JOURNAL OF BONE AND MINERAL RESEARCH

Volume 20, Number 9, 2005

Published online on May 23, 2005; doi: 10.1359/JBMR.050514
© 2005 American Society for Bone and Mineral Research

Advanced Glycation End-Products Attenuate Human Mesenchymal

Stem Cells and Prevent Cognate Differentiation Into Adipose Tissue,
Cartilage, and Bone*
Shinichiro Kume,1,2 Seiya Kato,2 Sho-ichi Yamagishi,3 Yosuke Inagaki,3 Seiji Ueda,4 Nobuyuki Arima,2
Takahiro Okawa,1 Masamichi Kojiro,2 and Kensei Nagata1

ABSTRACT: The impact of AGEs on human MSCs was studied. AGEs inhibited the proliferation of MSCs,
induced apoptosis, and prevented cognate differentiation into adipose tissue, cartilage, and bone, suggesting
a deleterious effect of AGEs in the pathogenesis of musculoskeletal disorders in aged and diabetic patients.
Introduction: Advanced glycation end-products (AGEs) are accumulated on long-lived proteins of various
tissues in advanced age and diabetes mellitus and have been implicated in chronic complication, including
musculoskeletal disorders. Human mesenchymal stem cells (MSCs) potentially differentiate into mature mus-
culoskeletal tissues during tissue repair, but the pathogenetic role of AGEs on MSCs is unclear.
Materials and Methods: AGEs were prepared by incubating BSA with glucose, glyceraldehydes, or glycol-
aldehyde (designated as AGE-1, AGE-2, or AGE-3, respectively). Proliferation, apoptosis, and reactive
oxygen species (ROS) generation were assayed in AGE-treated cells. The expression of the receptor for AGE
(RAGE) was examined by immunohistochemistry and Western blotting. Involvement of RAGE-mediated
signaling was examined using a neutralizing antiserum against RAGE. Differentiation into adipose tissue,
cartilage, and bone were morphologically and biochemically monitored with specific markers for each.
Results: AGE-2 and AGE-3, but not control nonglycated BSA and AGE-1, reduced the viable cell number
and 5-bromo-2’deoxyuridine (BrdU) incorporation with increased intracellular ROS generation and the per-
centage of apoptotic cells. MSCs expressed RAGE and its induction was stimulated by AGE-2 and AGE-3.
These AGEs inhibited adipogenic differentiation (assayed by oil red O staining, lipoprotein lipase production,
and intracellular triglyceride content) and chondrogenic differentiation (assayed by safranin O staining and
type II collagen production). On osteogenic differentiation, AGE-2 and AGE-3 increased alkaline phospha-
tase activity and intracellular calcium content; however, von Kossa staining revealed the loss of mineralization
and mature bone nodule formation. The antiserum against RAGE partially prevented AGE-induced cellular
events.
Conclusion: AGE-2 and AGE-3 may lead to the in vivo loss of MSC mass and the delay of tissue repair by
inhibiting the maturation of MSC-derived cells. The AGE–RAGE interaction may be involved in the del-
eterious effect of AGEs on MSCs.
J Bone Miner Res 2005;20:1647–1658. Published online on May 23, 2005; doi: 10.1359/JBMR.050514

Key words: mesenchymal stem cells, advanced glycation end-products, receptor for advanced glycation end-
products, proliferation, differentiation

INTRODUCTION reversibly cross-linked and fluorescent moieties.(1–3) The

A DVANCED GLYCATION END - PRODUCTS (AGEs) are
formed by reducing sugars nonenzymatically with the
amino groups of proteins to initiate a complex series of
rearrangements and dehydrations to produce a class of ir-
*Part of this work was presented as abstract form at the 25th
Congress of the International Academy of Pathology, Brisbane,
Australia, October 10–15, 2004.
The authors have no conflict of interest.
formation and accumulation of AGEs are a characteristic
feature of tissues in aged people, especially in patients with
diabetes mellitus, and these products have also been
strongly implicated in the pathogenesis of age-related and
diabetic complications.(4,5) It has been reported that AGEs
are involved in musculoskeletal diseases such as osteoar-
thritis (OA), which is the most common chronic disabling
disorder for aged people.(6) Accumulation of AGEs in the
articular cartilage in OA patients biochemically affects cel-

1
Department of Orthopaedic Surgery, Kurume University School of Medicine, Kurume, Japan; 2Department of Pathology, Kurume
University School of Medicine, Kurume, Japan; 3Department of Internal Medicine III and the Cardiovascular Research Institute, Kurume
University School of Medicine, Kurume, Japan; 4Department of Nephrology, Kurume University School of Medicine, Kurume, Japan.

1647
1648
lular characteristics and increases stiffness and brittleness of
the tissue.(7) Accumulation of AGEs increases stiffness of
the collagen network in the bone as well, which may explain
some of the age-related increase in skeletal fragility and
fracture risk.(8) Indeed, decreased bone strength and the
increased risk of fracture have been shown in human and
rodent models of diabetes, as well as of diabetic nephrop-
athy.(9–11) In the craniotomy model in type 1 diabetic mice,
the degree of bone healing is reported to be ∼40% of that
in nondiabetic animals, and the receptor for AGEs
(RAGE) is expressed at higher levels in the healing bone
tissue in diabetic than in control animals.(12) Taken to-
gether, these findings suggest that AGEs may play a role in
the pathogenesis of diabetic osteopenia as well as that of
various bone and joint disorders in nondiabetic patients;
however, the details of such deleterious mechanisms remain
to be clarified.
Mesenchymal stem cells (MSCs) are multipotent cells
present in various tissues during human development and
are capable of being isolated from adult bone marrow and
to be differentiating into bone, cartilage, muscle, and adi-
pose tissues.(13,14) In injured adults, MSCs are involved in
the formation, remodeling, and repair of musculoskeletal
tissues, suggesting the potential for therapeutic approaches
using MSCs in orthopedic and regenerative medicine.(15,16)
In bone formation, pre-osteoblasts, osteoblasts, and osteo-
cytes all originate from MSCs.(17) Defects in the number
and proliferative potential of MSCs have been suggested to
underlie age-related defects in osteoblast number and func-
tion.(18,19) In streptozotocin-induced diabetic rats, AGE-
modified type I collagen dose-dependently inhibits the phe-
notypic expression of osteoblasts.(20) It has also been
reported that AGEs influence osteoblast development
through specific receptors, including RAGE.(21,22) In hu-
man articular cartilage, an increase in AGE levels nega-
tively affects the proteoglycan synthesis, thereby reducing
cellular turnover and repair capacity and contributing to the
degradation of tissue.(23) It has been reported that CD36, a
cell surface glycoprotein, functions as a receptor for AGEs
in the adipogenic differentiation of mouse 3T3-L1 cells.(24)
These reports may suggest that AGEs have the ability to
regulate proliferation and differentiation of musculoskele-
tal lineage cells, but there have been no studies investigat-
ing whether AGEs directly affect the function of MCSs. In
this context, we studied the effects of AGEs on the prolif-
eration and differentiation of cultured human MSCs in
vitro. Here we first report that AGEs inhibited prolifera-
tion of MSCs with apoptosis and prevented cognate differ-
entiation into adipose tissue, cartilage, and bone with the
partial involvement of AGE–RAGE interaction.
MATERIALS AND METHODS
Cell culture condition of human MSCs
Human MSCs (Poietics) and mesenchymal stem cell
growth medium (MSCGM) were purchased from BioWhit-
taker (Walkersville, MD, USA). The cells were cultured at
37°C in a humidified 5% CO2–95% air mixture and were
kept at a subconfluent density. Cells passaged less than five
times were used in this study.
KUME ET AL.
Preparation of AGEs
AGEs were prepared as described previously.(25) Briefly,
50 mg/ml of BSA (Sigma) was incubated under sterile con-
ditions with D-glucose for 8 weeks or 0.1 M D-glyceralde-
hyde or D-glycolaldehyde in 0.2 M NaPO4 buffer (pH 7.4)
for 7 days. The unincorporated sugars were removed by
dialysis against 0.2 M PBS (pH 7.4). These products of
AGE proteins were designated as AGE-1, AGE-2, or
AGE-3, respectively. Nonglycated BSA was incubated un-
der the same conditions except for the absence of D-
glucose, D-glyceraldehyde, or D-glycolaldehyde as a nega-
tive control. Preparations were tested for endotoxin using
an Endospecy ES-20S system (Seikagaku Co., Tokyo, Ja-
pan), and no endotoxin was detectable.
Proliferation assay
Total accumulation of viable cells was estimated by hand
counting with a hemocytometer after Trypan blue exclu-
sion. For the measurement of proliferating cells, 5-bromo-
2’deoxyuridine (BrdU) incorporation(26) was performed us-
ing a cell proliferation ELISA kit (Roche) following the
manufacturer’s protocol. Briefly, MSCs were seeded on a
96-well multititer plate at 5 × 103 cells/well. After incuba-
tion under a serum-starved condition for 24 h, the growing
medium was applied, and MSCs were further cultured for
24 h. At the end of the culture course, 10 ␮l/well of BrdU
diluted with PBS was added for 4 h. Absorbance at 450–690
nm was measured using a scanning multiwell spectropho-
tometer (Eimax A-4; Fujirebio, Tokyo, Japan).
Semiquantitative analysis of apoptosis
The level of apoptosis was tested by the terminal deoxy-
nucleotidyl transferase (TdT)-mediated deoxyuridine tri-
phosphate (dUTP) nick end labeling (TUNEL) method.
The multitude of newly generated 3⬘-OH ends by DNA
fragmentation was identified visually using an in situ cell
death detection kit (Roche). A positive control was estab-
lished by incubating the specimens in DNase I, RNase-free
(Roche) for 10 minutes before the reaction with TdT en-
zyme. For the assay, the cells inoculated on a coverslip were
washed with PBS, fixed in 4% paraformaldehyde in PBS,
and permeabilized with 0.2% Triton X-100 in citrate buffer.
Samples were incubated with TdT and fluorescein-labeled
dUTP, counterstained with propidium iodide, and observed
with a fluorescence microscope (Leica). Percentages of
apoptotic cells were estimated by counting a total of 300
cells from random fields.
Measurement of intracellular reactive oxygen species
generation
The intracellular formation of reactive oxygen species
(ROS) was detected using a fluorescent probe CM-
H2DCFDA (Molecular Probes) as described previously.(27)
Briefly, cells were plated at 5 × 103 cells/well in a 96-well
plate. The cells were loaded with 10 ␮⌴ CM-H2DCFDA,
incubated for 60 minutes at 37°C, and analyzed using an
EZS-FL fluorescent plate reader (Asahi Techno Glass, To-
kyo, Japan) using the program EZScan-FL for Windows.
EFFECTS OF AGES ON MESENCHYMAL STEM CELLS
Western blotting
The cells were lysed in TNE buffer (20 mM Tris-HCl, pH
7.6, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1 mM
Na3VO4, 1 mM phenylmethanesulfanyl fluoride (PMSF), 5
mM 2-mercaptoethanol, 0.8 ␮M of aprotinin, 20 ␮M of leu-
peptin, 50 ␮M of pepstatin, 10 ␮M of pepstatin A, and 1%
NP-40) at 4°C. The cell lysate, which was determined to
contain 30 ␮g of protein using a Bradford protein assay kit
(Bio-Rad), was further dissolved in SDS-PAGE sample
buffer (25 mM Tris-HCl, pH 6.8, 5% glycerol, 1% SDS,
0.01% bromophenol blue, 50 mM DTT). The sample was
subsequently resolved by 12% SDS-PAGE. The gels were
electronically transferred to polyvinylidene fluoride
(PVDF) membranes (Millipore) followed by incubation
with a goat polyclonal anti-RAGE antibody (clone C-20;
Santa Cruz), at a dilution of 1:500 for 2 h at room tempera-
ture. Blots were visualized by enhanced chemiluminescence
(ECL; Amersham), using horseradish peroxidase–
conjugated secondary antibodies (Zymed). To confirm that
equal amounts of the proteins were subjected to Western
blotting analysis, the membranes were reprobed with a
mouse monoclonal antibody against ␤-actin (Sigma). Den-
sitometric analysis was performed with NIH image soft-
ware, and the relative ratio to ␤-actin expression was cal-
culated in each sample.
Immunofluorescence microscopy
MSCs were grown on the Laboratory-Tek chamber
(NUNC). At the end of the culture period, the cells were
fixed with 4% paraformaldehyde in PBS (pH7.4) for 10
minutes and permeabilized with 0.2% Triton X-100. The
cells were incubated with a 1:200 dilution of polyclonal an-
tibody for RAGE (Santa Cruz) for 1 h, followed by incu-
bation with a 1:1000 dilution of FITC-conjugated secondary
antibodies (Bio-Rad). After counterstaining with 0.1 ␮g/ml
of propidium iodide (Sigma), the specimens were observed
with a fluorescence microscope (Leica). Negative control
staining in which a primary antibody was omitted was also
tested.
Immunoelectron microscopy
The distribution of RAGE on the cell surface of MSCs
was also examined by electron microscopy using the immu-
nogold technique. Briefly, MSCs were fixed with 4% para-
formaldehyde in PBS for 1 h. After washing with PBS, cells
were rinsed with 0.5% BSA and 0.02% sodium azide
(Sigma) for 10 minutes followed by the incubation with a
1:200 dilution of anti-RAGE antibody (Santa Cruz) for 1 h
at room temperature. Positive signals were visualized using
an immunogold staining kit (EY Laboratories, San Mateo,
CA, USA) with 15-nm Protein A-gold colloidal particles.
Labeled cells were fixed with 1% glutaraldehyde, mounted
on polylysin-coated coverslips, and postfixed with 1% os-
mium tetroxide in PBS. Samples were examined with a Hi-
tachi S-800 field emission scanning electron microscope
equipped with a detector for backscattered electrons.
Neutralizing antibody for RAGE
Antiserum against human RAGE was prepared for neu-
tralizing assays as described previously.(28)
1649
Adipogenic differentiation of MSCs
Adipogenic differentiation was induced with commer-
cially available adipogenic differentiation medium (Bio-
Whittaker). Briefly, confluent MSCs were cultured with
medium containing 1 ␮M dexamethasone, 10 ␮g/ml bovine
insulin, 0.2 mM indomethacin, 0.5 mM isobutylmethylxan-
thine, and 10% FCS for a period of 3 days followed by the
removal of dexamethasone, indomethacin, and isobutyl-
methylxanthine, and additional culturing for 2 days. Adipo-
genic differentiation was terminated after 2–3 weeks in cul-
ture. For the oil red O staining, cells were fixed in 10%
neutral buffered formalin, washed in water, and stained
with a 0.6% (w/v) oil red O solution (60% isopropanol,
40% water) for 1 h at room temperature, followed by wash-
ing with water to remove unbound dye. The concentration
of lipoprotein lipase (LPL) in the conditioned medium was
measured using an enzyme-linked immunosorbent assay
(ELISA) kit (Daiichi Pure Chemicals, Tokyo, Japan). For
the measurement of intracellular triglyceride (TG) content,
cells were scraped off into 25 mM Tris-HCl (pH 7.5) con-
taining 1 mM EDTA. After homogenization, TG was ex-
tracted using chloroform-methanol (2:1, vol/vol) and quan-
tified enzymatically using a commercially available TG test
kit (WAKO, Tokyo, Japan).
Chondrogenic differentiation of MSCs
Chondrogenic differentiation was induced with a com-
mercially available chondrogenic differentiation medium
(BioWhittaker). Briefly, incomplete chondrogenic differen-
tiation medium (ICDM) containing 0.1 ␮M dexametha-
sone, 0.17 mM ascorbic acid 2-phosphate, 6.25 ␮g/ml bovine
insulin, 6.25 ␮g/ml transferrin, and an additional 10 ng/ml of
TGF-␤3 (PeproTech) was added for the complete chondro-
genic differentiation medium (CCDM). MSCs suspended in
ICDM (∼7.5 × 105 cells/ml) were centrifuged at 150g for 5
minutes, and ICDM was aspirated and replaced by CCDM.
MSCs resuspended in CCDM (5 × 105 cells/ml) were cul-
tured in polypropylene tubes (Corning). The cell pellets
were fed every 3 days by completely replacing the CCDM
and harvested after 2–4 weeks in culture. The clusters of the
cells were fixed with 10% neutral buffered formalin, and
paraffin-embedded sections were stained with the safranin
O method for glycosaminoglycan synthesis. Type II colla-
gen production into the media was measured by ELISA
using a native type II collagen detection kit (Chodrex, Red-
mond, WA, USA).
Osteogenic differentiation of MSCs
Osteogenic differentiation was induced with a commer-
cially available osteogenic differentiation medium (Bio-
Whittaker) containing 0.1 ␮M dexamethasone, 0.05 mM
ascorbic acid 2-phosphate, and 10 nM ␤-glycerophosphate.
MSCs were seeded at 5 × 103 cells/cm2 and were cultured up
to 3 weeks with replacement of the fresh osteogenesis in-
duction medium every 4 days. For the detection of osteo-
genic differentiation, bone nodule formation was examined
under a phase-contrast microscope (Nikon) after von Kossa
staining as previously described.(29) For alkaline phospha-
tase (ALP) activity, MSCs were washed three times with
1650 KUME ET AL.

PBS and harvested with 0.2% NP-40 (Sigma) plus 2 mM

MgCl2 in PBS. After centrifugation at 1500g for 15 minutes,
the supernatant was subjected to for ALP activity assay
with p-nitrophenol phosphate as a substrate using an
Osteolinks-BAP kit (a diagnostic enzyme immunoassay kit
for bone-specific ALP; Smitomo Pharmaceuticals Co., To-
kyo, Japan). For the measurement of intracellular calcium
content, the cells dissolved in NP-40 lysis buffer were ap-
plied to a Clinical analyzer (Hitachi), and the assay was
performed by the method of Connerty.(30) Type I collagen
production into the medium was measured using a human
type I capture ELISA kit (Chodrex).

Statistical analysis
Experimental groups were compared by ANOVA, and
when appropriate, with Scheffe’s test for multiple compari-
sons. All data are expressed as mean ± SD. A level of p <
0.05 was accepted as statistically significant.

RESULTS
Effects of AGEs on proliferation and apoptosis
of MSCs
Cultured MSCs were incubated with 100 ␮g/ml of non-
glycated BSA (as a control), AGE-1, AGE-2, or AGE-3,
which dosage has been shown to be sufficient for inducing
biological events in vascular endothelial cells(31) and retinal
pericytes.(32) The conditioned medium was changed every 3
days with fresh AGEs, and the viable cell number was
counted up to 5 days after the initial addition. As shown in
Fig. 1A, the growth curve of MSCs was shifted downwards
by the treatments with AGE-2 and AGE-3. At day 5, the FIG. 1. Growth suppression of human MSCs by AGE-2 and
AGE-3. (A) Cell counting assay. MSCs were seeded at a density
viable cell numbers were decreased by 45 ± 3.5% with of 1 × 103 cells/cm2 on 6-well culture dishes at day 0. The cells
AGE-2 (p < 0.01) and by 41 ± 7.0% with AGE-3 (p < 0.05). were cultured with MSCGM containing 100 ␮g/ml of nonglycated
Both AGE-2 and AGE-3, but not AGE-1, significantly re- BSA, AGE-1, AGE-2, or AGE-3. The cell counting was per-
duced DNA synthesis, which was measured by the BrdU formed at days 1–5. Data are shown as the mean ± SD (n ⳱ 3).
incorporation assay, in a dose-dependent fashion (Fig. 1B). (B) BrdU incorporation assay. MSCs were seeded on a 96-well
culture plate at a density of 1 × 103 cells/well. After 24 h of serum
Next, MSCs were incubated with AGEs for 3 days, and the starvation, cells were cultured with MSCGM containing 10–100
level of apoptosis was measured by semiquantitative ␮g/ml of nonglycated BSA (open bars), AGE-1 (hatched bars),
TUNEL staining. The cells treated with AGE-2 and AGE-2 (gray bars), or AGE-3 (filled bars), respectively. The val-
AGE-3 showed a dose-dependent increase in apoptotic cell ues of A450-A690 are shown as the mean ± SD (n ⳱ 6). *p < 0.05.
These data are representative of one of two independent experi-
death (Fig. 2). In contrast, AGE-1 did not show the inhibi- ments performed under the same conditions.
tory effect on the growth of MSC in terms of proliferation
and apoptosis.

Effects of AGEs on ROS generation in MSCs RAGE expression in MSCs

We have previously shown that cellular events mediated
by AGEs were associated with an enhancement of intracel-
lular ROS generation.(31,32) Thus, ROS generation was
tested in AGE-treated MSCs. As shown in Fig. 3, both
AGE-2 and AGE-3, but not AGE1, stimulated ROS gen-
eration in a dose-dependent fashion. The ROS generation
stimulated by 100 ␮g/ml of these AGEs was about 3-fold
higher than the control levels (p < 0.01). The following
experiments were performed with AGE-2 and AGE-3 at a
concentration of 100 ␮g/ml, because this dosage has been
shown to be sufficient to induce biological events against
MSCs.
The immunohistochemical study showed RAGE expres-
sion in MSCs (Fig. 4A). We further used an immunoelec-
tron microscopic imaging with the immunogold technique
to show the cell surface expression of RAGE (Fig. 4B).
Negative control experiments performed without anti-
RAGE antibody but with Protein-A gold conjugate did not
display any specific gold label (data not shown). Recently,
we have shown that AGEs upregulate RAGE expression in
endothelial cells,(31) retinal pericytes,(32) and renal mesan-
gial cells.(33) Also in MSCs, Western blotting showed that
AGE-2 as well as AGE-3 upregulated RAGE expression
about 1.5-fold (Fig. 4C).
EFFECTS OF AGES ON MESENCHYMAL STEM CELLS
FIG. 2. Apoptosis of human MSCs induced by AGE-2 and
AGE-3. MSCs were seeded on a coverslip and cultured with
MSCGM containing 10–100 ␮g/ml of nonglycated BSA (open
bars), AGE-1 (hatched bars), AGE-2 (gray bars), or AGE-3
(filled bars). After 24 h of cultivation, an in situ TUNEL assay for
semiquantitation of apoptosis was performed, and the percent-
ages of apoptotic cells were estimated by counting a total of 300
cells from random fields. Data are shown as the mean ± SD (n ⳱
3). *p < 0.05. These data are representative of one of two inde-
pendent experiments performed under the same conditions.
FIG. 3. Intracellular ROS generation of human MCSs induced
by AGE-2 and AGE-3. MSCs were seeded on a 96-well culture
plate at a density of 2 × 104 cells/well and were cultured for 24 h
with MSCGM containing 10–100 ␮g/ml of nonglycated BSA
(open bars), AGE-1 (hatched bars), AGE-2 (gray bars), or
AGE-3 (filled bars). Data are shown as the mean ± SD (n ⳱ 6).
*p < 0.05; **p < 0.01. These data are representative of one of two
independent experiments performed under the same conditions.
Effect of neutralizing anti-RAGE antibody in
AGEs-treated MSCs
Next, we tested the potential involvement of AGE-
RAGE interaction in the regulation of MSC growth. A cell
counting assay revealed that the growth inhibition by
1651
FIG. 4. Expression of RAGE in human MSCs. (A) Immunoflu-
orescence microscopy. MSCs were seeded on the Laboratory-Tek
chamber (NUNC), and immunohistochemical staining was carried
out for the expression of RAGE. (B) Immunoelectron micro-
graphs showing immunoreactive gold particles (15 nm in diam-
eter, arrowheads) against a goat anti-RAGE antibody at the cell
surface of MSCs. (a) Low magnification of a secondary electron
image, (b) high magnification of a backscattered electron image,
and (c) the same area of b with a secondary electron image are
shown. (C) Upregulation of RAGE expression by AGE-2 and
AGE-3. MSCs were cultured with MSCGM containing 100 ␮g/ml
of nonglycated BSA, AGE-2, or AGE-3 for 48 h. The results are
also expressed in relative densitometric units (RAGE/␤-actin,
n ⳱ 2).
AGE-2 or AGE-3 was partly recovered by the treatment
with anti-RAGE neutralizing antibody (Fig. 5A). The ROS
generation induced by these AGEs was also reduced by the
treatment with RAGE-neutralizing antibody (Fig. 5B).
Effects of AGEs on adipogenic differentiation
of MSCs
We further studied whether AGE-2 and AGE-3 could
influence the differentiation of MSCs. First, the differentia-
tion of MSCs into adipocyte was induced with dexametha-
sone-containing medium, and lipid droplets in the cells
1652
FIG. 5. AGE-RAGE interaction in MSCs. (A) Cell counting
assay. MSCs were seeded as shown in Fig. 1 and cultured with
MSCGM containing 100 ␮g/ml of nonglycated BSA (open bars),
AGE-1 (hatched bars), AGE-2 (gray bars), or AGE-3 (filled bars)
in the presence or absence of a neutralizing anti-RAGE anti-
serum (0.1% in concentration) for 48 h. The results of cell count-
ing were expressed as percentages in relation to the control non-
glycated BSA-treated cells. The values shown are the mean ± SD
(n ⳱ 3). *p < 0.05. (B) Intracellular ROS generation. MCSs were
seeded as shown in Fig. 3 and cultured with MSCGM containing
100 ␮g/ml of nonglycated BSA (open bars), AGE-2 (gray bars), or
AGE-3 (filled bars) in the presence or absence of a neutralizing
anti-RAGE anti-serum (0.1% in concentration) for 24 h. The
measured ROS values were expressed as percentages in relation
to the control nonglycated BSA-treated cells. Data are shown as
the mean ± SD (n ⳱ 6). *p < 0.05. These data are representative
of one of two independent experiments performed under the
same conditions.
were visualized by oil red O staining. Treatment with
AGE-2 or AGE-3 reduced the accumulation of lipid drop-
lets in MSCs (Fig. 6A). Consistently, reduced LPL expres-
sion and intracellular TG content were revealed in cells
treated with AGEs, and the addition of anti-RAGE anti-
body did not influence AGE-mediated inhibition of adipo-
genesis (Figs. 6B and 6C).
KUME ET AL.
FIG. 6. Effects of AGEs on adipogenic differentiation of MSCs.
(A) Oil red O staining. MSCs were induced adipogenic differen-
tiation for 3 weeks by treatment with 100 ␮g/ml of nonglycated
BSA, AGE-2, or AGE-3. (B) ELISA for lipoprotein lipase (LPL)
concentration and (C) the measurement of intracellular triglycer-
ide (TG). MSCs were induced to adipogenic differentiation by
treatment with 100 ␮g/ml of nonglycated BSA (open bar), AGE-2
(gray bars), or AGE-3 (filled bars) in the presence or absence of
a neutralizing anti-RAGE antiserum (0.1% in concentration).
Data are shown as the mean ± SD (n ⳱ 6). *p < 0.05; **p < 0.01;
ns, not statistically significant. These data are representative of
one of two independent experiments performed under the same
conditions.
Effects of AGEs on chondrogenic differentiation
of MSCs
As a result of chondrogenic differentiation, MSCs
formed a cluster of hypertrophic polygonal cells, within
which proteoglycan deposition was apparent by staining
with the safranin O method. However, only a faint staining
was found in AGE-2– or AGE-3–treated cells (Fig. 7A).
ELISA showed that the production of type II collagen, a
chondrocyte-specific matrix component, was reduced al-
most by one-half by treatment with theses AGEs, and this
reduction was partially recovered by addition of anti-
RAGE antibody by 20% in AGE-2–treated cells or by 30%
in AGE-3–treated cells (Fig. 7B).
Effects of AGEs on osteogenic differentiation
of MSCs
Under osteogenic stimulation, bone nodule formation
was seen in the control culture, but very little was observed
in AGE-treated cells (Fig. 8, left panels). Von Kossa stain-
EFFECTS OF AGES ON MESENCHYMAL STEM CELLS
FIG. 7. Effects of AGEs on chondrogenic differentiation of
MSCs. (A) Safranin O staining. MCSs were induced chondrogenic
differentiation for 3 weeks by addition of 100 ␮g/ml of nongly-
cated BSA, AGE-2, or AGE-3. (B) ELISA for type II collagen
synthesis. MCSs were induced to chondrogenic differentiation by
addition of 100 ␮g/ml of nonglycated BSA (open bar), AGE-2
(gray bars), or AGE-3 (filled bars) in the presence or absence of
a neutralizing anti-RAGE antiserum (0.1% in concentration).
The values of A490 are shown as the mean ± SD (n ⳱ 6). *p <
0.05; **p < 0.01; ns, not statistically significant. These data are
representative of one of two independent experiments performed
under the same conditions.
ing revealed a large mass of calcium deposition in accor-
dance with the bone nodule formation in controls; however,
in AGE-treated cells, the loss of mineralization was ob-
served (Fig. 8, right panels). The intracellular calcium con-
tents of AGE-2– or AGE-3–treated cells were increased
1.3-fold in each condition (p < 0.05), and these increases
were completely inhibited by the neutralizing anti-RAGE
antibody (Fig. 9A). The activity of bone-specific ALP was
upregulated 2.7-fold in AGE-2–treated cells (p < 0.01) and
3.3-fold in AGE-3–treated cells (p < 0.01), and these up-
regulations were partially recovered by the neutralizing
anti-RAGE antibody (Fig. 9B). On the other hand, type I
collagen synthesis was downregulated by about 20–30% in
both AGE-2– and AGE-3–treated cells (p < 0.01), and
these downregulations were slightly but significantly recov-
ered by the neutralizing anti-RAGE antibody (p < 0.05; Fig.
9C). In addition, we performed time-course analysis for
osteogenic differentiation. In control cells (untreated cells
and control-BSA–treated cells), intracellular calcium con-
tent increased linearly with time in culture, and upregula-
tion of ALP activity was temporarily induced in association
with elevated type I collagen synthesis. In AGE-2– and
AGE-3–treated cells, intracellular calcium content and
ALP activity were higher than those in control cells at the
end of the culture period; however, type I collagen synthesis
remained at a low level (Fig. 10).
1653
DISCUSSION
AGEs on MSCs growth regulation
It has been shown that AGEs inhibit cell growth in sev-
eral cell lines. In a previous study, we showed that pericyte
loss is an important aspect of the early stage of diabetic
retinopathy, and AGE-2 inhibits proliferation of cultured
bovine retinal pericytes in addition to promoting apoptosis
through the downregulation of expression ratio of bcl-2/
bax.(34) Apoptosis of retinal pericytes has been shown to be
associated with intracellular diacylglycerol/ceramide pro-
duction and activation of the caspase-10 pathway.(35) In rat
Schwann cells, reduced cell replication and induction of
apoptosis by AGE-2 and AGE-3, but not by AGE-1, have
been shown, suggesting that AGEs may be implicated in
diabetic neuropathy.(36) On the other hand, AGEs have the
ability to promote proliferation of cells under specific con-
ditions. In bovine retinal endothelial cells, AGEs enhance
the ROS generation, protein kinase C activation, and vas-
cular endothelial cell growth factor expression, implicating
the excess vascular formation in diabetic retinopathy.(37,38)
The AGE-RAGE interaction mediates proliferation of vas-
cular smooth muscle cells, which plays a role in neointimal
hyperplasia.(39–41) Furthermore, it has been reported that
low concentrations (<1 ␮M) of AGEs stimulate mesangial
cell proliferation, whereas higher concentrations (>10 ␮M)
reduce it.(42) In osteoblastic cell lines, AGE-collagen has
been reported to have the opposite effect on the prolifera-
tion and the number of surviving cells, dependent on the
stage of osteoblastic differentiation.(43) In this study, we
observed that relatively higher doses (1–100 ␮g/ml) of ex-
tracellular AGE-2 and AGE-3 attenuated MSCs with inhi-
bition of proliferation and increased apoptosis (Figs. 1 and
2). Because the bimodal effects of AGEs presumably vary
according to the source of AGEs, the type of cells, and
culture conditions, further study will be needed to clarify
the details of the mechanism of AGE-mediated growth
control of MSCs, particularly studies modeling the in vivo
microenvironment for the developmental or disease pro-
cesses.
AGE–RAGE interaction and its relationship with
ROS in MSCs
Among various subtypes of AGEs, it has been shown
that AGE-2 (glyceraldehyde-derived AGEs) and AGE-3
(glycolaldehyde-derived AGEs) are the main structures of
AGEs that are detectable in the serum of diabetic patients,
and both display highly toxic bioactivities on vascular cells,
mesangial cells, Schwann cells, melanoma cells, and cortical
neurons.(44) In this study, we consistently found that
AGE-2 and AGE-3, but not control BSA or AGE-1 (glu-
cose-derived AGE), significantly suppressed the growth of
MSCs. Our results may further support the idea that
AGE-2 and AGE-3 function as toxic AGEs and play a
central role in the pathophysiological processes associated
with AGEs formation.(44) AGEs have been considered as a
possible contributor mainly in the diabetic vascular compli-
cations, and now, AGEs play a central role in the patho-
physiological processes not only in diabetic but also in non-
1654
diabetic patients such as Alzheimer’s disease and chronic
alcoholism.(44) Collagenous proteins are major structural
component of connective tissues and play important roles
in the regulation of cellular function, suggesting that AGE-
modified collagen affects MSCs proliferation and differen-
tiation.(20) In this study, we focused on the effects of AGE-2
and AGE-3 that have been recognized as toxic(44); how-
ever, further study is needed to understand the effects of
AGE-modified collagen on the cellular function of MSCs.
AGE-2 and AGE-3 may be recognized by RAGE, and
the neutralizing anti-RAGE antibody successfully abolishes
AGE-mediated cellular events.(28,33) It has been shown that
the cellular event induced by AGE–RAGE interaction is
mediated by intracellular ROS generation.(33,45) In this
study, we first showed that RAGE is expressed by MSCs
(Fig. 4). The addition of anti-RAGE neutralizing antibody
completely inhibited the ROS generation induced by AGEs
(Fig. 5). Thus, the ROS generation observed in MSCs may
be largely dependent on RAGE signaling. On the other
hand, the treatment with neutralizing anti-RAGE antibody
partially recovered the growth inhibition of MSCs. There
are several molecules that have been reported to act as
AGE receptors in addition to RAGE, including OST-48
(ARE-R1)/80K-H/galectin-3,(46) scavenger receptor class
AI/AII(SR-A),(47) scavenger receptor class B type I (SR-
BI),(48) CD36,(49) lectin-like oxidized low density lipopro-
tein receptor-1,(50) and FEEL-1,-2.(51) It may be possible
that AGEs regulate the growth of MSCs in both RAGE-
dependent and RAGE-independent manners. We found
that AGE-2 and AGE-3 upregulated RAGE expression
(Fig. 4C). It has been shown that AGEs themselves have
KUME ET AL.
FIG. 8. Effects of AGEs on osteogenic dif-
ferentiation of MSCs (Part I). Phase-contrast
microscopy (left panels) and von Kossa stain-
ing (right panels). MSCs were induced osteo-
genic differentiation for 3 weeks by treat-
ment with 100 ␮g/ml of nonglycated BSA,
AGE-2, or AGE-3. The arrows indicate bone
nodule formation, and the high magnifica-
tion inset is also shown.
the ability to stimulate RAGE expression in osteoblastic
cell lines.(22) Augmentation of RAGE expression by such a
positive feedback mechanism may enhance the biological
significance of AGEs, which may exacerbate the disease
process. Further studies are needed to determine the ex-
pression pattern of AGE receptors and their downstream
signaling in many cell types, including MSCs.
Differentiation of MSCs and AGEs
There has been little information about the influence of
AGEs on the differentiation process of MSCs, and this
study was the first to report that AGE-2 and AGE-3 inter-
fered with differentiation of MSCs into adipocytes, chon-
drocytes, and osteocytes. The treatment with anti-RAGE
neutralizing antibody revealed that AGE-RAGE interac-
tion was involved in chondrogenic and osteogenic differen-
tiation but not in adipogenic differentiation of MSCs (Figs.
6–8). It has been reported that a series of gene expressions
are involved in adipogenic differentiation of human
MSCs,(52) and that CD36, a cell surface glycoprotein, func-
tions as a receptor for AGEs in the adipogenic differentia-
tion of mouse 3T3-L1 cells.(53) Although we do not provide
direct evidence, AGE receptors other than RAGE may
contribute to the modification of adipogenesis. Although
the clinical implication of the impact of AGEs on adipo-
genesis is still unclear, we conjecture that AGEs may play a
role in soft tissue remodeling, obesity, or insulin-resistance,
through the functional alteration of MSCs.
MSCs have been shown to be present in adult human
articular cartilage other than bone marrow tissue.(54) Our in
EFFECTS OF AGES ON MESENCHYMAL STEM CELLS
FIG. 9. Effects of AGEs on osteogenic differentiation of MSCs
(Part II). (A) Intracellular calcium content, (B) Bone ALP activ-
ity, and (C) type I collagen synthesis. MSCs were induced osteo-
genic differentiation for 3 weeks by addition of 100 ␮g/ml of non-
glycated BSA (open bar), AGE-2 (gray bars), or AGE-3 (filled
bars) in the presence or absence of a neutralizing anti-RAGE
anti-serum (0.1% in concentration) and analyzed. Data are shown
as the mean ± SD (n ⳱ 6). *p < 0.05; **p < 0.01. These data are
representative of one of two independent experiments performed
under the same conditions.
vitro data may support the idea that excess accumulation of
AGEs inhibits the intrinsic repair capacity based on the
chondrogenesis of MSCs. Consistent with this hypothesis, it
has been reported that an increase in AGE levels nega-
tively affects the proteoglycan synthesis and degradation of
articular cartilage in OA patients.(55) Further studies will be
1655
FIG. 10. Effects of AGEs on osteogenic differentiation of MSCs
(Part III). (A) Intracellular calcium content, (B) Bone ALP ac-
tivity, and (C) type I collagen synthesis. MSCs were induced os-
teogenic differentiation for 3 weeks in the presence or absence
(untreated) of 100 ␮g/ml of nonglycated BSA (control-BSA),
AGE-2, or AGE-3. Samples were collected every week and were
analyzed. Data are shown as the mean ± SD (n ⳱ 3). *p < 0.05;
**p < 0.01 against the value obtained in untreated cells. These
data are representative of one of two independent experiments
performed under the same conditions.
needed to determine whether there is a causative link be-
tween accumulation of AGEs and the disease process and
to develop the appropriate therapies.
Several steps have been identified in osteogenesis, such
as cell proliferation, extracellular matrix production, and
mineralization.(17,56) Proliferation of osteoblasts with the
production of type I collagen and fibronectin occurs ini-
tially, followed by enhanced expression of ALP and calcium
deposition.(56) It has been reported that the enhanced ex-
pression of ALP metabolizes calcium phosphate into in-
soluble phosphate salts, thereby mediating the calcifica-
tion.(57) The pattern of the changes in osteogenic markers
in control MSCs was almost compared with those described
in the previous report using more mature osteoblastic cells
(Figs. 8–10).(56–58) In this study, AGE-2 and AGE-3 inhib-
ited bone nodule formation (Fig. 8). It has been indicated
that von Kossa staining is not a sufficient marker for the in
1656
vitro mineralization of osteoblastic cells.(58) However, we
also observed changes in the other mineralization markers
such as ALP between controls and AGE-treated cells (Figs.
9 and 10). Thus, dystrophic mineralization may occur in
AGE-2– and AGE-3–treated cells. Interestingly, intracellu-
lar calcium contents and ALP activity were rather increased
in AGE-2– and AGE-3–treated cells at the later culture
period, without successful induction of type I collagen (Figs.
9 and 10). The treatment with anti-RAGE antibody par-
tially recovered the AGE-mediated inhibition of osteogen-
esis, and thus AGE-RAGE interaction may be involved in
the intracellular mechanism (Fig. 9). These data may sup-
port the hypothesis that AGE-2 and AGE-3 exert harmful
influence on the differentiation of MSCs through the
AGE–RAGE interaction.
Several specific signaling have been identified in osteo-
genesis, such as MAP kinase(59) and Notch(60) signaling.
Bone-specific gene expression such as osteocalcin has been
influenced by specific transcriptional factors such as Runx2/
Cbfa1.(61) Further studies would be needed to determine
how AGEs influence the differentiation of MSCs in terms
of intracellular signaling pathways. It has been shown that
aldehyde dehydrogenase (ALDH) activity is elevated in
hematopoietic stem cells.(62) Betaine aldehyde dehydroge-
nase (ALDH-9) is able to catalyze the oxidation of 2-oxo-
aldehyde methylglyoxal, a precursor of AGEs.(63) Al-
though the role of ALDH-like activity in MSCs has not
been clearly described, further study should be needed to
identify the endogenous regulatory factors for AGE-
mediated cellular response in these cells.
In conclusion, in this study, AGEs, especially AGE-2 and
AGE-3, attenuated MSC mass in association with an inhi-
bition of their proliferation and an increase in the number
of apoptotic cells. These AGEs also prevented MSCs from
differentiating into adipocytes, chondrocytes, and osteo-
cytes. Thus, we showed an impact of AGEs in the cellular
behavior of MSCs, which may further indicate the delete-
rious effects of AGEs in the pathogenesis of musculoskel-
etal diseases in aged and diabetic patients.
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chromatin remodeling and co-regulatory protein interactions. Address reprint requests to:
Connect Tissue Res 44(Suppl 1):141–148. Seiya Kato, MD, PhD
62. Hess DA, Meyerrose TE, Wirthlin L, Craft TP, Herrbrich PE, Department of Pathology
Creer MH, Nolta JA 2004 Functional characterization of Kurume University School of Medicine
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67 Asahi-machi
lated according to aldehyde dehydrogenase activity. Blood
104:1648–1655. Kurume 830-0011, Japan
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E-mail: seikato@med.kurume-u.ac.jp
lism and diabetic complications: Roles of aldose reductase, gly-
oxalase-I, betaine aldehyde dehydrogenase and 2-oxoaldehyde Received in original form November 30, 2004; revised form May
dehydrogenase. Chem Biol Interact 143-144:341–351. 11, 2005; accepted May 20, 2005.