposit of the reaction product in expressing cells,
represented by an intracytoplasmatic vesicle of unknown origin (data not
shown). Hence, positive cells can be identified in electron microcopy by the
presence of these electron-dense vesicles
(Fig. 3B, arrow). In
heterozygous lacZ-expressing embryos of stage E10.5 these deposits
were exclusively detected in PVCs closely associated with capillaries, but not
in endothelial cells. The characteristic common basement membrane of
endothelial cells and pericytes of functional blood vessels is not yet visible
at these stages and therefore cannot be used for identification. These data
support the idea that Anxa5-lacZ represents a novel,
specific marker for PVCs.
|
|
To test the limits of the method, we also isolated FDG+ cells
from E10.5 and E8.5 embryos. Embryos at E10.5 were similarly processed and
Anxa5-lacZ+ cells (2.5%) could be purified from
individual embryos (Fig. 4E).
By contrast, pools of heterozygous E8.5 embryos had to be used because of the
lower total cell numbers (Fig.
4D). Out of five heterozygous embryos (E8.5), only 15,000 cells
were isolated with a small subset (300 cells) representing
Anxa5-lacZ+ cells. Typically, isolations of cells
from different embryonic stages (E8.5-E12.5) resulted in a relative yield of
2-4% viable Anxa5-lacZ+ cells.
This experimental procedure could also be used for isolation of Anxa5-lacZ+ cells from adult mice (Fig. 4F). Vasculature of the meninges is easily accessible and represents an appropriate source of vascular-associated cells because of the low complexity of this tissue. Therefore, single cell suspensions of isolated meninges of the dura, arachnoid and pia mater were pooled from several Anxa5-lacZ+ mice, stained with FDG and sorted for ß-gal activity. About 1% of cells were positive and could be sorted from the pool of vital cells. Typically, about 7000 Anxa5-lacZ+ cells can be isolated from the meninges of an individual mouse. Depending on the gating stringency, an enrichment of up to 99% lacZ-expressing cells was achieved after sorting.
The purification of Anxa5-lacZ+ cells allowed
the analysis of the expression profile of these murine PVCs by qualitative
RT-PCR (Fig. 5). Sorted cells
from whole embryos at E10.5, isolated brains of E16.5 embryos and brain
meninges of adult animals were used for RNA isolation. Total RNA from E10.5
embryos represented the positive control. These RNAs were tested by RT-PCR for
the expression of NG2 and PDGFRß, which represent characteristic markers
for pericytes (Gerhardt and Betsholtz,
2003). Purified PVCs were clearly positive for these markers. By
contrast, markers for muscle satellite stem cells (Pax7) or differentiating
muscle cells (Myod1) were not expressed. Therefore, purified
Anxa5-lacZ+ cells display typical markers of
vascular pericytes.
Anxa5-lacZ+ cells may represent a stem cell-like population
Surprisingly, sorted cells from E10.5, E16.5 and adult meninges also
expressed the stem cell markers Flk1, Kit, Sca1, CD34 and low amounts of
VE-cadherin (Fig. 5A-C). None
of the sorted cells produced CD45 mRNA, a marker common to hematopoietic cells
(Asakura et al., 2002). The
expression profiles of sorted lacZ+ cells from various
developmental stages and tissues were highly homologous
(Fig. 5). A reproducible,
quantitative difference was seen for the relative expression of Sca1 and Kit,
with Kit being strongly expressed at E10.5 and E16.5, whereas only low levels
were seen in adult PVCs. By contrast, Sca1 was highly expressed in adult PVCs
but was detected at low amounts in purified cells from embryos (E10.5, E16.5).
These data indicate that the Anxa5-lacZ marker defines a
homogenous pool of cells from embryonic and adult vasculature, which resembles
characteristics of pericytes as well as stem cell populations
(Minasi et al., 2002
).
Expression profiles indicated that isolated
Anxa5-lacZ+ PVCs may represent a pool of cells
with stem cell-like character. For testing their potential differentiation
capabilities isolated cells were cultured in vitro. Single PVCs displayed a
stellate morphology, as it was described for pericytes
(Canfield et al., 2000).
Subsequently, PVCs started to align and after reaching confluence, the
formation of multilayered areas is observed (data not shown).
|
Mesenchymal stem cells are capable of differentiating into adipocytes; this
is the least understood pathway of mesenchymal stem cells differentiation
(Caplan and Bruder, 2001).
Confluent layers of isolated PVCs have therefore been cultivated in medium
that induces adipogenesis in mesenchymal stem cells or adult
trabecular-derived bone cells (Nuttall et
al., 1998
), but as yet, no increased expression of specific
adipogenic markers, such as lipoprotein lipase (LPL) and
proliferator-activated receptor
2 (PPAR
2), has been detected by
RT-PCR. Additionally, no excessive deposition of lipid droplets can be seen
using Oil red O staining (Nuttall et al.,
1998
) (data not shown). Surprisingly, a differentiation into
adipocyte-like cells is detectable in aggregates cultured in chondrogenic
medium. A strong staining with Oil Red O, which is indicative of synthesis of
neutral lipids, is detectable on sections of these aggregate cultures
(Fig. 6M). This differentiation
event was never observed in cells cultured in confluent monolayer cultures.
Additionally, these cells express the adipocyte-specific protein PPAR
2,
as shown by immunostaining (Fig.
6N,O) (Chawla et al.,
1994
). Therefore, we conclude that the cultured
Anxa5-lacZ+ population retain the potential to
differentiate into early chondrogenic, osteogenic and adipogenic cells in
three-dimensional aggregate cultures. A similar differentiation capacity has
been seen previously with isolated bovine pericytes
(Canfield et al., 2000
).
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Anxa5-lacZ+ perivascular cells do not exhibit hematopoietic potential
Adult stem cells have been characterized in many tissues and some
populations have revealed the potential for hematopoietic differentiation
(De Angelis et al., 1999;
Jackson et al., 2002
).
Consequently, we addressed the issue of whether some of these
Anxa5-lacZ+ cells exhibit a capacity for
reconstitution of the hematopoietic system, as has been shown for murine
muscle fractions (Howell et al.,
2002
). Therefore, we injected
Anxa5-lacZ+ PVCs from adult meninges into
lethally irradiated C57BL/6 mice, which expressed the polymorphic allele Ly5.1
on all nucleated hematopoietic cells (Shen
et al., 1986
). Isolated PVCs (1 x105) from 14
adult brain meninges of heterozygous, Ly5.2 expressing mice were mixed with
bone marrow cells (9 x105 cells) from C57BL/6-Ly5.1 mice to
support the regeneration of the bone marrow. This mixture was injected into
the tail vein of lethally irradiated Ly5.1 recipient mice. As a positive
control, 1 x105 bone marrow cells from Ly5.2-positive mice
were injected in a mixture with 9 x105 bone marrow cells from
Ly5.1 mice. Four weeks after injection, Ly5.1 and Ly5.2 expression was
analyzed in the peripheral blood of bone marrow reconstituted mice
(Fig. 8). As expected, no
Ly5.2+ cells were detectable in untreated C57BL/6-Ly5.1 mice. In
control experiments about 18% of blood cells were detected to be Ly5.2
positive (Fig. 8B). No
Ly5.2+ cells could be detected in the peripheral blood of
recipients that received
Anxa5-lacZ+/Ly5.2+ PVCs
(Fig. 8C). Therefore, purified
Anxa5-lacZ+ PVCs do not exhibit the capacity to
reconstitute cells of the hematopoietic system in vivo.
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Discussion |
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It has been suggested that pericytes and vSMC are phenotypic variants of a
continuous population of PVCs and have the potential to give rise to each
other, but may differentiate to chondrocytes, osteoblasts and adipocytes
(Canfield et al., 2000;
Nehls and Drenckhahn, 1993
).
The lack of adequate markers during early development of vasculature causes a
major problem for analyzing the role of PVCs in this process. During growth,
remodeling and differentiation of the vasculature, expression of specific
markers like SMA and NG2-proteoglycan become indicative for PVCs only at later
stages (Ozerdem et al., 2001
).
Three lines of evidence now clearly indicate that Anxa5-lacZ
expression represents a novel and highly specific marker for these PVCs during
development and in the adult. First, Anxa5-lacZ+
cells are exclusively detected in close proximity to PECAM-positive
endothelial cells and show colocalization with the markers SMA or NG2 in many
cells. Second, electron microscopy detects deposits of the
lacZ-product in cells directly contacting the endothelium. This can
be seen even in early embryos, where the characteristic common basement
membrane is not yet developed, as well as in pericytes of adult blood vessels
defined by their typical basement membrane
(Fig. 3). The third and most
important argument is seen with the expression analysis of isolated
Anxa5-lacZ+ cells. As previously shown, the
presence of NG2, SMA and PDFGRß is highly indicative for pericytes or
pericyte-related cell populations
(Gerhardt and Betsholtz,
2003
). All these markers are also significantly expressed in
purified Anxa5-lacZ+ cells from various sources.
The expression profile of these markers was identical among populations
isolated from E10.5, E16.5 and even adult meninges, which defines a common
conserved expression pattern at different stages of development. These data
clearly show that Anxa5-lacZ+ expression
correlate with typical markers of PVCs, even at very early stages of
development.
|
During vasculogenesis, the expression of the Anxa5-lacZ
reporter gene was detected in cells forming the primary capillary plexus in
the yolk sac of E7.5 to E8.5 embryos. This staining resembles the angioblast
appearing at onset of vasculogenesis. This expression is reflected by Flk1
expression in the purified Anxa5-lacZ+ cells (not shown),
representing a characteristic marker of angioblasts of the yolk sac mesoderm
(Shalaby et al., 1995). Later
during angiogenic remodeling, expression of the Anxa5-lacZ
marker defines the population of PVCs. Many studies have indicated that PVCs
are recruited from stromal cells by mutual contacts with endothelial cells
(reviewed by Gerhardt and Betsholtz,
2003
). Alternatively, trans-differentiation from endothelial cells
was discussed to contribute to the PVC cell pool. Flk1+ cells were
also identified in ES-cell cultures, which can serve as a progenitor for
endothelial cells and SMCs in vitro and in vivo
(Ema et al., 2003
;
Yamashita et al., 2000
).
Therefore, we assume that Anxa5 expression may represent a novel marker for a
subset of the mesenchymal stem cell compartment
(Dennis and Charbord, 2002
)
that differentiates into angioblast and later strictly correlates with the
transition to PVCs. Although `it takes two to make blood vessels
endothelial cells and pericytes' (Gerhardt
and Betsholtz, 2003
), the understanding of PVCs is rather limited
in comparison to endothelial cells. Therefore, the purification of
Anxa5-lacZ+ PVCs represents a novel and unique
tool with which to characterize this cell type by in vitro and in vivo studies
in the mouse system.
The isolated PVCs clearly reflect a pericyte-like phenotype that is
indicated by their morphology, by the detection of NG2 and SMA protein in
cultured cells, and by their differentiation into adipogenic, early
chondrogenic and osteogenic cells. Earlier studies have indicated that bovine
derived pericytes, mesenchymal stem cells and adult trabecular-derived bone
cells are able to differentiate into chondrogenic cells and adipocytes
(Canfield et al., 2000;
Nuttall et al., 1998
).
Interestingly, isolated cell populations were found to express different stem
cell markers, such as Flk1, Kit, Sca1 and CD34 (see
Fig. 5). This pattern reflects
the phenotype of aorta-derived multipotent progenitors
(Minasi et al., 2002
), as both
pools express the `hemangioblast' markers CD34, Kit and Flk1. Additionally,
the stem cell marker Sca1 is expressed. It has been discussed that
Sca1+-expression in muscle defines a vascular-associated pool of
adult stem cells in skeletal muscle with a high degree of phenotypic
plasticity (Asakura et al.,
2002
; Cao et al.,
2003
; De Angelis et al.,
1999
; Tamaki et al.,
2002
). Surprisingly, this marker was also detected in
Anxa5-lacZ+ PVCs from early embryos and was even
upregulated in adult meninges. Moreover, Flk1 and VE-cadherin are expressed in
purified PVCs, two markers also detected in embryoid body-derived precursor
blast colonies of the hematopoietic as well as the endothelial cell lineage
(Kennedy et al., 1997
).
Interestingly, the progeny of cultured ES-cells with hemangioblast potential
also express Flk1 and VE-cadherin, but not CD45
(Nishikawa et al., 1998
).
In vivo regeneration experiments of muscle clearly show that isolated PVCs
are specifically incorporated into columnar structures associated with blood
vessels located between muscle fibers, but not integrated into newly formed
muscle fibers. This correlates with the finding that
Anxa5-lacZ+ cells do not express the
characteristic marker Pax7 for satellite cells, the major myogenic stem cells
(Seale et al., 2000) cannot be
detected. Therefore, Anxa5-lacZ+ PVCs may not
retain myogenic differentiation capacity in vivo. Nevertheless, we cannot
exclude the possibility that Anxa5-lacZ reporter expression
is inactivated in differentiated muscle fibers, as we never detected
expression of the reporter gene in skeletal muscle cells.
Multiple stem cell populations have been defined in skeletal muscle. The
side population (SP) represents a well characterized cell pool
(Goodell et al., 1997), which
is found associated to the vasculature and displays hematopoietic potential in
vitro (Asakura et al., 2002
).
Muscle adult stem cells also exhibit hematopoietic capacity in vivo
(Gussoni et al., 1999
;
Howell et al., 2002
;
Jackson et al., 1999
). The
markers CD34 and Sca1 are characteristically expressed by the SP cells
(Goodell et al., 1997
),
although the correlation of CD34 expression and stem cell potential still
remains unclear (Parmar et al.,
2003
). Sca1 expression is used as a marker for potential
hematopoietic cells between muscle fibers
(Asakura et al., 2002
).
Although these markers are expressed in
Anxa5-lacZ+ PVCs and injected PVCs were found in
Sca-1+/CD34+ areas in regenerating muscle, we could not
define a capacity for hematopoietic differentiation of
Anxa5-lacZ+ PVCs isolated from adult meninges by
bone marrow reconstitution experiments.
Interestingly, the Anxa5-lacZ reporter is highly active
in mesenchymal condensations at E13.5, preceding formation of cartilage and
bone during development, but also later in all differentiated skeletal
elements (Brachvogel et al.,
2001). Additionally, isolated PVCs had the potential to develop
into early chondrogenic, osteogenic and adipogenic lineages. Recently, it was
shown that the embryonic dorsal aorta of E10.5 embryos contains progenitor
cells termed meso-angioblast, which are able to participate in the development
of perichondrium and cartilage, connective tissue, smooth muscle and cardiac
muscle (Minasi et al., 2002
).
It became obvious that a mesenchymal stem cell compartment may exist that is
represented by a spectrum of related cells with capacity for phenotypic
differentiation into stroma cells, adipocytes, chondrocytes or bone cells
(Caplan, 1991
;
Dennis and Charbord, 2002
).
This model differs from the standard paradigm that postulates hierarchical
lineages during development, as in the hematopoietic system, by a high degree
of plasticity during lineage progression and transition
(Theise et al., 2003
). We
propose that Anxa5-lacZ expression defines a subset of the
mesenchymal stem cell compartment and may reflect some aspects of various
differentiation pathways during development.
In this paper, we describe for the first time the use of Anxa5 expression for the isolation of a novel cell population resembling perivascular cells (pericytes and vSMCs), from mouse tissues, characterized by the expression of pericyte-specific markers. In future, this unique method will help to define the capacities of the poorly characterized PVCs. Our data show that these cells additionally display markers of mesenchymal stem cells and have the capacity to differentiate into adipocytes and osteoblastic cells. Therefore, expression of Anxa5 defines a novel subset of the mesenchymal stem cell compartment that reflects variable lineage progression and phenotypic plasticity.
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ACKNOWLEDGMENTS |
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