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Functional mesenchymal stem cell niches in adult mouse knee joint synovium in vivo.

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ARTHRITIS & RHEUMATISM
Vol. 63, No. 5, May 2011, pp 1289–1300
DOI 10.1002/art.30234
© 2011, American College of Rheumatology
Functional Mesenchymal Stem Cell Niches in
Adult Mouse Knee Joint Synovium In Vivo
Tobias B. Kurth,1 Francesco Dell’Accio,2 Vicki Crouch,1 Andrea Augello,1
Paul T. Sharpe,3 and Cosimo De Bari1
heterogeneous and located in topographically distinct
niches in the lining layer and the subsynovial tissue.
Twelve days after injury, double nucleoside–labeled
cells within synovium were embedded in cartilagespecific metachromatic extracellular matrix and
costained positive for the chondrocyte-lineage markers
Sox9 and type II collagen.
Conclusion. Our findings provide the first evidence of the existence of resident MSCs in the knee joint
synovium that undergo proliferation and chondrogenic
differentiation following injury in vivo.
Objective. We previously reported that human
synovium contains cells that, after culture expansion,
display properties of mesenchymal stem cells (MSCs).
The objective of this study was to identify MSCs in
native synovium in vivo.
Methods. To identify stem cells in the synovium in
vivo, a double nucleoside analog cell-labeling scheme
was used in a mouse model of joint-surface injury. For
labeling of slow-cycling cells, mice received iododeoxyuridine (IdU) for 30 days, followed by a 40-day washout
period. For labeling of cells that proliferate after injury,
mice underwent knee surgery to produce an articular
cartilage defect and received chlorodeoxyuridine (CIdU)
for 4 days, starting at multiple time points after surgery.
Unoperated and sham-operated joints served as controls. Knee joint paraffin sections were analyzed by
double and triple immunostaining to detect nucleoside
analogs, conventional MSC markers, and chondrocytelineage markers.
Results. Long-term–retaining, slow-cycling IdUpositive cells were detected in the synovium. At 4 days
and 8 days after injury, there was marked proliferation
of IdU-positive cells, which costained for CIdU. IdUpositive cells were nonhematopoietic, nonendothelial
stromal cells, were distinct from pericytes, and stained
positive for MSC markers. MSCs were phenotypically
The synovial membrane is a tissue that lines the
joint cavity of synovial joints and consists of a lining layer
of macrophage-like (type A) and fibroblast-like (type B)
synoviocytes and a loose sublining tissue. In the healthy
joint, type A synoviocytes act in innate immunologic
defense and support adaptive immunity, while type B
synoviocytes function to regulate the release of nutrients
and molecules, including hyaluronan, into the synovial
fluid (1).
In response to injury of various types, including
trauma, the synovial membrane rapidly becomes hyperplastic (2,3). It is commonly believed that synovial
hyperplasia is sustained mainly by stromal cells including
type B (fibroblast-like) synoviocytes, also called synovial
fibroblasts, with infiltration of blood-borne inflammatory and immune cells particularly in inflammatory joint
diseases such as rheumatoid arthritis (4). The biologic
function of synovial stromal cell proliferation is likely to
depend on the nature of the injury, but it is believed to
have a pivotal role in joint homeostasis and disease.
Deregulation of this process is thought to contribute to
the formation of pannus, which in rheumatoid arthritis
causes destruction of cartilage and bone (4). Despite the
high frequency and remarkable biologic and clinical
relevance of synovial hyperplasia, very little is known
Supported by MRC grant G108/620. Dr. De Bari is a Fellow
of the Medical Research Council, UK.
1
Tobias B. Kurth, PhD, Vicki Crouch, BSc, Andrea Augello,
PhD, Cosimo De Bari, MD, PhD: University of Aberdeen, Aberdeen,
UK; 2Francesco Dell’Accio, MD, PhD: Queen Mary University of
London, London, UK; 3Paul T. Sharpe, PhD: King’s College London
and Biomedical Research Centre, Guy’s and St. Thomas National
Health Service Foundation Trust, London, UK.
Drs. Kurth and Dell’Accio contributed equally to this work.
Address correspondence to Cosimo De Bari, MD, PhD,
Institute of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, UK. E-mail: c.debari@abdn.ac.uk.
Submitted for publication July 28, 2010; accepted in revised
form December 30, 2010.
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KURTH ET AL
about the identity of the synovial cells that proliferate
following injury.
We provided the first evidence that the synovial
membrane in adult humans contains cells that, after release and culture expansion, display features of multipotent mesenchymal/stromal stem cells (MSCs) (5–7).
MSCs are defined as fibroblast-like cells that undergo
sustained in vitro growth and have the capacity to form
mesenchymal tissues such as cartilage and bone (8).
After culture expansion, synovial membrane MSCs express the conventional markers of cultured MSCs including CD44, CD73, CD90, and CD105 but do not
express hematopoietic or endothelial markers (9,10).
Despite extensive in vitro characterization, equivalent
cells within their native synovial tissue in vivo have not
been identified because of a lack of specific markers.
In adults, as reported for tissues and organs such
as the hair follicle and the intestinal mucosa (11,12),
stem cells are quiescent slow-cycling cells that, through
asymmetric cell division, generate transit-amplifying
cells that, after a proliferative burst, differentiate to
replace mature cells that are lost due to physiologic
turnover. Following a stress stimulus such as an injury,
this system rapidly intensifies, and the slow-cycling cells
undergo intense proliferation. In this study, we took
advantage of these functional features of adult stem cells
and adapted an in vivo scheme of double nucleoside
analog cell-labeling (13) to positively detect stem cells
within the synovium of knee joints, using a validated
mouse model of traumatic joint-surface injury (14).
Here, we provide the first evidence of the existence in
vivo, within postnatal knee joint synovium, of nonhematopoietic, nonendothelial stromal stem cells with a phenotype compatible with MSCs, distinct from pericytes,
which proliferated following articular cartilage injury
and, under specific experimental conditions, differentiated into chondrocytes in areas of cartilage metaplasia
within the synovium.
MATERIALS AND METHODS
Animals and nucleoside analog administration. All
animal experiments were approved by the UK Home Office.
Three-week-old male C57BL/6 mice received the artificial
nucleoside iododeoxyuridine (IdU; Sigma) with the drinking
water for 30 days, at a concentration of 1 mg/ml. IdU administration was then stopped for the next 40 days. Thereafter,
mice underwent knee surgery to produce an articular cartilage
injury in the patellar groove of the left femur, while the right
leg was sham operated as detailed below. Mice received
the artificial nucleoside chlorodeoxyuridine (CIdU; Sigma)
either directly after surgery or on day 4 or day 8 after
surgery, via subcutaneous injection of 200 ␮l at a concentration
of 10 mg/ml in sterile phosphate buffered saline (PBS) followed by administration of CIdU at a concentration of 1 mg/ml
in the drinking water for 4 days. At all time points, control
mice received identical treatment with IdU and CIdU but
underwent no surgical procedure. In another experiment,
transgenic mice expressing the LacZ gene in pericytes and
smooth muscle cells (15) received IdU as described above,
without undergoing surgery. These mice were killed at time
points similar to those at which treated mice were killed, but
they did not receive CIdU.
Surgery. Mice were subjected to surgery to produce a
joint-surface injury, as previously described (14). Briefly, mice
were anesthetized, and a medial parapatellar arthrotomy was
performed under a dissection microscope. The joint was
extended, and the patella was dislocated laterally. The joint
was then fully flexed, and a longitudinal full-thickness injury
was made in the patellar groove, using a custom-made device.
The patellar dislocation was then reduced. The joint capsule
and the skin were sutured in separate layers. The contralateral
knee was subjected to arthrotomy and patellar dislocation
without cartilage injury (sham-operated control).
Histologic assessment. The mice were killed at the end
of CIdU administration. The knee joints were dissected and
fixed in 2% paraformaldehyde and 0.05% glutaraldehyde in
PBS for 1 hour at room temperature. After decalcification for
2 weeks in 4% EDTA in PBS, knee joints were dehydrated and
embedded in paraffin. Five-micrometer–thick sections were
placed on Superfrost Plus slides (Menzer-Gläser). Sections
were rehydrated and stained with hematoxylin and eosin or 1%
toluidine blue, according to routine protocols.
Immunohistochemical analysis. Paraffin sections were
immunostained using protocols that were optimized for each
antigen. After dewaxing and rehydration were performed,
antigen retrieval was either enzyme based, using porcine
pepsin (Sigma) in 0.2N HCl at concentrations from 0.5 mg
to 3 mg for 45 minutes at 37°C, or was performed by heating
sections in an EDTA-based buffer solution (pH 9) for
30 minutes at 100°C in an autostainer (Leica).
For immunohistochemical analysis, endogenous peroxidase was quenched for 10 minutes with 3% H2O2 in water,
followed by blocking of endogenous avidin and biotin, using
the Avidin/Biotin Blocking Kit (Vector) according to the
manufacturer’s instructions. Nonspecific binding was blocked
with the immunoglobulin-blocking reagent and protein concentrate contained in the Mouse on Mouse (M.O.M.) Basic Kit
(Vector). Primary antibodies for IdU or CIdU were applied for
1 hour at room temperature in 500 units of DNase (Sigma).
Biotinylated anti-mouse IgG and anti-rat IgG antibodies were
applied for 30 minutes. Detection of the signal was performed
by incubation with the avidin⫺biotin⫺peroxidase reagent included in the Vectastain Elite ABC Kit (Vector) followed by
development with 3,3⬘-diaminobenzidine peroxidase substrate
including nickel solution to produce black staining. Following
dehydration, sections were mounted in Depex mounting medium and analyzed with an Axioskop 40 microscope connected
to an Axiocam (Zeiss).
For immunofluorescence analysis, autofluorescence
MSC NICHES IN THE SYNOVIUM IN VIVO
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Table 1. Antibodies used in the study, suppliers, working dilutions, and methods of antigen retrieval*
Antibody
Code
Supplier
Optimal dilution
Antigen retrieval method†
Mouse anti-IdU
Rat anti-CIdU
Rabbit anti-CD73
Rabbit anti-Sox9
Rabbit anti–type II collagen
Rabbit anti-PDGFR␣
Rabbit anti-vimentin
Rat anti-CD45
Rat anti–Sca-1
Rabbit anti-vWF
Rabbit anti-p75
Rabbit anti–␤-galactosidase
Goat anti-CD105
Goat anti-p75
Goat anti-CD146
Rat anti-CD44
Goat anti–c-Kit
Biotinylated rabbit anti-rat
Biotinylated anti-mouse
Alexa Fluor 488 goat antimouse
Alexa Fluor 647 chicken
anti-mouse
Alexa Fluor 488 goat antirat
Alexa Fluor 594 goat antirat
Alexa Fluor 594 goat antirabbit
Alexa Fluor 594 chicken
anti-goat
Alexa Fluor 488 donkey
anti-goat
ab8955
ab6326
ab71822
ab59265
ab21291
ab61219
ab45939
550539
557403
A-0082
AB1554
AB1211
AF1320
sc-6188
sc-18940
sc-18849
sc-1494
BA-4001
MKB-2225
A-11029
Abcam
Abcam
Abcam
Abcam
Abcam
Abcam
Abcam
BD PharMingen
BD PharMingen
Dako
Chemicon
Chemicon
R&D Systems
Santa Cruz
Santa Cruz
Santa Cruz
Santa Cruz
Vector
Vector
Invitrogen
1:100
1:100
1:50
1:50
1:200
1:50
1:200
1:20
1:200
1:200
1:100
1:300
1:20
1:50
1:100
1:100
1:100
1:200
1:250
1:500
B or P, 0.5–3 mg/ml
B or P, 0.5–3 mg/ml
B
B
P, 3 mg/ml
B
B
B
P, 0.5 mg/ml
B or P, 3 mg/ml
B
P, 0.5 mg/ml
B
B
B
B
P, 0.5 mg/ml
–
–
–
A-21463
Invitrogen
1:200
–
A-11006
Invitrogen
1:500
–
A-11007
Invitrogen
1:500
–
A-11037
Invitrogen
1:500
–
A-21468
Invitrogen
1:500
–
A-11055
Invitrogen
1:100
–
* Includes primary and secondary antibodies used for immunohistochemical analysis and immunofluorescence staining, with
optimal dilutions. IdU ⫽ iododeoxyuridine; CIdU ⫽ chlorodeoxyuridine; PDGFR␣ ⫽ platelet-derived growth factor receptor
␣; Sca-1 ⫽ stem cell antigen 1; vWF ⫽ von Willebrand factor; Santa Cruz ⫽ Santa Cruz Biotechnology.
† Either boiling (B) for 30 minutes in a pH 9 buffer or incubation at 37°C for 45 minutes with pepsin (P).
was quenched with two 5-minute washes with 50 mM NH4CI,
and nonspecific binding was blocked with 1% bovine serum
albumin for 45 minutes. Sections were incubated with primary
antibodies overnight at 4°C, and antigens were detected by
incubation with Alexa Fluor secondary antibodies for 1 hour at
room temperature. Table 1 lists the antibodies used in this
study as well as the suppliers, the working dilutions, and the
methods of antigen retrieval. After nuclear counterstaining
using either TO-PRO-3 (Invitrogen) or Sytox green, sections
were mounted with Mowiol and analyzed with a Zeiss 510
META Laser Scanning Confocal Microscope. Images were
obtained with an Axiocam.
Quantification of fluorescence intensity. For each condition, 3 sections per joint from 3 mice underwent immunofluorescence staining for IdU. From every section, images of
different areas of the synovium and articular cartilage were
obtained with an Axioscope 40 microscope connected to an
Axiocam, and the fluorescence intensity of IdU-positive cells
was measured using ImageJ software (NIH Image, National
Institutes of Health, Bethesda, MD; online at: http://
rsbweb.nih.gov/ij/). Values were expressed as the percentage of
IdU-positive synovial cells normalized to IdU-positive chondrocytes within each section, counting a minimum of 100 cells
of each cell type per section (n ⫽ 3). Chondrocytes were not
shown to proliferate; therefore, they were used as an intrasection reference for internal control to eliminate intersection
variability.
Quantification of labeled cells. For each condition,
3 sections per joint from 3 mice underwent immunofluorescence staining for IdU and CIdU. Nuclei were counterstained
with 4⬘,6-diamidino-2-phenylindole (DAPI). From every section, 3 images of different areas of the synovium were obtained. Cells were counted for single positivity of IdU or CIdU
or for double positivity. Values were expressed as the percentage of positive cells compared with the total number of
DAPI-counterstained cells, counting at least 1,000 cells per
condition (n ⫽ 3).
For quantification of cells positive for IdU and MSC
markers, 3 sections per joint from 3 uninjured control mice
were double-stained for IdU and the corresponding MSC
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KURTH ET AL
Figure 1. Proliferation of slow-cycling cells following joint-surface injury. A, Double nucleoside analog cell-labeling scheme. B, Expected labels at
different time points after injury: a, Iododeoxyuridine (IdU)–positive cells do not proliferate and retain IdU (green); b, IdU-positive cells proliferate
and acquire chlorodeoxyuridine (CIdU) (red) while gradually losing IdU, hence appearing yellow, orange, or red; c, unlabeled IdU-negative cells
(white) proliferate and acquire CIdU (red). C, Hematoxylin and eosin–stained sections of an uninjured control joint and a representative injured
joint. F ⫽ femur; P ⫽ patella; S ⫽ synovium. Asterisk indicates articular cartilage injury. Bars ⫽ 200 ␮m. D, Left and middle, Higher-magnification
views of boxed areas in C, showing single immunohistochemical staining for IdU and CIdU. c ⫽ uninjured control. Bars ⫽ 100 ␮m. Right, Double
immunofluorescence staining for IdU and CIdU in sections corresponding to the boxed areas shown in the left column. Bars ⫽
10 ␮m. E, Fluorescence intensity of IdU-positive synovial cells normalized to the fluorescence intensity of IdU-positive articular chondrocytes within
each section in uninjured controls. F, Double immunofluorescence staining for IdU and CIdU in the subchondral bone marrow of the patellar groove
region. G, Proportions of cells positive for IdU, CIdU, and both IdU and CIdU on days 4, 8, and 12. Values in E and G are the mean ⫾ SEM results
from 3 mice per group. 4dpi ⫽ 4 days after injury; 4dps ⫽ 4 days after sham operation.
MSC NICHES IN THE SYNOVIUM IN VIVO
marker. From every section, images of different areas of the
synovium were obtained. Cells were counted for single positivity of IdU or MSC markers or for double positivity.
Values were expressed as the percentage of double-positive
cells compared with the total number of IdU-positive cells or
the total number of MSC marker–positive cells, counting at
least 100 IdU-positive cells or MSC marker–positive cells per
joint (n ⫽ 3).
RESULTS
Proliferation of slow-cycling cells in synovium in
response to joint-surface injury. In well-studied systems
such as the hair follicle and the intestinal crypt, postnatal
stem cells are quiescent, slow-cycling cells in resting
conditions but undergo a burst of proliferation following
injury (16). To investigate whether adult joint synovium
has cells that display the functional behavior of stem
cells, we adapted a previously reported double nucleoside cell-labeling scheme (13) in a standardized mouse
model of joint-surface injury (14) (Figures 1A and C).
We anticipated the following scenarios. During
the initial labeling period with IdU, all dividing cells
including stem cells and proliferative progenitors prior
to differentiation would become labeled. During the
40-day washout period, the IdU label would be diluted
and become undetectable in rapidly dividing cells while
being retained by slow-cycling (stem) cells or by cells
that had stopped dividing, e.g., as a consequence of
differentiation. During the labeling period with CIdU,
all dividing cells would incorporate CIdU, but only
slow-cycling long-term–retaining IdU–positive cells
would become double labeled. The IdU-labeled cells
that did not divide after injury would not take up CIdU
(Figure 1B).
In uninjured control mice, slow-cycling longterm–retaining IdU–positive cells were detected in the
synovium of the knee joints, while CIdU-positive cells
were infrequent and rarely costained for IdU (Figure
1D). On day 4 and day 8 after injury, there was marked
accumulation of IdU and CIdU double-positive cells;
this accumulation was still detectable 12 days after
injury, although at a lower degree (Figure 1G). Of
note, the intensity of fluorescence of IdU-positive cells
within synovium became weaker over time (Figure 1E),
presumably as an effect of repeated cell divisions
following injury. These results demonstrate that the
adult knee joint synovial membrane contains slowcycling cells, and that following articular cartilage injury,
at least a subset of these cells proliferate to generate
a pool of transit-amplifying cells in vivo. Notably,
under our experimental conditions, no difference in
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nucleoside labeling was detected in the subchondral
bone marrow of the patellar groove region between
uninjured controls and injured mice, at all time points
tested (Figure 1F).
MSC-like phenotype of nucleoside-labeled cells.
We next investigated the phenotype of IdU-positive cells
in the synovial tissue, using multiple immunofluorescence stainings in situ. In uninjured mice, IdU-positive
cells were not of endothelial or hematopoietic origin,
because they consistently stained negative for the endothelial marker von Willebrand factor (vWF) (Figures 2A
and B), the panhematopoietic marker CD45 (Figures 2C
and D), and the hematopoietic progenitor cell marker
c-Kit (17) (Figures 2I and J). In injured mice, both CD45
and c-Kit were detected in an increasing number of cells,
but these cells did not costain for IdU (results not
shown). In contrast, most IdU-positive cells stained
positive for vimentin, thus confirming the mesenchymal
nature of the slow-cycling long-term–retaining cells (Figures 2E and F).
Stem cell antigen 1 (Sca-1) is described as a
positive selection marker for MSCs in fluorescenceactivated cell sorting when using C57BL/6 mouse bone
marrow (18). In uninjured knee joint synovium, we
detected isolated Sca-1–positive cells that costained for
IdU (Figures 2G and H). Because Sca-1 is also known to
be expressed in conjunction with c-Kit by hematopoietic
progenitors (19), we carried out triple immunofluorescence staining for Sca-1, c-Kit, and IdU. We detected
cells positive for IdU and Sca-1 but negative for c-Kit
(Figures 2I and J). These data confirmed that the
slow-cycling long-term–retaining IdU–positive cells in
synovium were nonhematopoietic in nature, with subsets having a Sca-1–positive, c-Kit–negative phenotype
compatible with that of MSCs. IdU-positive cells also
stained for platelet-derived growth factor receptor ␣
(PDGFR␣) (Figures 2K and L), a putative marker of
murine bone marrow MSCs in vivo (20).
We then investigated the expression of markers
that are known to be associated with human MSCs in
culture (21). In uninjured mice, subsets of IdU-positive
cells stained positive for established MSC markers such
as the hyaluronan receptor CD44 (22) (Figures 2M and
N), CD73 (23) (Figures 2O and P), and low-affinity
nerve growth factor receptor p75, which is reported to be
expressed by uncultured prospective MSCs in human
bone marrow (24) (Figures 2Q and R). In contrast,
CD105 was expressed by endothelium and scattered cells
within synovium but did not colocalize with IdU-positive
cells (Figures 2S and T). Taken together, these findings
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KURTH ET AL
Figure 2. Mesenchymal stem cell (MSC)–like phenotype of iododeoxyuridine (IdU)–positive cells in synovium. A–T, Representative images of
immunofluorescence staining for IdU (green) and lineage markers (red and light blue) in uninjured joints. Nuclei are counterstained in dark blue.
Enlarged images of the boxed areas are shown in the adjacent panels. IdU-positive cells (dashed arrows) were negative for the endothelial marker
von Willebrand factor (vWF) (B) (arrowhead) and the panhematopoietic marker CD45 (D) (arrowheads), whereas they were positive for the
mesenchymal/stromal cell marker vimentin (F) (arrows). A subset of stem cell antigen 1 (Sca-1)–positive cells costained for IdU (H) (arrow), while
other Sca-1–positive cells were IdU negative (H) (arrowhead). Cells positive for both IdU and Sca-1 (J) (arrow) were negative for c-Kit (J)
(arrowhead). IdU-positive cells costained for the MSC markers platelet-derived growth factor receptor ␣ (PDGFR␣) (L) (arrow), CD44 (N)
(arrow), CD73 (P) (arrows), and p75 (R) (arrows). Arrowheads in L, N, P, and R indicate cells positive for MSC markers but negative for IdU.
CD105 stained the endothelium and scattered cells (T) (arrowhead), which were negative for IdU. Bars ⫽ 10 ␮m. U and V, Proportions of cells
positive for IdU and MSC markers (IdU⫹M⫹) in relation to total IdU-positive cells (U) or to total MSC marker–positive cells (V). Values are the
mean ⫾ SEM results from 3 mice per group.
indicate that the synovium of mice contains slow-cycling
mesenchymal cells that stain positive for known markers
of MSCs.
We next determined the proportions of cells
within the IdU-positive cell pool that costained for MSC
markers and the proportions of cells within MSC
MSC NICHES IN THE SYNOVIUM IN VIVO
1295
Figure 3. Topography, relationship with pericytes, and phenotypic heterogeneity of MSC-like IdU-positive cells. Representative images of immunofluorescence staining for IdU (green) and lineage markers (red and blue) in uninjured joints are shown. A, A subset of IdU-positive cells costained for p75
and vimentin (arrows), while some cells were solely IdU positive (arrowheads). B, Some cells in the subsynovial tissue were positive for IdU, p75, and CD73
(arrows), and some were positive only for IdU and p75 (arrowheads). C, In the lining layer, cells positive for IdU, p75, and CD44 (arrows) were observed
beside cells that were positive only for IdU and p75 (arrowheads). D, Cells positive for both IdU and CD44 were confirmed in the lining layer (arrows),
whereas cells positive for IdU and CD73 were observed in subsynovial tissue (arrowheads). E, Triple immunofluorescence staining for IdU, CD146, and
␤-galactosidase (␤-gal) in knee sections from transgenic mice expressing ␤-galactosidase in pericytes is shown. F, A higher-magnification view of the boxed
area in E showed that CD146 is a pericyte marker (arrow) and confirmed that pericytes were IdU negative (dashed arrow). G, Triple immunofluorescence
staining for IdU, CD146, and vWF is shown. H, A higher-magnification view of the boxed area in G showed that IdU-positive cells (dashed arrow) are
negative for the pericyte marker CD146 (arrow) and the endothelial marker vWF. I, Triple immunofluorescence staining for IdU, CD146, and p75 is shown.
J–L, Higher-magnification views of the boxed areas in I showed that pericytes are negative for IdU but positive for p75 (arrowheads), while cells positive
for IdU and p75 that are located close to blood vessels (arrows) are negative for CD146. Merged images for IdU and p75 (J) and for IdU and CD146 (K),
and a triple-channel merged image (L) are shown. Bars ⫽ 10 ␮m. See Figure 2 for other definitions.
marker–labeled cell populations that were positive for
IdU. As shown in Figures 2U and V, none of the
markers tested was specific for IdU-positive cells, because there were cells that were positive for MSC
1296
markers but negative for IdU. In addition, none of the
individual MSC markers labeled all IdU-positive cells,
thus suggesting a heterogeneous phenotype of the slowcycling IdU-positive cells in synovium.
Topographic location and phenotypic heterogeneity of slow-cycling MSC-like cell populations within
synovium of uninjured joints. We next investigated the
topographic distribution of IdU-positive cells in the
synovium across the knee joint and also analyzed their
phenotype by triple immunofluorescence staining, using
multiple combinations of MSC markers in conjunction
with IdU. IdU-positive cells were relatively evenly distributed in the synovium. They were scattered and often
grouped in small clusters of 3–6 cells, with no specific
topographic location. Notably, IdU-positive cells were
observed in both the synovial lining and the subsynovial
connective tissue. Most IdU-positive cells costained for
vimentin and p75 in both synovial layers (Figure 3A).
CD73 colocalized with IdU exclusively in the subsynovial
tissue, where some of the IdU-positive cells that were
positive for CD73 also stained positive for p75 (Figure
3B). IdU-positive cells that costained for CD44 were
observed in the synovial lining, and some of these cells
were also positive for p75 (Figure 3C). The topographic segregation of IdU-positive cell subsets within
the synovium was further confirmed with triple immunofluorescence staining for IdU, CD44, and CD73.
While cells that were positive for both IdU and CD73
were located exclusively in the sublining tissue, cells
positive for IdU and CD44 were observed mainly in the
lining layer or juxtaposed to it; however, these 2 MSC
markers, CD44 and CD73, appeared to be mutually
exclusive (Figure 3D). These results indicate the presence of topographically distinct and phenotypically heterogeneous IdU-positive MSC-like cell subsets in both
layers of the synovium.
Relationship between MSCs and pericytes in the
knee synovium. Pericytes were recently shown to display
MSC-like properties in multiple tissues (25). In our
study, subsets of IdU-positive cells were located close to
blood vessels; hence, we investigated the relationship
between perivascular IdU-positive cells and pericytes.
To this end, we used a transgenic mouse in which the
reporter ␤-galactosidase (␤-gal) is expressed in pericytes
and smooth muscle cells (15). CD146, a known pericyte
marker (26,27), colocalized with ␤-gal in cells with
topography compatible with pericytes, thus validating
this transgenic mouse postnatally (Figures 3E and F).
IdU-positive cells were observed close to ␤-gal–positive
pericytes in uninjured mice but did not colocalize with
KURTH ET AL
␤-gal or CD146 (Figures 3E–L). Because CD146 stained
a greater number of perivascular (␤-gal–positive and
␤-gal–negative) cells, we also performed immunofluorescence staining for IdU, CD146, and vWF (Figures 3G
and H). Under our experimental conditions, in all mice
tested, IdU-positive cells were distinct from CD146positive pericytes and vWF-positive endothelial cells.
Similar results were obtained when using other pericyte
markers such as NG2, PDGFR␤, and ␣-smooth muscle
actin (results not shown).
Pericytes in tissues such as retina and liver are
known to express p75 (27,28). To investigate the relationship between cells positive for both IdU and p75 and
pericytes positive for p75, we performed triple immunofluorescence staining to codetect IdU, p75, and CD146
in uninjured mice. In a subset of blood vessels, p75 was
expressed by CD146-positive pericytes, all of which were
negative for IdU. We detected cells that were double
positive for IdU and p75 but were negative for CD146;
these cells had a perivascular location that was not
compatible with the topography of pericytes (Figures
3I–L). Of note, in the synovium of injured mice, at all 3
time points postinjury, we observed proliferation of
p75-positive cells that costained for both IdU and CIdU,
but we never detected proliferation of CD146-positive
pericytes (results not shown). Taken together, our findings indicate that the IdU-positive slow-cycling MSClike cells in the synovium are distinct from pericytes.
Chondrogenic fate of nucleoside-labeled cells in
vivo. After undergoing joint surgery, 5–10% of animals
accidentally undergo dehiscence of the sutures. This
results in patellar dislocation with secondary cartilage
metaplasia within the synovium. Twelve days after injury
(but not at earlier time points), in mice with patellar
dislocation, toluidine blue staining revealed large areas
of metachromasia within the synovium, mainly adjacent
to the femoral and patellar articular cartilage (Figure
4K). Similar but minute areas of weak metachromasia
were detected in injured mice without patellar dislocation at 12 days after injury (Figure 4J). The metachromasia in synovium shown by toluidine blue staining was
observed neither in uninjured control mice (Figure 4A)
nor in injured mice at earlier time points (results not
shown).
We took advantage of the complication of patellar dislocation in injured mice to provide proof of
concept that double nucleoside–labeled cells in synovium have another typical stem cell feature, i.e., the
ability to differentiate into mature cells such as chondrocytes. To this end, sections were costained for IdU,
MSC NICHES IN THE SYNOVIUM IN VIVO
1297
Figure 4. Detection of cells positive for both iododeoxyuridine (IdU) and chlorodeoxyuridine (CIdU) in the area of ectopic cartilage metaplasia
within the synovium of injured mice. A, J, and K, Toluidine blue staining for metachromasia in the synovium was negative in control mice (A) but
was positive 12 days after injury in nondislocated joints (J) (arrows) and dislocated joints (K). B and F, Higher-magnification images of the
boxed area in A are shown. L and P, Higher-magnification images of the boxed area in K are shown. B–I, No Sox9 (B–E) or type II collagen (Col2)
(F–I) was detectable in the synovium of uninjured joints. C–E, Merged images for IdU and Sox9 (C) and for CIdU and Sox9 (D), and a triple-channel
merged image (E) are shown, all of which were enlarged from the boxed area in B. G–I, Merged images for IdU and type II collagen (G) and for
CIdU and type II collagen (H), and a triple-channel merged image (I) are shown, all of which were enlarged from the boxed area in F. L–S, At 12
days postinjury, Sox9 (L–O) and type II collagen (P–S) were expressed by cells mostly positive for both IdU and CIdU (arrows) or for only IdU
(dashed arrows). M–O, Merged images for IdU and Sox9 (M) and for CIdU and Sox9 (N), and a triple-channel merged image (O) are shown, all
of which were enlarged from the boxed area in L. Q–S, Merged images for IdU and type II collagen (Q) and for CIdU and type II collagen (R),
and a triple-channel merged image (S) are shown, all of which were enlarged from the boxed area in P. Bars ⫽ 10 ␮m.
CIdU, and the early chondrocyte lineage marker Sox9, a
transcription factor required for cartilage formation
(29). Although no Sox9 was detectable in the synovium
of uninjured joint sections at all time points tested
(Figures 4B–E), a number of cells expressed the marker
of early chondrogenesis 12 days after injury (Figures
4L–O), with positive cells located in the area of metachromasia and most also being positive for IdU and
CIdU. Notably, no Sox9-positive cells stained only for
CIdU, while some Sox9-positive cells stained only for
IdU, suggesting either that IdU-positive cells had differentiated without proliferation or that they had undergone very few cell cycles prior to day 8 postinjury.
Type II collagen, a chondrocyte-specific marker,
was detected in consecutive sections mostly associated
with cells showing an enlarged rounded nucleus (Figures
1298
KURTH ET AL
4P–S). As observed for Sox9, the majority of cells
positive for type II collagen costained for both IdU and
CIdU, but a number of cells costained only for IdU.
Type II collagen was not detectable in the synovium at
either 4 days or 8 days after injury (data not shown) or
in knee joint sections obtained from uninjured mice
(Figures 4F–I). Colocalization of IdU and CIdU with
Sox9 and type II collagen was also observed 12 days after
injury in the minute metachromatic areas of synovial
tissue in nondislocated joints (data not shown). These
data indicate that double nucleoside–labeled cells within
metachromatic synovium display a chondrocyte-like
phenotype.
DISCUSSION
Synovial hyperplasia is a very common phenomenon following joint injuries, but the identity of the
underpinning proliferating stromal cells is not known.
Similar to bone marrow and most connective tissues
(30–33), the stroma of the synovial tissue contains cells
that after release and culture expansion display features
of MSCs (5). However, despite extensive in vitro characterization, the equivalent cells within their native
tissue in vivo have not been identified because of a lack
of specific markers.
In the current study, we adapted to our mouse
model of traumatic knee joint–surface injury a double
nucleoside analog–labeling scheme that was recently
used to positively detect neural stem cells in vivo (13).
We identified and characterized, in the mouse knee joint
synovium, a population of quiescent, slow-cycling nonhematopoietic, nonendothelial, MSC-like stromal cells,
present in both the lining layer and sublining tissue, that
proliferated following injury to produce synovial hyperplasia (Figure 5).
Although IdU-positive cells expressed conventional MSC markers, none of the MSC markers tested
was sufficient to label all IdU-positive cells, and all MSC
markers also labeled cells that were negative for IdU.
Moreover, IdU-positive cells were heterogeneous in
their phenotype, possibly reflecting the coexistence of
functionally distinct cell subsets.
IdU-positive cells were negative for CD105, an
established marker of human MSCs in culture. We
cannot exclude the possibility that CD105 would identify
MSCs in humans but not in mice, because variations in
MSC phenotype and biology across species are known
(34). In addition, acquisition of CD105 might occur as a
result of phenotypic rearrangements during ex vivo cell
Figure 5. Schematic representations of cell phenotypes identified
using the double nucleoside analog cell-labeling strategy. A, Schematic
drawing of an uninjured control synovial joint. B, Details of the dashed
box in A, showing cell populations in the synovium of uninjured joints.
Iododeoxyuridine (IdU)–positive cells (green) were located in both the
synovial lining (SL) and the subsynovial tissue (SST). Subsets of
IdU-positive cells displayed a mesenchymal stem cell phenotype.
IdU-negative cells (blue) included hematopoietic lineage cells (HC),
endothelial cells (EC), pericytes (PC), and other cell types of unknown
phenotype. C, Schematic drawing of a synovial joint 12 days after
articular cartilage injury (arrowhead). D, Details of the dashed box in
C, showing cell populations in the synovium. Proliferating cells were
detected in both the synovial lining and the subsynovial tissue and
were either double positive for IdU and chlorodeoxyuridine (CIdU;
orange) or single positive for CIdU (red). Subsets of cells positive for
IdU and CIdU and cells positive only for IdU (green) expressed
chondrocyte lineage markers. The boxed areas in B and D show cell
phenotypes. B ⫽ bone; C ⫽ cartilage; SM ⫽ synovial membrane;
␤-gal ⫽ ␤-galactosidase; SC ⫽ synovial cavity (see Figure 2 for other
definitions).
manipulations (35). Indeed, markers observed on cells in
vitro cannot be directly extrapolated to the equivalent
native cells in vivo. For this reason, we used an unbiased
approach, based on nucleoside labeling, to detect stem
cells within the synovium in vivo.
IdU-positive cells were detected in both the
lining layer and the sublining tissue. In the lining layer,
IdU-positive cells were negative for CD45, thus excluding their monocytic nature (36), but were positive for the
MSC markers PDGFR␣, p75, and CD44. However,
CD44 is also known to be expressed by synovial fibroblasts (37). IdU-positive cells in the lining layer
costained for cadherin 11 (results not shown), a known
marker of synovial fibroblasts (38). The relationship
between MSCs and fibroblasts in the synovium is currently being investigated in our laboratory.
Pericytes are adventitial cells located around
MSC NICHES IN THE SYNOVIUM IN VIVO
small vessels of connective tissues, and data indicate that
a subset of MSCs could be or could derive from pericytes
(27,39). In a recent study (25), pericytes in human tissues
were reported to be positive for the MSC markers CD44,
CD73, CD90, and CD105 and displayed mesenchymal
multipotency in vitro (25). However, no evidence of
differentiation of pericytes within their environments in
vivo by lineage-tracking experiments was provided. In
addition, joint tissues were not investigated, and therefore the paradigm of the pericyte origin of the cultured
MSCs cannot be extrapolated to synovial tissue. Our
findings indicate that the MSC population we identified
in the synovium in vivo is phenotypically and functionally distinct from pericytes.
Following traumatic injury to the joint surface,
we observed synovial hyperplasia that was sustained by
proliferation of mesenchymal cells. Although we cannot
exclude the contribution to synovial hyperplasia of incoming cells from bone marrow or from the circulation
via the bloodstream, the observations that quiescent
long-term–retaining IdU-positive cells were detected
within the synovium of uninjured mice, that following
injury, the number of IdU-positive and CIdU–doublepositive cells increased in parallel with a decrease in the
number of IdU-positive cells, and that the intensity of
fluorescence of IdU-positive cells in synovium decreased
over time following injury make a strong case in support
of in situ proliferation of quiescent resident MSCs within
synovium.
Twelve days after injury, cells within the synovial
cartilage that were positive for both IdU and CIdU
expressed the chondrocyte lineage markers Sox9 and
type II collagen (Figures 5C and D). Although most cells
with a chondrocyte phenotype were double positive for
IdU and CIdU, a few scattered cells were positive only
for IdU. This suggests that either proliferation was not a
prerequisite for chondrogenic differentiation or that
IdU-labeled cells had divided very few times before
CIdU was administered 8 days after surgery, so that they
retained IdU beyond the period of proliferation but
were negative for CIdU. Although we cannot exclude
the possibility that IdU-positive chondrocytes migrated
out of the articular cartilage into the synovium, our study
indicated an obvious contribution of synovial cells to the
process of cartilage metaplasia in synovium, in keeping
with previous observations that treatment of synovial
tissue explants in vitro with chondrogenic growth factors
induced cartilage formation (40,41). Cartilage metaplasia was observed throughout the knee joint synovium but
was much more pronounced at the transition zone where
1299
the synovium connects to the articular cartilage, mimicking the chondrophytes observed in osteoarthritis.
We did not observe any cartilage repair in the
C57BL/6 mice used in this study. An identical experimental injury to the joint surface in young adult DBA/1
mice resulted in full healing of the lesion (13). We are
currently investigating whether the resident MSCs in
synovium are able to contribute to cartilage repair in a
permissive mouse strain such as DBA/1. Knowledge of
the cellular and molecular mechanisms underlying joint
tissue homeostasis, remodeling, and repair in health and
disease will help modulate MSC niches pharmacologically to achieve joint tissue regeneration and, in the
broader picture, to influence outcomes of joint disorders
and restore joint homeostasis.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. De Bari had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Kurth, Dell’Accio, Sharpe, De Bari.
Acquisition of data. Kurth, Crouch, De Bari.
Analysis and interpretation of data. Kurth, Dell’Accio, Augello, De
Bari.
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