First published online 27 April 2005
doi: 10.1242/dev.01846
Development 132, 2657-2668 (2005)
Published by The Company of Biologists 2005
Perivascular cells expressing annexin A5 define a novel mesenchymal stem cell-like population with the capacity to differentiate into multiple mesenchymal lineages
Bent Brachvogel1,*,
Helga Moch2,
Friederike Pausch2,
Ursula Schlötzer-Schrehardt3,
Clementine Hofmann4,
Rupert Hallmann5,
Klaus von der Mark2,
Thomas Winkler6 and
Ernst Pöschl2
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
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Fig. 1. Expression of Anxa5 in early embryonic development is restricted to the
primary capillary plexus, dorsal aorta and extra-embryonic tissues.
Whole-mount embryos heterozygous for Anxa5-lacZ fusion gene
were stained for ß-galactosidase activity at E7.0 (A), E7.5 (B), E8.5
(C,D) and E9.0 (F). Sections of E8.5 (E) and E9.0 (G-I) embryos were
counterstained with Eosin after X-gal staining. Strong expression is detected
in the ectoplacental cone (A,B, arrowheads). Expression of
Anxa5-lacZ reporter in lining angioblasts of the visceral
yolk sac mesoderm (D; E, arrows), dorsal aorta (B,C,H arrows) and in
intersegmental vessels (I, arrows) is marked. Scale bar: 50 µm in E,H,I;
200 µm in A-D,F,G.
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Fig. 2. Anxa5-lacZ reporter is specifically expressed in the
vasculature of E10.5 embryos. Sagittal sections (A-D,F-I) of heterozygous
embryos were stained for ß-galactosidase activity (A,F) followed by
immunodetection of SMA (B,G), PECAM (C,H) and nuclear staining by DAPI (D,I).
In the overlays (E,J), X-gal deposits are indicated by white spots.
Anxa5-lacZ+ cells are restricted to
SMA+/PECAM+ dorsal aorta and PECAM+
capillaries (E). At higher magnification, only a subset of PECAM-associated
cells was positive for lacZ, as shown by DAPI staining (J, arrows).
(K-N) Anxa5-lacZ+ cells are located in regions
positive for the pericyte-specific marker NG2-proteoglycan in embryonic brain
at E13.5. Transverse sections of capillaries were stained for
ß-galactosidase activity (K, arrows). Positive areas appear black because
of the monochrome character of the camera system. In parallel, PECAM (L) and
NG2-proteoglycan (M) were detected by immunostaining. After merging,
colocalization of PECAM and NG2 appears orange (N). X-gal deposits are shown
as white spots (N, arrows). Scale bar: 50 µm.
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Fig. 3. Anxa5-lacZ+ cells are found associated with,
but are not identical to, the endothelium in capillaries of E10.5 embryos and
adult vasculature. (A) Sagittal sections of heterozygous embryos at E10.5 were
stained for lacZ activity and immunostained for PECAM. After merging,
Anxa5-lacZ+ regions appear red (arrows).
Anxa5-lacZ+ cells are located parallel to the
lining endothelial cells. (B) Ultrastuctural localization of
Anxa5-lacZ+ expression in heterozygous embryos at
E10.5 by electron microscopy. An electrondense deposit of X-gal product is
detected (arrow) exclusively in PVCs. (C,D) Sections from adult kidney tested
for lacZ activity (C) and SMA expression (D) showed colocalization in
the vascular walls of large blood vessels. The relative intensity of
fluorescence was reduced by X-gal staining. (E) Expression of the
Anxa5-lacZ reporter in brain of adult mice is found in the
pial vasculature. (F) X-gal staining (arrow) is restricted to cytoplasmic
vesicles in pericytes, characterized by the common basement membrane
(arrowhead) with endothelial cells. Capillaries (C), endothelial cells (EC)
and pericytes (P) are marked (B,F). Scale bars: 50 µm (A,C); 1 mm (E).
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Fig. 4. Detection and purification of lacZ+ cells by
fluorescence-activated cell sorting. Single cell suspensions of heterozygous
embryos at E12.5 were stained with the fluorescent lacZ substrate FDG
and cell death was detected by adding propidium iodide (PI). In FACS analysis,
only vital cells in gate R1 (FSC/SSC) were considered for data analysis (A).
Cells from wild-type embryos were used as control and almost no events were
detected in gate R2 (B). About 3% of the heterozygous cells analyzed,
represent vital Anxa5-lacZ+ cells (C). Similar
amounts of Anxa5-lacZ+ cells were detected in
embryos at E8.5 (D) and E10.5 (E) as well as in adult brain meninges (F). The
percentages of cells within the gate are depicted, genotypes (wild type, wt;
heterozygous, +/–) are indicated.
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Fig. 5. Expression profiles of isolated Anxa5-lacZ+
cells. Cells were sorted from heterozygous embryos at E10.5 (A), dissected
brains from E16.5 embryos (B), adult brain meninges (C) and RNA was isolated.
RNA isolated from total embryos at E10.5 represented the positive control (D).
RT-PCR was used to detect the mRNA of specific markers for stem cells (Flk1,
Kit, Sca1, CD45, CD34, VE-cadherin), myogenic cells (Myod1) as well as
myogenic satellite cells (Pax7) and pericytes (PDGFRß, NG2).
Anxa5 was tested as a positive control and reactions without mRNA (E)
were used as negative controls. GAPDH was used for standardization.
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Fig. 6. Anxa5-lacZ+ cells differentiate into early
chondrogenic, osteogenic and adipocyte lineages in vitro. Aggregate cultures
of PVCs were cultivated in chondrogenic medium (A-F,M-O). By day 14
(B,C,E,F,M-O) or 21 (A,D), cryosections were performed and strong staining was
seen for collagen VI (Col6; C) and collagen IX (Col9; B), whereas collagen II
was barely detectable (Col2; A). Aggregate sections were stained for neutral
fat deposits using oil Red O staining (red droplets) and Hematoxylin (M) as
well as for PPAR 2 (N). Additionally, isolated PVCs were cultivated in
osteogenic medium for 21 days (G-L). Ca2+ deposits in cryosections
of these aggregates were detected by Alizarin Red (H) as well as van Kossa
staining (I). Collagen I was detected by immunostaining (G). Aggregates of
PVCs were cultivated in proliferating medium as negative control for the
staining of Ca2+ deposits (K,L). Secondary antibodies were used as
negative control for the immunostaining (D-F,J,O). Scale bars: 50 µm.
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Fig. 7. Purified PVCs participate in muscle regeneration in vivo. Isolated
Anxa5-lacZ+ PVCs of adult brain meninges were injected
into regenerating areas of crushed tibialis anterior (TA) muscle of
immunodeficient NOD/SCID mice. By day 13, consecutive muscle sections were
analyzed for ß-galactosidase activity by X-gal staining and expression of
desmin (B,E), PECAM (C) as well as CD34 (F) by immunohistochemistry. Injected
Anxa5-lacZ+ cells were detected at day 13 in the
zone of regeneration (arrows, A,D) in columnar structures associated with
intact desmin-positive muscle fibers (B,E). No staining was seen in intact
fibers. lacZ-expressing cells were found in areas positive for
CD34+ (D,F) but not in areas positive for PECAM (A,C). Scale bars:
50 µm.
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Fig. 8. Purified Anxa5-lacZ+ PVCs do not have
hematopoietic capacity in vivo. Purified
Anxa5-lacZ+ PVCs from adult brain meninges
(Ly5.2+, 1 x105 cells) were mixed with bone marrow
cells (Ly5.1+, 9 x105) and injected intravenously
into recipient mice (Ly5.1+) after lethal irradiation. After 28
days, peripheral blood of recipients was analyzed by flow cytometry for the
presence of Ly5-markers. (A) No Ly5.2+ cells were detectable in
blood of the negative control mice, represented by non irradiated wild-type
Ly5.1+ mice without transplantation. (B) In the positive control,
18% of the blood cells derived from the injected Ly5.2+ BMCs. (C)
No Ly5.2+ cells could be detected after administration of
Anxa5-lacZ+/Ly5.2+ donor PVCs.
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© The Company of Biologists Ltd 2005
