Extracted Text
Jeong_et_al-2013-STEM_CELLS.pdf
REGENERATIVEMEDICINE
Thrombospondin-2 Secreted by Human Umbilical Cord
Blood-Derived Mesenchymal Stem Cells Promotes
Chondrogenic Differentiation
SANGYOUNGJEONG,
a,b
DONGHYUNKIM,
a
JUEUNHA,
a
HYEJINJIN,
a
SOON-JAEKWON,
a
JONGWOOKCHANG,
a
SOOJINCHOI,
a
WONILOH,
a
YOONSUNYANG,
a
GONHYUNGKIM,
c
JAESUNGKIM,
d
JUNG-ROYOON,
e
DONGHYUNGCHO,
b
HONGBAEJEON
a
a
Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Seoul, Republic of Korea;
b
Graduate School of
East-West Medical Science, Kyung Hee University, Yongin, Gyeonggi-Do, Republic of Korea;
c
Laboratory of
Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic
of Korea;
d
Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul,
Republic of Korea;
e
Department of Orthopedic Surgery, Seoul Veterans Hospital, Seoul, Republic of Korea
Key Words:Human umbilical cord blood-derived mesenchymal stem cells•Paracrine action•Osteoarthritis•Secretome•Synovial
fluid•Thrombospondin-2
ABSTRACT
Increasing evidence indicates that the secretome of mesen-
chymal stem cells (MSCs) has therapeutic potential for the
treatment of various diseases, including cartilage disorders.
However, the paracrine mechanisms underlying cartilage
repair by MSCs are poorly understood. Here, we show that
human umbilical cord blood-derived MSCs (hUCB-MSCs)
promoted differentiation of chondroprogenitor cells by
paracrine action. This paracrine effect of hUCB-MSCs on
chondroprogenitor cells was increased by treatment with
synovial fluid (SF) obtained from osteoarthritis (OA)
patients but was decreased by SF of fracture patients, com-
pared to that of an untreated group. To identify paracrine
factors underlying the chondrogenic effect of hUCB-MSCs,
the secretomes of hUCB-MSCs stimulated by OA SF or
fracture SF were analyzed using a biotin label-based anti-
body array. Among the proteins increased in response to
these two kinds of SF, thrombospondin-2 (TSP-2) was spe-
cifically increased in only OA SF-treated hUCB-MSCs. In
order to determine the role of TSP-2, exogenous TSP-2 was
added to a micromass culture of chondroprogenitor cells.
We found that TSP-2 had chondrogenic effects on chondro-
progenitor cells via PKCa, ERK, p38/MAPK, and Notch
signaling pathways. Knockdown of TSP-2 expression on
hUCB-MSCs using small interfering RNA abolished the
chondrogenic effects of hUCB-MSCs on chondroprogenitor
cells. In parallel with in vitro analysis, the cartilage regen-
erating effect of hUCB-MSCs and TSP-2 was also demon-
strated using a rabbit full-thickness osteochondral-defect
model. Our findings suggested that hUCB-MSCs can stimu-
late the differentiation of locally presented endogenous
chondroprogenitor cells by TSP-2, which finally leads to
cartilage regeneration.STEMCELLS2013;31:2136–2148
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
Cartilage is a highly differentiated and avascular tissue and
consequently has a low self-regeneration capacity; full regener-
ation of damaged cartilage, such as in osteoarthritis (OA), is
therefore challenging. Many research groups have attempted to
increase the regeneration potential of damaged cartilage using
mesenchymal stem cells (MSCs) including our phase I/IIa clin-
ical trial using MSCs for cartilage repair (Clinical Trials Gov
Identifier: NCT01733186) under approval of the Food and
Drug Administration.
MSCs can be isolated from various tissues and can differ-
entiate into chondrogenic lineage cells in vitro and in vivo
[1,2]. Human umbilical cord blood-derived MSCs (hUCB-
MSCs) are an alternative stem cell source with several advan-
tages, including noninvasive collection methods, hypoimmuno-
genicity, superior tropism, and differentiation potential [3–5].
Many studies have reported the therapeutic activity of the
MSC secretome. The paracrine effects of MSCs promote
Author contributions: S.Y.J.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript
writing; D.H.K., J.E.H., and H.J.J.: collection and/or assembly of data; S.-J.K., J.W.C., W.I.O., and D.H.C.: data analysis and interpreta-
tion; S.J.C.: provision of study and patient material; Y.S.Y.: data analysis and interpretation and financial support; G.H.K.: collection
and/or assembly of data and data analysis and interpretation; J.S.K.: conception and design, collection and/or assembly of data, and data
analysis and interpretation; J.-R.Y.: provision of study and patient material; H.B.J.: conception and design, data analysis and interpreta-
tion, manuscript writing, and final approval of manuscript.
Correspondence: Hong Bae Jeon, Ph.D., Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul 137–874, Republic of Korea. Tele-
phone:182-2-3465-6772; Fax:182-2-475-1991; e-mail: jhb@medi-post.co.kr Received November 29, 2012; accepted for publication
May 20, 2013; first published online inSTEMCELLSEXPRESSJuly 10, 2013.VCAlphaMed Press 1066-5099/2013/$30.00/0 doi: 10.1002/
stem.1471
STEMCELLS2013;31:2136–2148 www.StemCells.com
fracture healing and restore new bone formation [6], and fac-
tors released from MSCs recruit macrophages and endothelial
lineage cells into the wound, thus enhancing wound healing
[7]. Furthermore, autologous MSC transplantation attenuates
left ventricular remodeling and improves cardiac performance
via the paracrine action of the engrafted cells [8]. Moreover,
we have shown that hUCB-MSCs reduce neuronal cell death
by secreting galectin-3 [9] and decrease Abplaques by induc-
ing neprilysin [10]. Such paracrine actions of MSCs can
affect cartilage repair [11–15].
Despite these possible beneficial effects, the mechanism
by which MSCs and their secreted factors influence cartilage
repair remains unclear, necessitating analysis of the hUCB-
MSC secretome. However, in clinical trials, the effector mole-
cules produced by MSCs under in vitro conditions are often
not related to the therapeutic effects observed under clinical
conditions [16]. Such discrepancies between in vitro and in
vivo effects of MSCs may result from their interactions with
different microenvironments. Synovial fluid (SF) acquired
from osteoarthritic joints (OA SF) can represent the in vivo
disease microenvironment [17,18]; OA SF modulates MSC
activity in vitro in a manner representative of the clinical con-
dition [19]. Therefore, experiments that closely mimic disease
microenvironments, such as OA SF, should be useful.
Here, we demonstrated a chondrogenic differentiation effect
via the paracrine action of hUCB-MSCs using a coculture sys-
tem of chondrogenic progenitor cells (CPC) derived from
mouse limb buds. To determine whether hUCB-MSC paracrine
molecules responded to SF, we analyzed the secretome profile
of media from hUCB-MSCs conditioned with SF from OA and
fracture patients, using a biotin label-based antibody array. One
of the differentially expressed proteins, thrombospondin-2
(TSP-2), was selected and its effect on chondrogenic differen-
tiation of progenitor cells was validated using micromass cul-
ture and an in vivo osteochondral-defect model. This is the
first report to identify factors secreted from hUCB-MSCs in
response to OA SF and to demonstrate that TSP-2 is a hUCB-
MSC paracrine factor with chondrogenic effects.
MATERIALS AND METHODS
Culture of hUCB-MSCs
This study was approved by the Institutional Review Board of
MEDIPOST Co., Ltd. Neonatal hUCB was collected from umbili-
cal veins, with informed maternal consent. Mononuclear cells
were isolated from hUCB by centrifugation on a Ficoll-Hypaque
gradient (density: 1.077 g/cm
3
; Sigma, St. Louis, MO). Separated
mononuclear cells were washed, suspended ina-minimum essen-
tial medium (a-MEM; Gibco, Carlsbad, CA, http://www.invitro-
gen.com) supplemented with 10% (v/v) fetal bovine serum
(FBS; Gibco) and 50mg/mL gentamicin (Gibco), and seeded at
a concentration of 5310
5
cells per centimeter square in cul-
ture flasks. Cultures were maintained at 37
C in a humidified
5% CO
2atmosphere with a twice-weekly medium change.
After 1–3 weeks, when the monolayer of fibroblast-like adher-
ent cell colonies had reached 80% confluence, the cells were
detached with TrypLE Express (Gibco), washed, resuspended
in culture medium (a-MEM supplemented with 10% FBS and
50mg/ml gentamicin), and subcultured. In all experiments,
hUCB-MSCs used were at passage 6.
Acquisition and Treatment of Cells with SF
All patients provided appropriate written informed consent. SF
was withdrawn from the knee joints of four female and two male
patients (mean [SD]: 60.5 [11.5] years) diagnosed with OA, grade
4, and a knee joint of two female patients (age: 31 and 36 years),
each of which had fractured a knee in an accident. Radiographs
were taken of all OA knees and scored for Kellgren and Law-
rence (KL) grading (0–4). SF samples were centrifuged at 300g
for 5 minutes to sediment macromolecules and cell debris, and
the supernatants were stored at280
C until required for analysis.
hUCB-MSCs were cultured ina-MEM containing 10% (v/v) FBS
and 50mg/mL gentamicin. For the antibody array, the culture
medium from hUCB-MSCs at 80% confluence was replaced with
10% (v/v) SF diluted with serum-freea-MEM containing 50mg/
mL gentamicin, and the cells were cultured for 6 hours. To elimi-
nate SF contamination and to confirm the increased expression of
TSP-2, culture medium from hUCB-MSCs at 80% confluence
was replaced with 0.2% (v/v) SF diluted with serum-freea-MEM
containing 50mg/mL gentamicin, and cells were cultured for 6,
12, and 24 hours.
Micromass Culture of Chondroprogenitor Cells
Chondroprogenitor cells were isolated from the limb buds of 11.5
dpc ICR mouse embryos and maintained as micromass cultures
to induce chondrogenesis. Isolated chondroprogenitor cells were
suspended, without expansion (passage 0), in Dulbecco’s modi-
fied Eagle’s medium (DMEM, Gibco), containing 2% (v/v) FBS
and 50mg/mL gentamicin at 2.0310
7
cells per milliliter and
were spotted as 15-mL drops onto culture dishes to induce chon-
drogenesis for 6 days. Human recombinant TSP-2 (R&D Sys-
tems, Minneapolis, MN, http://www.rndsystems.com) was
prepared and applied to chondroprogenitor cells in serum-free
DMEM. As positive control, insulin-like growth factor-1 (100 ng/
mL; Merck Millipore, Billerica, MA, http://www.millipore.com)
was added to micromass culture medium [20].
Chondrogenesis was determined by staining of sulfated pro-
teoglycans and examining the expression of collagen type II,
aggrecan, hyaluronan and proteoglycan like protein 1 (HAPLN1),
and Sox-9 using Western blotting. Accumulation of sulfated pro-
teoglycans was detected by Alcian blue staining. During cocul-
ture, hUCB-MSCs or human embryonic kidney (HEK-293) cells
were cultured in the upper phase of transwells. hUCB-MSCs or
HEK-293 (9310
4
cells per 2 milliliter) were added to the upper
chamber, and 2 mL of DMEM containing 10% serum was added
to the lower chamber. After 24 hours of incubation at 37
C and
5% CO
2, cells on the upper surface of the chamber (which had
not penetrated the insert) were applied to progenitor cells in a
different culture plate. Alcian blue-stained cells were extracted
and quantified by measuring absorbance at 590 nm.
Gross Findings from Osteochondral-Defect Animal
Model
The animal experiment protocol was approved by the Institutional
Animal Care and Use Committee of Chungbuk National Univer-
sity. Thirty 10-week-old male New Zealand White (NZW) rab-
bits, each weighing 2–2.5 kg, were used. Knee joints of rabbits
were sterilely draped and were opened through a parapatellar
arthrotomy with general anesthesia. The patella was dislocated
laterally, and full-thickness articular osteochondral defects (5-mm
diameter; 5-mm depth) were induced in the trochlear groove by
carefully perforating the respective areas on the cancellous bone
using a biopsy punch. After elimination of cartilage, bone frag-
ments, and thrombi, the border of the perforated site was trimmed
using a surgical knife and cleansed from blood. Each group was
untreated, transplanted with hyaluronic acid (HA), HEK-293,
hUCB-MSCs, TSP-2 protein, and hUCB-MSCs treated with TSP-
2 small interfering RNA (siRNA), respectively. Mixtures of
hUCB-MSCs (1310
6
cells per 200mL) or HEK-293 (1310
6
cells per 200mL) with 4% sodium HA gel composite were
applied into the area of the full-thickness defect using a syringe.
TSP-2 (25mg/200mL) was also administered into the defect area
along with 4% sodium HA gel composite. The untreated group
received only a osteochondral defect. Finally, in all animals, the
patellar retinaculum and the overlaying skin were sutured. An
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Figure 1.Enhanced differentiation of chondroprogenitor cells by coculture with hUCB-MSCs. Mouse limb bud chondroprogenitor cells were
prepared from 11.5 dpc mouse embryos and maintained as micromass culture in 2% fetal bovine serum-containing medium for 6 days. hUCB-
MSCs and chondroprogenitor cells were separately cocultured by a transwell approach in a culture plate. Effects of hUCB-MSCs on differentia-
tion of chondroprogenitor cells were monitored by synthesis of sulfated proteoglycans and expression of chondrogenic markers.(A):Chondropro-
genitor cells that had differentiated into mature cartilage were stained by Alcian blue dye.(B):The stained dye was extracted and quantified by
absorbance at 590 nm.(C):The expression of the chondrogenic markers, collagen type II, aggrecan, HAPLN1, and chondrogenic transcription
factor, Sox-9 was determined by Western blotting and quantified(D)using ImageJ software.(E):The photo of chondrogenic differentiated
ATDC5 pellets was represented.(F):Relative size difference among the pellets was measured using IMT i-SolutionTM software.(G):The frozen
pellets in OCT compound were cut and stained with Safranin O and anticollagen type II antibody.(H):The sulfated glycosaminoglycan contents
were extracted and determined. Data are shown as the mean (SD) of at least six independent experiments. *,p<.05. Scale bars5250mm.
Abbreviations: hUCB-MSC, human umbilical cord blood-derived mesenchymal stem cell; OA, osteoarthritis; HAPLN1, hyaluronan and proteo-
glycan link protein 1; SF, synovial fluid.
but expressions of cartilage-specific markers were lower than
those in the presence of TSP-2 (Fig. 4E–4H). These results
clearly demonstrated that TSP-2 has chondrogenic effects, which
are mediated through PKCa/ERK, p38/MAPK, and Notch sig-
naling pathways.
Knockdown of TSP-2 Abolishes Chondrogenic
Effects of hUCB-MSC on Differentiation
of Chondroprogenitor Cells
Since we had observed chondrogenic effects of TSP-2 in
hUCB-MSCs, we attempted to block expression of TSP-2 for
further validation. After TSP-2 siRNA treatment, expression
of TSP-2 was completely blocked (Fig. 5E) and knockdown
of TSP-2 was confirmed up to 28 days (Supporting Informa-
tion Fig. 4). siRNA-mediated knockdown of TSP-2 expression
led to decreased synthesis of sulfated proteoglycans (Fig. 5A,
5B) and expression of collagen type II, aggrecan, HAPLN1,
and Sox-9 (Fig. 5C, 5D), indicating reduced chondrogenesis.
However, use of HEK-293 cells as non-stem cells in coculture
did not replicate the findings in hUCB-MSCs (Fig. 5A–5D).
Collectively, these results suggested that the chondrogenic
effects of hUCB-MSCs are mediated via TSP-2.
Gross Findings and Histological Observations
of Effects of hUCB-MSCs and TSP-2 in an
Osteochondral-Defect Animal Model
To further validate the role of TSP-2 in cartilage regeneration
in an in vivo system, we made osteochondral defects in the
trochlear groove of rabbits and applied either hUCB-MSCs or
only TSP-2 into the defects. In addition, given our previous
in vitro data that hUCB-MSCs can promote differentiation of
endogenous chondroprogenitor cells, we used a full-thickness
cartilage defect model that exposed chondroprogenitor cells
from the marrow [28]. At 10 weeks after surgery, the gross
morphology of joints was observed and sections were stained
with hematoxylin and eosin and Safranin O. Upon gross
examination, the margins of the defects of untreated or HA-
treated groups were clearly recognizable and their surfaces
were irregular (Fig. 6Aa, 6Ab). The surfaces of defects trans-
planted with HEK-293 were depressed and most parts of the
defects were not filled with any tissue (Fig. 6Ac). The repara-
tive tissues of untreated and HA-treated groups were filled
with fibrous tissue and remained concave (Fig. 6Ag, m, 6Ah,
n). In the HEK-293-treated group, thin fibrous tissue was
observed on the subchondral bone (Fig. 6Ai, 6Ao). In con-
trast, the hUCB-MSC-treated group showed good lateral inte-
gration, macroscopically (Fig. 6Ae), and a hyaline-like
cartilage matrix was evident with Safranin O. The tissue was
hyaline-like, with good integration, thickness, and surface reg-
ularity. An enlarged image of the regenerated tissue showed
the lacunae structure (Fig. 6Ak, 6Aq). There was marked
improvement in the quality of the repaired tissue seen in the
hUCB-MSC-treated group compared with the untreated, HA,
and HEK-293-treated groups.
The surfaces of regenerated tissue in the TSP-2-treated
group were mostly not depressed, and the defects were cov-
ered with white opaque tissue compared with the hUCB-
MSC-treated group (Fig. 6Ad). Histologically, a magnified
view of the repaired tissue represented a partially hyaline-like
Figure 3.TSP-2 expression by hUCB-MSCs responded to treatment with SF from fracture and OA patients.(A):Western blots of cell lysates
from hUCB-MSCs treated with 10% SF from two fracture patients and four OA patients for 6 hours.(B):ELISA analysis on expression of TSP-
2 in culture media from hUCB-MSCs treated with 0.2% SF samples from two fracture patients and four OA patients for 6, 12, and 24 hours.
(C):TSP-2 concentrations in supernatants of micromass culture media. Data are shown as the mean (SD) of at least four independent experi-
ments. *,p<.05. Abbreviations: hUCB-MSC, human umbilical cord blood-derived mesenchymal stem cell; OA, osteoarthritis; SF, synovial fluid;
TSP-2, thrombospondin-2.
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Figure 4.Effects of exogenous TSP-2 on differentiation of chondroprogenitor cells.(A):Alcian blue staining of mouse limb bud chondroproge-
nitor cells which had been maintained as micromass culture for 6 days in the presence of TSP-2 (0.1–100 ng/mL) under serum-free conditions.
IGF-1 (100 ng/mL) was used as positive control for induction of chondrogenic differentiation.(B):Alcian blue-stained cells were extracted and
quantified by measuring absorbance.(C):Expression levels of collagen type II, aggrecan, HAPLN1, and Sox-9 protein were determined by West-
ern blotting;b-actin was used as loading control.(D): Expression of collagen type II protein was quantified by ImageJ software.(E):Chondro-
progenitor cells were maintained as micromass culture up to 6 days in the absence or presence of TSP-2 (100 ng/mL). Cells were concurrently
exposed to 1mM Go to inhibit PKCa,10mM of PD to inhibit ERK, 10mM of SB to inhibit p38/MAPK, or 50 nM DAPT to inhibitc-secretase.
Accumulation of sulfated proteoglycans was detected by Alcian blue staining.(F):Alcian blue-stained cells were extracted and quantified by
measuring absorbance.(G):Effects of inhibitors on expression of collagen type II, aggrecan, HAPLN1, and Sox-9 protein were detected by
Western blotting.b-actin was used as loading control.(H):Expressions of collagen type II, aggrecan, HAPLN1, and Sox-9 protein were quanti-
fied by ImageJ software. Data are presented as the mean (SD) result from four independent experiments. *,p<.05. Abbreviation: DAPT,N-[N-
(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycinet-butyl ester; HAPLN1, Hyaluronan and proteoglycan link protein 1; Go, Go6976; IGF-1,
insulin-like growth factor-1; PD, PD98059; SB, SB203580; TSP-2, thrombospondin-2.
structure (Fig. 6Aj, 6Ap). In defects transplanted with hUCB-
MSCs treated with TSP-2 siRNA, the surfaces were irregular
(Fig. 6Af). Defects showed dense fibrous tissue rather than
cartilage tissue (Fig. 6Al, 6Ar). Upon immunohistochemical
analysis, untreated, HA-treated, and HEK-293-transplanted
groups rarely expressed collagen type II (Fig. 6As–6Au),
whereas collagen type II was distributed throughout the neo-
cartilage in the hUCB-MSC-treated group (Fig. 6Aw). Defects
treated with TSP-2 were well repaired in deep zone of carti-
lage, but the superficial zone was partly unrepaired (Fig.
6Av). However, collagen type II expression in defects trans-
planted with hUCB-MSCs treated with TSP-2 siRNA was
observed at periphery of the defect sites (Fig. 6Ax). Histologi-
cal observations were quantified according to the modified
O’Driscoll scoring scale [21]. The mean modified O’Driscoll
scores of each experimental group correlated well with both
macroscopic and histological analyses (Fig. 6B). Both TSP-2-
and hUCB-MSC-treated groups had higher scores than those
of the control groups, including the untreated, HA-treated,
and HEK-293-treated groups (p<.01). The TSP-2-treated
group achieved a lower score compared with the hUCB-
MSC-treated group (p<.05). The score in the hUCB-MSCs
Figure 5.Reduced differentiation of chondroprogenitor cells by inhibition of TSP-2 in hUCB-MSCs. hUCB-MSCs were transfected for 24
hours with negative control siRNA (25 nM) or TSP-2 siRNA (25 nM). After a 24-hour incubation, transfected hUCB-MSCs were applied to
chondroprogenitor cells in a coculture system. Chondroprogenitor cells were cultured with hUCB-MSCs or HEK-293 (non-stem cells) cells up to
6 days in 2% fetal bovine serum-containing medium.(A):Chondroprogenitor cells stained with Alcian blue at day 6.(B):Alcian blue stain was
extracted from the cells and quantified by measuring absorbance.(C):Protein levels of collagen type II, aggrecan, HAPLN1, and Sox-9 were
determined by Western blotting;b-actin was used as loading control.(D):Expression of collagen type II protein was quantified by ImageJ soft-
ware.(E):TSP-2 concentrations were measured in the supernatants of each culture condition. Data are shown as the mean (SD) of at least four
independent experiments. *,p<.05. Abbreviations: hUCB-MSCs, human umbilical cord blood derived mesenchymal stem cells; HAPLN1, hya-
luronan and proteoglycan link protein 1; siRNA, small interfering RNA; TSP-2, thrombospondin-2.
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Figure 6.Macroscopic and histological analysis and immunostaining of hUCB-MSC-treated and TSP-2-injected knees in a full-thickness osteo-
chondral-defect rabbit model.(A):Six experimental groups, including an untreated group (n55), and groups treated with 4% HA (n55), a com-
posite of HEK-293 (5310
6
cells/ml) and 4% HA (n55), TSP-2 (25mg) and 4% HA (n55), a composite of hUCB-MSCs (5310
6
cells/ml)
and 4% HA (n55), and a composite of hUCB-MSCs treated with TSP-2 siRNA (5310
6
cells/ml) and 4% HA (n55) were used. At 10 weeks
postoperatively, animals were sacrificed and the defect area was observed and stained for analysis. Gross findings are shown in(A–F). Femoral
condyles were sectioned and stained with hematoxylin and eosin(G–L)and Safranin-O(M–R). For immunohistochemical analysis, collagen type
II was detected as brown color(S–X). The untreated group (saline treatment) showed poor healing (a, g, m, and s). The defects in the HA-treated
group were rarely recovered (b, h, n, and t). The defects treated with HEK-293 exhibited irregular surfaces similar to the HA-treated group (c, i,
o, and u). In the TSP-2-treated group, the regenerated tissue was slightly less recovered compared to the hUCB-MSCs-treated group but showed
better-integrated cartilage tissue than in the untreated, HA-treated, and HEK-293 groups (d, j, p, and v). The hUCB-MSCs-treated animals demon-
strated good defect filling, with a smooth surface and regenerated cartilage tissue (e, k, q, and w). The defects transplanted with hUCB-MSCs
treated with TSP-2 siRNA represented poorly regenerated tissue compared with TSP-2- and hUCB-MSCs-treated groups (f, l, r, and x). Scale
bars51 mm (G–X). (B): Using the modified O’Driscoll scoring scale, the integration rates were quantified in the six experimental groups at 10
weeks. Data are shown as the mean (SD) of each group (n55). Statistical significance was determined using Kruskal–Wallis test followed by
Mann–WhitneyUtest. *,p<.05 or **,p<.01. Abbreviations: hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells; HA,
Hyaluronate; siRNA, small interfering RNA; TSP-2, thrombospondin-2.
treated with TSP-2 siRNA decreased compared with that in
the hUCB-MSC-treated group (p<.01). Collectively, these
data indicated that hUCB-MSCs have cartilage regeneration
effects in an osteochondral defect model, which are mainly
mediated by the secreted TSP-2.
DISCUSSION
Here, we demonstrated that hUCB-MSCs promoted differen-
tiation of chondroprogenitor cells by paracrine action. This
chondrogenic effect was modulated by the surrounding envi-
ronment, such as OA or fracture SF. Through secretome array
analysis and in vitro evaluation, we found that hUCB-MSCs
secreted trophic factors that varied with the pathological con-
ditions, such as exposure to OA SF. Moreover, expression of
TSP-2 by hUCB-MSCs was specific to OA SF treatment and
enhanced cartilage regeneration in an osteochondral defect
animal model.
When MSCs are transplanted into cartilage lesions, they
are exposed to a complex microenvironment, including
inflammatory cytokines, proteases, and degraded extracellular
matrix secreted from damaged cartilage, which can modulate
paracrine activities of MSCs. For instance, the proinflamma-
tory cytokine, tumor necrosis factor-a, changes the secretome
of human adipose tissue-derived MSCs [29]. In this study,
OA SF-treated hUCB-MSCs increased the chondrogenic dif-
ferentiation of chondroprogenitor cells, compared to a non-
treated hUCB-MSC group, whereas fracture SF-treatment of
hUCB-MSCs decreased chondrogenic differentiation (Fig. 1).
Furthermore, secretome analysis of hUCB-MSCs induced by
these SFs resulted in distinct expression patterns (Fig. 2; Sup-
porting Information Table 2). These data indicated that the
pathological condition can modulate the paracrine action of
hUCB-MSCs. In elucidating this modulation, we found that,
among factors secreted from hUCB-MSCs, only TSP-2 was
specifically expressed upon OA SF-treatment (Fig. 3). Indeed,
TSP-2 levels also markedly increased in the SF of OA patient
two weeks after hUCB-MSCs transplantation (Supporting
Information Fig. 3C).
To date, TSP-2 was known as an antiangiogenic multi-
functional protein that interacts with diverse cellular regula-
tory factors [30,31]. In the context of skeletal function, the
role of TSP-2 in bone remodeling has been intensively stud-
ied by Hankenson’s group, who revealed that TSP-2 affects
proliferation and osteogenic differentiation of MSCs [32,33].
Mice lacking TSP-2 display increased bone formation and
MSCs numbers [32]. Moreover, TSP-2 is an autocrine inhibi-
tor of MSCs proliferation [33]. Thus, TSP-2 plays an impor-
tant role in bone homeostasis in several conditions, such as
fracture healing [34], ovariectomy [35], and mechanical load-
ing [36]. Although the biological processes of chondrogenesis
and osteogenesis are tightly coupled, few reports have directly
investigated the chondrogenic effects of TSP-2. Of the throm-
bospondin family, only TSP-2 and cartilage oligomeric matrix
protein (TSP-5) are expressed in chondrocytes [37] and a lack
of TSP-2 results in connective tissue abnormalities in mice
[38]. However, these previous reports did not fully address
the direct chondrogenic effects of TSP-2.
In our study, we confirmed that TSP-2 promoted differen-
tiation of chondroprogenitor cells in a concentration-
dependent manner (Fig. 4A–4D). Furthermore, treatment of
an osteochondral defect model with TSP-2 resulted in carti-
lage regeneration (Fig. 6). Thus these data showed direct evi-
dence of TSP-2-mediated chondrogenic effects. Interestingly,
two isoforms of TSP-2 (molecular weights of 200 kDa and
125 kDa) were recently reported; 200 kDa species are
secreted in osteoblasts [39]. In this study, we could not detect
200 kDa TSP-2, but lower sized TSP-2 was expressed in
hUCB-MSCs with or without OA SF (data not shown). There-
fore, the expression of the TSP-2 isoforms seems to be cell
type-specific.
To understand the mechanism of action of TSP-2, the var-
ious signaling pathways related to the chondrogenic effects of
TSP-2 must be investigated. This include the protein kinase
signaling pathways, of PKCa, p38/MAPK, and ERK [25,26],
as we also confirmed (Fig. 4E–4H); we showed that TSP-2
enhanced these signaling pathways synergistically. Recently,
there have been reports that TSP-2 initiates chondrogenic dif-
ferentiation of MSCs by potentiation of the Notch signaling
pathway [27,40]. In our data, expression of TSP-2 rapidly
increased during coculture between hUCB-MSCs and chon-
droprogenitor cells. Therefore, we examined and verified the
involvement of the Notch signaling pathway in the TSP-2-
mediated differentiation of chondroprogenitor cells. Addition-
ally, it is possible that TSP-2 can also potentiate chondrogenic
differentiation of hUCB-MSCs by autocrine action via the
Notch signaling pathway.
Our findings suggested that the secretome of hUCB-
MSCs, including TSP-2, can be regulated by the pathological
condition, which may modulate the chondrogenic effects of
transplanted hUCB-MSCs. Thus, finding factors in OA SF
associated with the specific TSP-2 expression pattern is
important for predicting the therapeutic effect of transplanted
hUCB-MSCs, which may also guide selection of patients who
have these factors related to TSP-2 expression.
There are two available transplantation routes for delivery
of MSC-based cartilage disease therapy, such as scaffold-
assisted application or direct intra-articular injection of MSCs
[41]. Here, we used 4% HA gel as a scaffold for delivery of
hUCB-MSCs into defect sites with exposed marrow; this
scaffold-assisted application of MSC is a prerequisite for the
surgical process. On the other hand, there are several studies
showing that delivery of MSCs in HA solution by intra-
articular injection into damaged joints enhances cartilage
regeneration [42–44]. These studies suggested that HA facili-
tates the migration and adherence of injected MSC into
injured sites. Based on these studies, it is possible that direct
intra-articular injection of hUCB-MSCs in HA solution allow
greater response to the microenvironment, resulting in carti-
lage repair by paracrine action.
Because MSCs differentiate into cells of a mesodermal
lineage, including cartilage, bone, tendon, and ligament [45],
most studies using MSCs for repair of cartilage defects have
focused on MSC-mediated cartilage formation [46].
Recently, it has been acknowledged that paracrine actions of
MSCs play a role in regeneration of damaged cartilage [11],
leading to trials for applying the paracrine action of MSCs
in cartilage repair [11–15]. There have been several reports
that the immunomodulatory effects of MSCs control expres-
sion of inflammatory cytokines in damaged cartilage tissue
[12,47–50]. However, it is uncertain whether the cartilage
newly formed after MSC transplantation is generated by
paracrine actions of MSCs in vivo. To address this issue, we
monitored the fate of transplanted hUCB-MSCs using
reverse transcriptase PCR of human gene and PKH 26 label-
ing during cartilage repair. As a result, transplanted hUCB-
MSCs were detected at 4 weeks, but not at 8 weeks, after
transplantation (Supporting Information Fig. 2A, 2B). Our
data indirectly indicated that the regenerated cartilage after
hUCB-MSCs transplantation was of recipient origin, imply-
ing the involvement of host cells, such as CPC [51,52], in
cartilage repair. Similarly, several studies demonstrated the
Jeong, Kim, Ha et al. 2145
www.StemCells.com
ACKNOWLEDGMENTS
This study was supported by a grant from the “Innovative
Research Institute for Cell Therapy,” (A062260) sponsored by
the Ministry of Health, Welfare & Family, Republic of Korea
DISCLOSURE OFPOTENTIAL
CONFLICTS OFINTEREST
The authors indicate no potential conflicts of interest.
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Jeong, Kim, Ha et al. 2147
www.StemCells.com
Thrombospondin-2 Secreted by Human Umbilical Cord
Blood-Derived Mesenchymal Stem Cells Promotes
Chondrogenic Differentiation
SANGYOUNGJEONG,
a,b
DONGHYUNKIM,
a
JUEUNHA,
a
HYEJINJIN,
a
SOON-JAEKWON,
a
JONGWOOKCHANG,
a
SOOJINCHOI,
a
WONILOH,
a
YOONSUNYANG,
a
GONHYUNGKIM,
c
JAESUNGKIM,
d
JUNG-ROYOON,
e
DONGHYUNGCHO,
b
HONGBAEJEON
a
a
Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Seoul, Republic of Korea;
b
Graduate School of
East-West Medical Science, Kyung Hee University, Yongin, Gyeonggi-Do, Republic of Korea;
c
Laboratory of
Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic
of Korea;
d
Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul,
Republic of Korea;
e
Department of Orthopedic Surgery, Seoul Veterans Hospital, Seoul, Republic of Korea
Key Words:Human umbilical cord blood-derived mesenchymal stem cells•Paracrine action•Osteoarthritis•Secretome•Synovial
fluid•Thrombospondin-2
ABSTRACT
Increasing evidence indicates that the secretome of mesen-
chymal stem cells (MSCs) has therapeutic potential for the
treatment of various diseases, including cartilage disorders.
However, the paracrine mechanisms underlying cartilage
repair by MSCs are poorly understood. Here, we show that
human umbilical cord blood-derived MSCs (hUCB-MSCs)
promoted differentiation of chondroprogenitor cells by
paracrine action. This paracrine effect of hUCB-MSCs on
chondroprogenitor cells was increased by treatment with
synovial fluid (SF) obtained from osteoarthritis (OA)
patients but was decreased by SF of fracture patients, com-
pared to that of an untreated group. To identify paracrine
factors underlying the chondrogenic effect of hUCB-MSCs,
the secretomes of hUCB-MSCs stimulated by OA SF or
fracture SF were analyzed using a biotin label-based anti-
body array. Among the proteins increased in response to
these two kinds of SF, thrombospondin-2 (TSP-2) was spe-
cifically increased in only OA SF-treated hUCB-MSCs. In
order to determine the role of TSP-2, exogenous TSP-2 was
added to a micromass culture of chondroprogenitor cells.
We found that TSP-2 had chondrogenic effects on chondro-
progenitor cells via PKCa, ERK, p38/MAPK, and Notch
signaling pathways. Knockdown of TSP-2 expression on
hUCB-MSCs using small interfering RNA abolished the
chondrogenic effects of hUCB-MSCs on chondroprogenitor
cells. In parallel with in vitro analysis, the cartilage regen-
erating effect of hUCB-MSCs and TSP-2 was also demon-
strated using a rabbit full-thickness osteochondral-defect
model. Our findings suggested that hUCB-MSCs can stimu-
late the differentiation of locally presented endogenous
chondroprogenitor cells by TSP-2, which finally leads to
cartilage regeneration.STEMCELLS2013;31:2136–2148
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
Cartilage is a highly differentiated and avascular tissue and
consequently has a low self-regeneration capacity; full regener-
ation of damaged cartilage, such as in osteoarthritis (OA), is
therefore challenging. Many research groups have attempted to
increase the regeneration potential of damaged cartilage using
mesenchymal stem cells (MSCs) including our phase I/IIa clin-
ical trial using MSCs for cartilage repair (Clinical Trials Gov
Identifier: NCT01733186) under approval of the Food and
Drug Administration.
MSCs can be isolated from various tissues and can differ-
entiate into chondrogenic lineage cells in vitro and in vivo
[1,2]. Human umbilical cord blood-derived MSCs (hUCB-
MSCs) are an alternative stem cell source with several advan-
tages, including noninvasive collection methods, hypoimmuno-
genicity, superior tropism, and differentiation potential [3–5].
Many studies have reported the therapeutic activity of the
MSC secretome. The paracrine effects of MSCs promote
Author contributions: S.Y.J.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript
writing; D.H.K., J.E.H., and H.J.J.: collection and/or assembly of data; S.-J.K., J.W.C., W.I.O., and D.H.C.: data analysis and interpreta-
tion; S.J.C.: provision of study and patient material; Y.S.Y.: data analysis and interpretation and financial support; G.H.K.: collection
and/or assembly of data and data analysis and interpretation; J.S.K.: conception and design, collection and/or assembly of data, and data
analysis and interpretation; J.-R.Y.: provision of study and patient material; H.B.J.: conception and design, data analysis and interpreta-
tion, manuscript writing, and final approval of manuscript.
Correspondence: Hong Bae Jeon, Ph.D., Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul 137–874, Republic of Korea. Tele-
phone:182-2-3465-6772; Fax:182-2-475-1991; e-mail: jhb@medi-post.co.kr Received November 29, 2012; accepted for publication
May 20, 2013; first published online inSTEMCELLSEXPRESSJuly 10, 2013.VCAlphaMed Press 1066-5099/2013/$30.00/0 doi: 10.1002/
stem.1471
STEMCELLS2013;31:2136–2148 www.StemCells.com
fracture healing and restore new bone formation [6], and fac-
tors released from MSCs recruit macrophages and endothelial
lineage cells into the wound, thus enhancing wound healing
[7]. Furthermore, autologous MSC transplantation attenuates
left ventricular remodeling and improves cardiac performance
via the paracrine action of the engrafted cells [8]. Moreover,
we have shown that hUCB-MSCs reduce neuronal cell death
by secreting galectin-3 [9] and decrease Abplaques by induc-
ing neprilysin [10]. Such paracrine actions of MSCs can
affect cartilage repair [11–15].
Despite these possible beneficial effects, the mechanism
by which MSCs and their secreted factors influence cartilage
repair remains unclear, necessitating analysis of the hUCB-
MSC secretome. However, in clinical trials, the effector mole-
cules produced by MSCs under in vitro conditions are often
not related to the therapeutic effects observed under clinical
conditions [16]. Such discrepancies between in vitro and in
vivo effects of MSCs may result from their interactions with
different microenvironments. Synovial fluid (SF) acquired
from osteoarthritic joints (OA SF) can represent the in vivo
disease microenvironment [17,18]; OA SF modulates MSC
activity in vitro in a manner representative of the clinical con-
dition [19]. Therefore, experiments that closely mimic disease
microenvironments, such as OA SF, should be useful.
Here, we demonstrated a chondrogenic differentiation effect
via the paracrine action of hUCB-MSCs using a coculture sys-
tem of chondrogenic progenitor cells (CPC) derived from
mouse limb buds. To determine whether hUCB-MSC paracrine
molecules responded to SF, we analyzed the secretome profile
of media from hUCB-MSCs conditioned with SF from OA and
fracture patients, using a biotin label-based antibody array. One
of the differentially expressed proteins, thrombospondin-2
(TSP-2), was selected and its effect on chondrogenic differen-
tiation of progenitor cells was validated using micromass cul-
ture and an in vivo osteochondral-defect model. This is the
first report to identify factors secreted from hUCB-MSCs in
response to OA SF and to demonstrate that TSP-2 is a hUCB-
MSC paracrine factor with chondrogenic effects.
MATERIALS AND METHODS
Culture of hUCB-MSCs
This study was approved by the Institutional Review Board of
MEDIPOST Co., Ltd. Neonatal hUCB was collected from umbili-
cal veins, with informed maternal consent. Mononuclear cells
were isolated from hUCB by centrifugation on a Ficoll-Hypaque
gradient (density: 1.077 g/cm
3
; Sigma, St. Louis, MO). Separated
mononuclear cells were washed, suspended ina-minimum essen-
tial medium (a-MEM; Gibco, Carlsbad, CA, http://www.invitro-
gen.com) supplemented with 10% (v/v) fetal bovine serum
(FBS; Gibco) and 50mg/mL gentamicin (Gibco), and seeded at
a concentration of 5310
5
cells per centimeter square in cul-
ture flasks. Cultures were maintained at 37
C in a humidified
5% CO
2atmosphere with a twice-weekly medium change.
After 1–3 weeks, when the monolayer of fibroblast-like adher-
ent cell colonies had reached 80% confluence, the cells were
detached with TrypLE Express (Gibco), washed, resuspended
in culture medium (a-MEM supplemented with 10% FBS and
50mg/ml gentamicin), and subcultured. In all experiments,
hUCB-MSCs used were at passage 6.
Acquisition and Treatment of Cells with SF
All patients provided appropriate written informed consent. SF
was withdrawn from the knee joints of four female and two male
patients (mean [SD]: 60.5 [11.5] years) diagnosed with OA, grade
4, and a knee joint of two female patients (age: 31 and 36 years),
each of which had fractured a knee in an accident. Radiographs
were taken of all OA knees and scored for Kellgren and Law-
rence (KL) grading (0–4). SF samples were centrifuged at 300g
for 5 minutes to sediment macromolecules and cell debris, and
the supernatants were stored at280
C until required for analysis.
hUCB-MSCs were cultured ina-MEM containing 10% (v/v) FBS
and 50mg/mL gentamicin. For the antibody array, the culture
medium from hUCB-MSCs at 80% confluence was replaced with
10% (v/v) SF diluted with serum-freea-MEM containing 50mg/
mL gentamicin, and the cells were cultured for 6 hours. To elimi-
nate SF contamination and to confirm the increased expression of
TSP-2, culture medium from hUCB-MSCs at 80% confluence
was replaced with 0.2% (v/v) SF diluted with serum-freea-MEM
containing 50mg/mL gentamicin, and cells were cultured for 6,
12, and 24 hours.
Micromass Culture of Chondroprogenitor Cells
Chondroprogenitor cells were isolated from the limb buds of 11.5
dpc ICR mouse embryos and maintained as micromass cultures
to induce chondrogenesis. Isolated chondroprogenitor cells were
suspended, without expansion (passage 0), in Dulbecco’s modi-
fied Eagle’s medium (DMEM, Gibco), containing 2% (v/v) FBS
and 50mg/mL gentamicin at 2.0310
7
cells per milliliter and
were spotted as 15-mL drops onto culture dishes to induce chon-
drogenesis for 6 days. Human recombinant TSP-2 (R&D Sys-
tems, Minneapolis, MN, http://www.rndsystems.com) was
prepared and applied to chondroprogenitor cells in serum-free
DMEM. As positive control, insulin-like growth factor-1 (100 ng/
mL; Merck Millipore, Billerica, MA, http://www.millipore.com)
was added to micromass culture medium [20].
Chondrogenesis was determined by staining of sulfated pro-
teoglycans and examining the expression of collagen type II,
aggrecan, hyaluronan and proteoglycan like protein 1 (HAPLN1),
and Sox-9 using Western blotting. Accumulation of sulfated pro-
teoglycans was detected by Alcian blue staining. During cocul-
ture, hUCB-MSCs or human embryonic kidney (HEK-293) cells
were cultured in the upper phase of transwells. hUCB-MSCs or
HEK-293 (9310
4
cells per 2 milliliter) were added to the upper
chamber, and 2 mL of DMEM containing 10% serum was added
to the lower chamber. After 24 hours of incubation at 37
C and
5% CO
2, cells on the upper surface of the chamber (which had
not penetrated the insert) were applied to progenitor cells in a
different culture plate. Alcian blue-stained cells were extracted
and quantified by measuring absorbance at 590 nm.
Gross Findings from Osteochondral-Defect Animal
Model
The animal experiment protocol was approved by the Institutional
Animal Care and Use Committee of Chungbuk National Univer-
sity. Thirty 10-week-old male New Zealand White (NZW) rab-
bits, each weighing 2–2.5 kg, were used. Knee joints of rabbits
were sterilely draped and were opened through a parapatellar
arthrotomy with general anesthesia. The patella was dislocated
laterally, and full-thickness articular osteochondral defects (5-mm
diameter; 5-mm depth) were induced in the trochlear groove by
carefully perforating the respective areas on the cancellous bone
using a biopsy punch. After elimination of cartilage, bone frag-
ments, and thrombi, the border of the perforated site was trimmed
using a surgical knife and cleansed from blood. Each group was
untreated, transplanted with hyaluronic acid (HA), HEK-293,
hUCB-MSCs, TSP-2 protein, and hUCB-MSCs treated with TSP-
2 small interfering RNA (siRNA), respectively. Mixtures of
hUCB-MSCs (1310
6
cells per 200mL) or HEK-293 (1310
6
cells per 200mL) with 4% sodium HA gel composite were
applied into the area of the full-thickness defect using a syringe.
TSP-2 (25mg/200mL) was also administered into the defect area
along with 4% sodium HA gel composite. The untreated group
received only a osteochondral defect. Finally, in all animals, the
patellar retinaculum and the overlaying skin were sutured. An
Jeong, Kim, Ha et al. 2137
www.StemCells.com
Figure 1.Enhanced differentiation of chondroprogenitor cells by coculture with hUCB-MSCs. Mouse limb bud chondroprogenitor cells were
prepared from 11.5 dpc mouse embryos and maintained as micromass culture in 2% fetal bovine serum-containing medium for 6 days. hUCB-
MSCs and chondroprogenitor cells were separately cocultured by a transwell approach in a culture plate. Effects of hUCB-MSCs on differentia-
tion of chondroprogenitor cells were monitored by synthesis of sulfated proteoglycans and expression of chondrogenic markers.(A):Chondropro-
genitor cells that had differentiated into mature cartilage were stained by Alcian blue dye.(B):The stained dye was extracted and quantified by
absorbance at 590 nm.(C):The expression of the chondrogenic markers, collagen type II, aggrecan, HAPLN1, and chondrogenic transcription
factor, Sox-9 was determined by Western blotting and quantified(D)using ImageJ software.(E):The photo of chondrogenic differentiated
ATDC5 pellets was represented.(F):Relative size difference among the pellets was measured using IMT i-SolutionTM software.(G):The frozen
pellets in OCT compound were cut and stained with Safranin O and anticollagen type II antibody.(H):The sulfated glycosaminoglycan contents
were extracted and determined. Data are shown as the mean (SD) of at least six independent experiments. *,p<.05. Scale bars5250mm.
Abbreviations: hUCB-MSC, human umbilical cord blood-derived mesenchymal stem cell; OA, osteoarthritis; HAPLN1, hyaluronan and proteo-
glycan link protein 1; SF, synovial fluid.
but expressions of cartilage-specific markers were lower than
those in the presence of TSP-2 (Fig. 4E–4H). These results
clearly demonstrated that TSP-2 has chondrogenic effects, which
are mediated through PKCa/ERK, p38/MAPK, and Notch sig-
naling pathways.
Knockdown of TSP-2 Abolishes Chondrogenic
Effects of hUCB-MSC on Differentiation
of Chondroprogenitor Cells
Since we had observed chondrogenic effects of TSP-2 in
hUCB-MSCs, we attempted to block expression of TSP-2 for
further validation. After TSP-2 siRNA treatment, expression
of TSP-2 was completely blocked (Fig. 5E) and knockdown
of TSP-2 was confirmed up to 28 days (Supporting Informa-
tion Fig. 4). siRNA-mediated knockdown of TSP-2 expression
led to decreased synthesis of sulfated proteoglycans (Fig. 5A,
5B) and expression of collagen type II, aggrecan, HAPLN1,
and Sox-9 (Fig. 5C, 5D), indicating reduced chondrogenesis.
However, use of HEK-293 cells as non-stem cells in coculture
did not replicate the findings in hUCB-MSCs (Fig. 5A–5D).
Collectively, these results suggested that the chondrogenic
effects of hUCB-MSCs are mediated via TSP-2.
Gross Findings and Histological Observations
of Effects of hUCB-MSCs and TSP-2 in an
Osteochondral-Defect Animal Model
To further validate the role of TSP-2 in cartilage regeneration
in an in vivo system, we made osteochondral defects in the
trochlear groove of rabbits and applied either hUCB-MSCs or
only TSP-2 into the defects. In addition, given our previous
in vitro data that hUCB-MSCs can promote differentiation of
endogenous chondroprogenitor cells, we used a full-thickness
cartilage defect model that exposed chondroprogenitor cells
from the marrow [28]. At 10 weeks after surgery, the gross
morphology of joints was observed and sections were stained
with hematoxylin and eosin and Safranin O. Upon gross
examination, the margins of the defects of untreated or HA-
treated groups were clearly recognizable and their surfaces
were irregular (Fig. 6Aa, 6Ab). The surfaces of defects trans-
planted with HEK-293 were depressed and most parts of the
defects were not filled with any tissue (Fig. 6Ac). The repara-
tive tissues of untreated and HA-treated groups were filled
with fibrous tissue and remained concave (Fig. 6Ag, m, 6Ah,
n). In the HEK-293-treated group, thin fibrous tissue was
observed on the subchondral bone (Fig. 6Ai, 6Ao). In con-
trast, the hUCB-MSC-treated group showed good lateral inte-
gration, macroscopically (Fig. 6Ae), and a hyaline-like
cartilage matrix was evident with Safranin O. The tissue was
hyaline-like, with good integration, thickness, and surface reg-
ularity. An enlarged image of the regenerated tissue showed
the lacunae structure (Fig. 6Ak, 6Aq). There was marked
improvement in the quality of the repaired tissue seen in the
hUCB-MSC-treated group compared with the untreated, HA,
and HEK-293-treated groups.
The surfaces of regenerated tissue in the TSP-2-treated
group were mostly not depressed, and the defects were cov-
ered with white opaque tissue compared with the hUCB-
MSC-treated group (Fig. 6Ad). Histologically, a magnified
view of the repaired tissue represented a partially hyaline-like
Figure 3.TSP-2 expression by hUCB-MSCs responded to treatment with SF from fracture and OA patients.(A):Western blots of cell lysates
from hUCB-MSCs treated with 10% SF from two fracture patients and four OA patients for 6 hours.(B):ELISA analysis on expression of TSP-
2 in culture media from hUCB-MSCs treated with 0.2% SF samples from two fracture patients and four OA patients for 6, 12, and 24 hours.
(C):TSP-2 concentrations in supernatants of micromass culture media. Data are shown as the mean (SD) of at least four independent experi-
ments. *,p<.05. Abbreviations: hUCB-MSC, human umbilical cord blood-derived mesenchymal stem cell; OA, osteoarthritis; SF, synovial fluid;
TSP-2, thrombospondin-2.
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Figure 4.Effects of exogenous TSP-2 on differentiation of chondroprogenitor cells.(A):Alcian blue staining of mouse limb bud chondroproge-
nitor cells which had been maintained as micromass culture for 6 days in the presence of TSP-2 (0.1–100 ng/mL) under serum-free conditions.
IGF-1 (100 ng/mL) was used as positive control for induction of chondrogenic differentiation.(B):Alcian blue-stained cells were extracted and
quantified by measuring absorbance.(C):Expression levels of collagen type II, aggrecan, HAPLN1, and Sox-9 protein were determined by West-
ern blotting;b-actin was used as loading control.(D): Expression of collagen type II protein was quantified by ImageJ software.(E):Chondro-
progenitor cells were maintained as micromass culture up to 6 days in the absence or presence of TSP-2 (100 ng/mL). Cells were concurrently
exposed to 1mM Go to inhibit PKCa,10mM of PD to inhibit ERK, 10mM of SB to inhibit p38/MAPK, or 50 nM DAPT to inhibitc-secretase.
Accumulation of sulfated proteoglycans was detected by Alcian blue staining.(F):Alcian blue-stained cells were extracted and quantified by
measuring absorbance.(G):Effects of inhibitors on expression of collagen type II, aggrecan, HAPLN1, and Sox-9 protein were detected by
Western blotting.b-actin was used as loading control.(H):Expressions of collagen type II, aggrecan, HAPLN1, and Sox-9 protein were quanti-
fied by ImageJ software. Data are presented as the mean (SD) result from four independent experiments. *,p<.05. Abbreviation: DAPT,N-[N-
(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycinet-butyl ester; HAPLN1, Hyaluronan and proteoglycan link protein 1; Go, Go6976; IGF-1,
insulin-like growth factor-1; PD, PD98059; SB, SB203580; TSP-2, thrombospondin-2.
structure (Fig. 6Aj, 6Ap). In defects transplanted with hUCB-
MSCs treated with TSP-2 siRNA, the surfaces were irregular
(Fig. 6Af). Defects showed dense fibrous tissue rather than
cartilage tissue (Fig. 6Al, 6Ar). Upon immunohistochemical
analysis, untreated, HA-treated, and HEK-293-transplanted
groups rarely expressed collagen type II (Fig. 6As–6Au),
whereas collagen type II was distributed throughout the neo-
cartilage in the hUCB-MSC-treated group (Fig. 6Aw). Defects
treated with TSP-2 were well repaired in deep zone of carti-
lage, but the superficial zone was partly unrepaired (Fig.
6Av). However, collagen type II expression in defects trans-
planted with hUCB-MSCs treated with TSP-2 siRNA was
observed at periphery of the defect sites (Fig. 6Ax). Histologi-
cal observations were quantified according to the modified
O’Driscoll scoring scale [21]. The mean modified O’Driscoll
scores of each experimental group correlated well with both
macroscopic and histological analyses (Fig. 6B). Both TSP-2-
and hUCB-MSC-treated groups had higher scores than those
of the control groups, including the untreated, HA-treated,
and HEK-293-treated groups (p<.01). The TSP-2-treated
group achieved a lower score compared with the hUCB-
MSC-treated group (p<.05). The score in the hUCB-MSCs
Figure 5.Reduced differentiation of chondroprogenitor cells by inhibition of TSP-2 in hUCB-MSCs. hUCB-MSCs were transfected for 24
hours with negative control siRNA (25 nM) or TSP-2 siRNA (25 nM). After a 24-hour incubation, transfected hUCB-MSCs were applied to
chondroprogenitor cells in a coculture system. Chondroprogenitor cells were cultured with hUCB-MSCs or HEK-293 (non-stem cells) cells up to
6 days in 2% fetal bovine serum-containing medium.(A):Chondroprogenitor cells stained with Alcian blue at day 6.(B):Alcian blue stain was
extracted from the cells and quantified by measuring absorbance.(C):Protein levels of collagen type II, aggrecan, HAPLN1, and Sox-9 were
determined by Western blotting;b-actin was used as loading control.(D):Expression of collagen type II protein was quantified by ImageJ soft-
ware.(E):TSP-2 concentrations were measured in the supernatants of each culture condition. Data are shown as the mean (SD) of at least four
independent experiments. *,p<.05. Abbreviations: hUCB-MSCs, human umbilical cord blood derived mesenchymal stem cells; HAPLN1, hya-
luronan and proteoglycan link protein 1; siRNA, small interfering RNA; TSP-2, thrombospondin-2.
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Figure 6.Macroscopic and histological analysis and immunostaining of hUCB-MSC-treated and TSP-2-injected knees in a full-thickness osteo-
chondral-defect rabbit model.(A):Six experimental groups, including an untreated group (n55), and groups treated with 4% HA (n55), a com-
posite of HEK-293 (5310
6
cells/ml) and 4% HA (n55), TSP-2 (25mg) and 4% HA (n55), a composite of hUCB-MSCs (5310
6
cells/ml)
and 4% HA (n55), and a composite of hUCB-MSCs treated with TSP-2 siRNA (5310
6
cells/ml) and 4% HA (n55) were used. At 10 weeks
postoperatively, animals were sacrificed and the defect area was observed and stained for analysis. Gross findings are shown in(A–F). Femoral
condyles were sectioned and stained with hematoxylin and eosin(G–L)and Safranin-O(M–R). For immunohistochemical analysis, collagen type
II was detected as brown color(S–X). The untreated group (saline treatment) showed poor healing (a, g, m, and s). The defects in the HA-treated
group were rarely recovered (b, h, n, and t). The defects treated with HEK-293 exhibited irregular surfaces similar to the HA-treated group (c, i,
o, and u). In the TSP-2-treated group, the regenerated tissue was slightly less recovered compared to the hUCB-MSCs-treated group but showed
better-integrated cartilage tissue than in the untreated, HA-treated, and HEK-293 groups (d, j, p, and v). The hUCB-MSCs-treated animals demon-
strated good defect filling, with a smooth surface and regenerated cartilage tissue (e, k, q, and w). The defects transplanted with hUCB-MSCs
treated with TSP-2 siRNA represented poorly regenerated tissue compared with TSP-2- and hUCB-MSCs-treated groups (f, l, r, and x). Scale
bars51 mm (G–X). (B): Using the modified O’Driscoll scoring scale, the integration rates were quantified in the six experimental groups at 10
weeks. Data are shown as the mean (SD) of each group (n55). Statistical significance was determined using Kruskal–Wallis test followed by
Mann–WhitneyUtest. *,p<.05 or **,p<.01. Abbreviations: hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells; HA,
Hyaluronate; siRNA, small interfering RNA; TSP-2, thrombospondin-2.
treated with TSP-2 siRNA decreased compared with that in
the hUCB-MSC-treated group (p<.01). Collectively, these
data indicated that hUCB-MSCs have cartilage regeneration
effects in an osteochondral defect model, which are mainly
mediated by the secreted TSP-2.
DISCUSSION
Here, we demonstrated that hUCB-MSCs promoted differen-
tiation of chondroprogenitor cells by paracrine action. This
chondrogenic effect was modulated by the surrounding envi-
ronment, such as OA or fracture SF. Through secretome array
analysis and in vitro evaluation, we found that hUCB-MSCs
secreted trophic factors that varied with the pathological con-
ditions, such as exposure to OA SF. Moreover, expression of
TSP-2 by hUCB-MSCs was specific to OA SF treatment and
enhanced cartilage regeneration in an osteochondral defect
animal model.
When MSCs are transplanted into cartilage lesions, they
are exposed to a complex microenvironment, including
inflammatory cytokines, proteases, and degraded extracellular
matrix secreted from damaged cartilage, which can modulate
paracrine activities of MSCs. For instance, the proinflamma-
tory cytokine, tumor necrosis factor-a, changes the secretome
of human adipose tissue-derived MSCs [29]. In this study,
OA SF-treated hUCB-MSCs increased the chondrogenic dif-
ferentiation of chondroprogenitor cells, compared to a non-
treated hUCB-MSC group, whereas fracture SF-treatment of
hUCB-MSCs decreased chondrogenic differentiation (Fig. 1).
Furthermore, secretome analysis of hUCB-MSCs induced by
these SFs resulted in distinct expression patterns (Fig. 2; Sup-
porting Information Table 2). These data indicated that the
pathological condition can modulate the paracrine action of
hUCB-MSCs. In elucidating this modulation, we found that,
among factors secreted from hUCB-MSCs, only TSP-2 was
specifically expressed upon OA SF-treatment (Fig. 3). Indeed,
TSP-2 levels also markedly increased in the SF of OA patient
two weeks after hUCB-MSCs transplantation (Supporting
Information Fig. 3C).
To date, TSP-2 was known as an antiangiogenic multi-
functional protein that interacts with diverse cellular regula-
tory factors [30,31]. In the context of skeletal function, the
role of TSP-2 in bone remodeling has been intensively stud-
ied by Hankenson’s group, who revealed that TSP-2 affects
proliferation and osteogenic differentiation of MSCs [32,33].
Mice lacking TSP-2 display increased bone formation and
MSCs numbers [32]. Moreover, TSP-2 is an autocrine inhibi-
tor of MSCs proliferation [33]. Thus, TSP-2 plays an impor-
tant role in bone homeostasis in several conditions, such as
fracture healing [34], ovariectomy [35], and mechanical load-
ing [36]. Although the biological processes of chondrogenesis
and osteogenesis are tightly coupled, few reports have directly
investigated the chondrogenic effects of TSP-2. Of the throm-
bospondin family, only TSP-2 and cartilage oligomeric matrix
protein (TSP-5) are expressed in chondrocytes [37] and a lack
of TSP-2 results in connective tissue abnormalities in mice
[38]. However, these previous reports did not fully address
the direct chondrogenic effects of TSP-2.
In our study, we confirmed that TSP-2 promoted differen-
tiation of chondroprogenitor cells in a concentration-
dependent manner (Fig. 4A–4D). Furthermore, treatment of
an osteochondral defect model with TSP-2 resulted in carti-
lage regeneration (Fig. 6). Thus these data showed direct evi-
dence of TSP-2-mediated chondrogenic effects. Interestingly,
two isoforms of TSP-2 (molecular weights of 200 kDa and
125 kDa) were recently reported; 200 kDa species are
secreted in osteoblasts [39]. In this study, we could not detect
200 kDa TSP-2, but lower sized TSP-2 was expressed in
hUCB-MSCs with or without OA SF (data not shown). There-
fore, the expression of the TSP-2 isoforms seems to be cell
type-specific.
To understand the mechanism of action of TSP-2, the var-
ious signaling pathways related to the chondrogenic effects of
TSP-2 must be investigated. This include the protein kinase
signaling pathways, of PKCa, p38/MAPK, and ERK [25,26],
as we also confirmed (Fig. 4E–4H); we showed that TSP-2
enhanced these signaling pathways synergistically. Recently,
there have been reports that TSP-2 initiates chondrogenic dif-
ferentiation of MSCs by potentiation of the Notch signaling
pathway [27,40]. In our data, expression of TSP-2 rapidly
increased during coculture between hUCB-MSCs and chon-
droprogenitor cells. Therefore, we examined and verified the
involvement of the Notch signaling pathway in the TSP-2-
mediated differentiation of chondroprogenitor cells. Addition-
ally, it is possible that TSP-2 can also potentiate chondrogenic
differentiation of hUCB-MSCs by autocrine action via the
Notch signaling pathway.
Our findings suggested that the secretome of hUCB-
MSCs, including TSP-2, can be regulated by the pathological
condition, which may modulate the chondrogenic effects of
transplanted hUCB-MSCs. Thus, finding factors in OA SF
associated with the specific TSP-2 expression pattern is
important for predicting the therapeutic effect of transplanted
hUCB-MSCs, which may also guide selection of patients who
have these factors related to TSP-2 expression.
There are two available transplantation routes for delivery
of MSC-based cartilage disease therapy, such as scaffold-
assisted application or direct intra-articular injection of MSCs
[41]. Here, we used 4% HA gel as a scaffold for delivery of
hUCB-MSCs into defect sites with exposed marrow; this
scaffold-assisted application of MSC is a prerequisite for the
surgical process. On the other hand, there are several studies
showing that delivery of MSCs in HA solution by intra-
articular injection into damaged joints enhances cartilage
regeneration [42–44]. These studies suggested that HA facili-
tates the migration and adherence of injected MSC into
injured sites. Based on these studies, it is possible that direct
intra-articular injection of hUCB-MSCs in HA solution allow
greater response to the microenvironment, resulting in carti-
lage repair by paracrine action.
Because MSCs differentiate into cells of a mesodermal
lineage, including cartilage, bone, tendon, and ligament [45],
most studies using MSCs for repair of cartilage defects have
focused on MSC-mediated cartilage formation [46].
Recently, it has been acknowledged that paracrine actions of
MSCs play a role in regeneration of damaged cartilage [11],
leading to trials for applying the paracrine action of MSCs
in cartilage repair [11–15]. There have been several reports
that the immunomodulatory effects of MSCs control expres-
sion of inflammatory cytokines in damaged cartilage tissue
[12,47–50]. However, it is uncertain whether the cartilage
newly formed after MSC transplantation is generated by
paracrine actions of MSCs in vivo. To address this issue, we
monitored the fate of transplanted hUCB-MSCs using
reverse transcriptase PCR of human gene and PKH 26 label-
ing during cartilage repair. As a result, transplanted hUCB-
MSCs were detected at 4 weeks, but not at 8 weeks, after
transplantation (Supporting Information Fig. 2A, 2B). Our
data indirectly indicated that the regenerated cartilage after
hUCB-MSCs transplantation was of recipient origin, imply-
ing the involvement of host cells, such as CPC [51,52], in
cartilage repair. Similarly, several studies demonstrated the
Jeong, Kim, Ha et al. 2145
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ACKNOWLEDGMENTS
This study was supported by a grant from the “Innovative
Research Institute for Cell Therapy,” (A062260) sponsored by
the Ministry of Health, Welfare & Family, Republic of Korea
DISCLOSURE OFPOTENTIAL
CONFLICTS OFINTEREST
The authors indicate no potential conflicts of interest.
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